Contributory presentations/posters, XIII International Biophysics Congress in New Delhi in 1999

CONTRIBUTORY PRESENTATIONS I POSTERS

A1 : Proteins : Structure and Dynamics

P1 CRYSTAL STRUCTURE OF WINGED BEAN ACIDIC LECTIN

N. Mano|, V.R. Srinivas, A. Surolia, M. Vijayan & K. Suguna Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, INDIA.

The acidic lectin from winged bean (Psophocarpus tetragonolobus), WBAI1, is an N-glycosylated dimeric protein of molecular weight 58kD. It binds strongly to the H-antigenic determinant on human erythrocytes and also to the T-antigenic disaccharide GaI-131,3-GalNAc. Crystals of WBAII in complex with methyl-ct-D-galactose were obtained in two different forms. The structures were solved by the molecular replacement method using the coordinates of the basic form of winged bean lectin (WBAt), and refined. Clear density for the bound carbohydrate could be seen in the Fourier maps. The strucure provided details of protein-carbohydrates interactions. A close examination of the binding site with modelled sugars revealed the structural basis for the strong affinity of WBAII for the H-antigen and other teminally monofucosyJated carbohydrates. It was also possible to explain the differences in the specificities of WBAII and WBAI, which binds only to the A- and B-type antigens but not to the H-antigen, from this analysis.

P 2 A COMPARATIVE STUDY OF CARBOHYDRATE BINDING IN GAL/GALNAC SPECIFIC LEGUME LECTINS

R. Ravishankar, K. Sueuna, A. Surolia and M. Vijayan, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India

Peanut lectin (PNA) is a 110kD, homotetrameric, legume lectin with specificity for galactose at the monosaccharide level, unlike other galactose binding legume lectins of known structure which also bind to N-acetylgalactosamine (GalNAc). X-ray analysis of PNA complexes with lactose (Gai[31-4Glc), N-acetyllactosamine (Gal~ 1-4GlcNAc), methyl-[~-galactose and T-antigen (Gal~ 1-3GalNAc) have resulted in a wealth of structural data on legume lectin carbohydrate interactions. There are 4 invariant hydrogen bonds involving galactose in all legume iectins, viz. Asp83 ODl - galactose 03, Asp83 OD2 - galactose 04, Glyl04 N - galactose 03 and Asnl27 ND2 - galactose 03. The complexes provide a qualitative structural rationale for differences in the binding strengths of PNA to these sugars. The water molecules in the combining site are substantially conserved in the four complexes. Nine water clusters were identified when all the binding sites were superposed. Some of the water molecules mimic the role of interacting sugar hydroxyls, when absent, not only at the primary combining site, but also at the site of the second sugar ring. A subsequent comparison of the binding sites of all Gal/GalNAc binding legume lectins of known structure shows that the similarity of peanut iectin carbohydrate interactions with those in other Gal/GalNAc specific lectins extend to a substantial degree, to water bridges as well. The comparative study also provides a structural explanation for the exclusive specificity of peanut lectin for galactose at the monosaccharide level as against that of the other lectins for galactose as well as N-acetylgalactosamine.

P 3 CRYSTAL STRUCTURE OF HUMAN APOLIPOPROTEIN H

R.Schwarzenbacher T, K.Zeth 2, K.Diederichs 2, G.M.Kostner 3, A.Gries, P.Laggner I and R.Prassl T qnstitute of Biophysics and X-ray Structure Research, Austrian Academy of Sciences, Graz, Austria, 2Faculty of Biology, University of Konstanz, Germany and 3Institute of Medical Biochemistry, University of Graz, Austria

Apolipoprotein-H (apo-H), also designated as 132-glycoprotein-I, is associated with circulating lipoproteins and preferentially binds to negatively charged phospholipids. Apo H acts as cofactor for the binding of antiphospholipid autoantibodies in patients with antiphospholipid syndrome.

Apo-H is a glycoprotein (Mr 43kDa) which contains four short consensus repeats (SCRs) and a fifth domain, which was suggested to act as a lipid binding region. SCR domains are modules of--60 amino acids with four invariant cysteins and a small number of conserved residues. SCRs are common in many proteins involved in the complement regulatory system.

The 2.9A crystal structure reveals an extended elongated spatial arrangement of the SCRs with some bend towards the fifth domain. The SCRs in Apo-H fold into the characteristic beth-barrel structure with high structural homology to each other. The fifth domain contains 3 disulfide linkages stabilizing a four stranded beta-spiral with extended loop-regions containing the surface exposed lysine rich lipid binding region.

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P4 The Unliganded Structure of the cAMP-Dependent Protein Kinase Catalytic Subunit

Nladhusudan ~, Pearl Akamine ~, Nguyen-huu Xuong 2 and Susan S. Taylor t ~Howard Huges Medical Institute, Department of Chemistry & Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0654, USA. 2Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA 92093-0359, USA.

The crystal structure of the apoenzyme of the recombinant murine catalytic subunit of the cAMP-dependent protein kinase has been refined to 3A, with R/Rfree values of 23/33%. Structure was solved by molecular replacement method using program AMoRe from CCP4 suite. A complete 2.9 A data set was collected at the Stanford Synchrotron Radiation Laboratory and further refinement is in progress. This unliganded form adopts a more open conformation relative to the other available structures suggesting that ligand binding results in significant conformational changes in the small lobe - which predominantly contributes to ATP binding. This correlates with the known biochemical data. The structural comparison of the apo form with substrateJinhibitor bound ternary complexes and nucleoside-binary complex will provide important insights into the catalytic cycle of the enzyme.

P5 STRUCTURAL ANALYSIS OF WILD TYPE E. coli URACIL DNA GLYCOSYLASE (ECUDG) AND

NEW CRYSTAL FORMS OF ITS COMPLEX WITH A PROTEINACEOUS INHIBITOR, Ugi.

M.Bidya Sagar, R.Ravishankar, K.Saikrishnan,S.RoyX,K.PurnapatreI,P.HandaI,U.Varshney I & M. Vijayan, Molecular Biophysics Unit and 1Department of Microbiology &Cell Biology, lndian Institute of Science,

Ban galore -560 012, India We had earlier reported the crystal structure of a complex between the DNA repair enzyme Uracil DNA

glycosylase (UDG) from E.coli and its proteinaceous inhibitor Ugi from the Bacillus subtilis bacteriophage, PBS-2. Subsequently, the structure of a different crystal form of the complex, the crystal structure of free Ugi and the structure of a mutant of UDG have been reported from other laboratories. We have now determined the structures of two new crystal forms of the UDG-Ugi complex and the crystals of free wild-type UDG. A comparative study of the 10 crystailographically independent molecules in the different structures of UDG-Ugi complex shows that the central B-sheet and the four major helices in the UDG molecule remain rigid while flexibility is confined to the other stretches of the polypeptide chain, particularly the loops. The four molecules in the asymmetric unit of the crystal of free UDG have very nearly the same structure. Somewhat larger differences however occur between the bound UDG and free UDG structures. The differences between the bound and the free Ugi molecules are still larger. As in the case of the bound enzyme, free UDG is structurally closer to the human enzyme than to the herpes enzyme.

P6 Plas t ic i ty and H y d r a t i o n o f l y sozyme . X- ray analysis o f a crys ta l fo rm g r o w n at nea r neu t ra l

p H and its low h u m i d i t y var ian t and a c o m p a r a t i v e s t u d y of known crystal s t ruc tu res . B. K. Biswal, N. S u k u m a r & M. Vi jayan

Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, INDIA. Most of the crystal forms of lysozyme are grown at acidic pH. We have recently determined the structures of the orthorhombic form grown at basic pH and its low humidity variant obtained through a water-mediated transformation in which the crystal undergoes a reversible transformation with change in solvent content when the environmental humidity is systematically varied. We have now analysed the structure of a form grown at pH 6.5 and its low humidity variant. The comparison of the relevant structures demonstrate that the variation in the molecular structure as a function of pH is much lower than caused by change in solvent content. This study led to a detailed comparative examination of 20 crystal structures of lysozyme, 8 determined in this laboratory, corresponding to widely different environmental conditions. This examination permits the delineation of the rigid and the flexible regions of the molecule in terms of main chain conformation and that of conformationally invariant and variant side chains. There is no strong correlation between variability in the main chain and side chains except at the binding site which is divided into" relatively rigid and flexible halves. The study leads to the identification of a few invariant water molecules in the hydration shell, most of which are involved in stabilizing the structure through intramolecular water bridges. It also provides insights into the relation between accessibility and hydration.

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P 7 Rabbi t muscle creatine kinase - Crystal structure and its implications. J.K. Mohana Rao,

Macromolecular Structure Laboratory, NCI - Frederick Cancer Research and Development Center, ABL - Basic Research Program, P. O. Box B, Frederick, MD 21702, USA.

The creatine kinase (CK) system is involved in energy buffeting as well as energy transport. The enzyme is a marker in several diseases such as myocardial infarction, brain tumors and certain types of cancer. The crystal structure of homodimeric rabbit muscle creatine kinase (rMCK) was elucidated at a resolution of 2.35 A [Rao, Bujacz and Wlodawer: FEBS Letters (1998) 439, 133-137]. Each CK monomer consists o f two domains: a predominantly helical one at the N-terminus and an antiparalle113 sheet and long ~ helices at the C-terminus. The active site, with bound sulphates making contacts with a cluster ofarginine residues, occurs in the C-terminal domain. The rMCK structure was compared with those of the octameric chicken mitochondrial CK and the related monomeric arginine kinase (AK). This study, to some extent, explains why octamers may not be present in rMCK and dimers in AK. These and other structural results on some mutants o f rMCK will be presented.

Research supported by the National Cancer Institute, DHHS, under contract with ABL.

P 8 Interesting Hydration Patterns and their Role in Trypsin Activity

A. Johnson and Vasantha Pattabhi. Department of Crystallography and Biophysics, University of Madras, Guindy Campus,

Guindy, Chennai 600 025. E-mail : [email protected]

The crucial role of waters in biological system is very well recognised. Protein structure is stabilized to a substantial extend by hydrophobic interactions in the aqueous medium. Here we report the interesting hydration patterns observed in the crystal structures o f porcine ct and 13 trypsins which were solved in P212~2) space group and refined up to 1.8 and 1.63 A respectively. Comparison of the native trypsin structure with the autolysates and inhibitor complexes reveals a complete network of functional waters from the recognition pocket to the active site entry for the first time. The binding inhibitor molecule replaces most of these water molecules. Small molecule inhibitors and protease inhibitors replace different regions of this functional water network. The water molecules that are necessary for the stability of the autolysis loop have also been identified. The relative orientation of His 57 and Ser 195, which is important for the activity of the enzyme, is different for liganded and unliganded trypsin molecules. Acetate ion binding results in an intermediate orientation for these active site residues in the native 13 trypsin. Details relating to these results will be presented.

P 9 Investigations on the structure and stability of physalis mottle tymovirus

S. Sri Krishna l, Mira Sastri 2 and H.S.Savithri 2, M. R. N. Murthy l IMolecular Biophysics Unit and 2Biochemistry Department

Indian Institute of Science, Bangaiore, 560 012, India. Physaiis mottle virus (PhMV) is a single stranded RNA plant tymovirus. The virus particles contain 3 subunits (A, B and C) in the icosahedral asymmetric unit. The X-ray crystal structure of the virus was determined to 3.8 A resolution by molecular replacement using the known structure of turnip yellow mosaic virus (TYMV) as the starting model. The final map accounts for all, except residues 1-10 of the A subunit and the N-terminai residue's of B and C subunits. The coat protein structure consists of a flexible N-terminal arm (residues 1-27) and an eight stranded antiparailel 13-barrel (residues 28-188). The ordered segment of the N-terminal residues of the A subunits, which constitute 12 Pentamers at the icosahedrai 5-fold axes interact with a hexamer formed by B and C subunits. In contrast, the N-terminal residues of B and C subunits have a very different conformation. The hexamers are held more strongly than pentamers and hexamer-bexamer contacts are more extensive than pentamer-hexamer contacts. The structure suggests a plausible mechanism for the release of nucleic acid and the formation of empty capsids triggered by a change in the conformation of the N-terminal arm. However, stability studies on a number of deletion and substitution mutants of PhMV suggest that the information for virus assembly might lie outside the N-terminal arm. Interestingly, some of the mutants assemble into a 19S aggregate, which is likely to be an intermediate in the assembly/disassembly of the virus.

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P I O 1,9A X-ray study reveals closed flaps in unlignnded HIV-I PR tethered dimmer

Bindu Pillai, K. K. Kannan and M. V. Hosur Condensed Matter Physics Division, B.A.R.C., Trombay, Mumbai - 400085.

Human Immunodeficiency Virus (HIV) is the causative agent of the fatal disease AIDS which is fast spreading into normal populations in India. The virus encodes its own protease enzyme which is absolutely required for the propagation of the virus. Therefore inhibitors of this enzyme are being developed as potential drugs against AIDS. An accurate three dimensional structure is required to ac tasa template for purposes of structure-based drug-design. We have undertaken to study structures of wild-type and mutants of tethered HIV-I PR. We have expressed this HIV-I protease in bacteria, and have purified the enzyme in large quantities for structural studies. Single crystals of the native tethered dimer belong to the hexagonal space group P61 with a=63.2A and c=88.4A, and contain one tethered dimer in the asynunetric unit. X-ray diffraction data collected on the inhouse RAXIS-IIC imaging plate diffractometer is more than 95% complete to 1.9A resolution, and has Rm of 5.5% on intensities. The structure has been solved by the molecular replacement method, and simulated annealing refinement has yielded an R of 19.7% for all data. Two major structural results are: (1) the flaps are in the closed conformation and (2) the SI/S 1' subsites in the active site are significantly smaller in size compared to the wild- type enzymes. Interestingly, the subsite sizes are very similar to the corresponding subsites in the SIV-protease.

P l l X-ray study of the Ribosome Inactivating Protein Saporin S-9

Mukesh Kumar, Swati Patwardhan, K. K. Kannan and M. V. Hosur* Condensed Matter Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbal - 400085.

Ribosome Inactivating Proteins are a class of plant proteins that are gaining wide interest as toxin component of immunotoxins used in the treatment of a variety of diseases such as AIDS, cancer, autoimmune and parasitic diseases etc. These proteins, presumed to have antiviral role in vivo, function by cleaving a specific glycosidic bond of 28S rRNA from eukaryotic species. Three dimensional structures of these proteins are likely to help in the preparation of more effective immunotoxins through conjugation at proper sites.

Saporins are RiPs present in plants belonging to the family of Saponaria O~cinalis. These proteins are extremely stable against either denaturation by urea or proteolytic digestion by protease. Therefore saporins are suggested to be ideal candidates in the preparation of immunotoxins. We have purified the $9 isoform of saporin from the seeds of the plant, using ion exchange chromatography on an FPLC system, to understand the reasons for their high stability. The purified protein has been crystallised and diffraction data to 2.6A resolution has been collected on the inhouse RAXIS IIC IP diffractometer. The orientation and position of molecules within the crystals has been determined by molecular replacement calculation. Interpretation of electron density map has been carried out using the software package O. X-ray refinement is being carried out using X-PLOR. The current R-factor to 2.6 A resolution is 29%. Further refinement and model building is in progress.

P 1 2 Crys~l Stmemm of [~-iactamme from ~ e b a c t ~ f l ' a m 8

Padmanabhu r B'., Sasaki, Sugio, S., Nukaga, M~., Matsu2aki, T. Yokohama Research Center, Mitsubishi Chemical Corporation, Yokohama, Japan. ~Faculty of Pharmaceutical Sciences, Cbiba University, Chiba 263, Japan. *Present address: IMCB, The University of Tokyo, Tokyo 113-0032, JaparL

I~-Ia~am antibiotics are effective and widely used compounds against pathogenic bacterial infections. However, tbeir effectiveness has been restricted by the ability of bacteria to produce [3-1actamas~, enzymm that catalyze the hydrolysis of the amide bond of the 13-1actam ~ that provides the major bacterial defense mechanism agal~ this type of antibiotic compounds. The I~-lactamases have been classified ir~o four classes, A-D. Classes A, C and D are collectively known as serine ~-lactamases which share an active-site serine residue, whereas class B ~-lactamase belong to a mettalo-13-1actamase family. We report here two crystal forms of the refined three dimensional stmcttues ofthe Class C I~-iactamase from Citrobactor

freund/i. The first isoform was crystallized in P61 space group with cell dimensions ofa--b= 115.85 A; v= 123.64 A; y= 120 ~ and was diffracted upto 2.5A. The second isoform was also crystallized in P61 s~_nece~_ group but slightly shorter along the c-axis. The cell parameters of the second isoform are a=b= 116.67A; c = 117.20 A ; T ffi 120~ and it was diffracted upto 3.0A. Both the isoforms were solved by the molecular replacement method using the [3-1actamase slructure from Enterobactor cloacae as a model. The structures were refined to the final R-factors of 19.0% and 17.3% at 2.5A and 3.0A resolutions, respectively. Details of the structure and comparisons with the known class C and class A ~-lactamase structures will he presented

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P13 STRUCTURAL VARIABILITY AND FUNCTIONAL CONVERGENCE IN LACTOFERRINS

S.Karthikeyan, S.Sharma, A.K.Sharma, M.Paramasivam, P.Kmnar, J.A.Khan, S.Yadav, A. Srinivasan and T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, New Delhi- 110 029, India

Three-dimensional structure analyses of lactoferrin from several species : human, bovine, buffalo and mare in different forms such as differic, dicupric, disamarium, oxalate substituted diferric, oxalate substituted dicupric and atx,-lactoferrin liave revealed various ways in which protein structure adapts to different structural and functional states. The lactoferrin molecule folds into two lobes, each of which is further divided into two domains. The metal saturated form of lactoferdn adopts a closed conformation in all the species whereas the metal free form (apo) of human lactoferrin has N-lobe in open conformation and the C-lobe is in closed conformation while in mare lactoferrin, both N- and C-lobes adopt closed conformations. On further extension to Iransfetrins, in duck apo-ovotransferrin, both lobes are found in open conformations. Comparison of the differic, dicupric, disamadum lactoferrins has shown that different metals can, through variations in the metal positions, have different stereochemistries and anion coordinations without significant changes in the protein structure. Substitutions of oxalate for carbonate as seen in the structure of diferric dioxalate mare lactoferrin and in a hybrid dicupric complex with oxalate in one site in human lactoferrin show that larger anions can be accomodated by small side chain movements in the binding site. The lactoferrin also binds two molecules of melanin monomer, indole-5,6-quinone specifically suggesting its role in the mechanism of melanin polymerization. The multidomain/ multilobe nature of lactoferrin also allows the rigid body movements.

P14 Crystal structure of the bifunctional inhibitor of trypsin and obamylase from ragi seeds at 2.2 A resolution. 3. GOURINATI~ NEELIMA ALAM, A. SR1NIVASAN AND T. P. SINGH Department of Biophysics, All India Institute of Medical sciences, New Delhi 110029. lndicL

The crystal structure of a bifunctional inhibitor of a-amylase and trypsin from .Ragi seeds (Indian finger millet : Eleusine coracana Gaertneri) (RATI) has been determined by X-ray diffraction method at 2.2A resolution. The inhibitor consists of 122 amino acids with 5 disulfide ts'idges and belongs to the plant ot-amylase / trypsin inhibitor family. This is the first crystal structure from this family. The crystals were grown by microdialysis method using ammonium sulfate as a precipitant. The crystals belong to orthorhombic space group P212~2 with-a=47.4 b=54.5 ~ 4 0 . 3 ~ and Z=4. The structure was determined by molecular replacement method using Corn Hageman factor inhibitor (C-74H) as model. It has been refined to an R-factor of 21.9%. RATI folds into a pear shaped structure, with four ot-helices and two short anti parallel ~5-sheets. Both trypsin binding site and amylase binding site are completely different from known inhibitors. It shows an r.m.s deviation of 2.0 ,~, for the Cot atoms when compared with its NMR structure whereas the corresponding value with CHFI if found to be 1.4/~. RATI also shows structural similarity with non-specific lipid transport protein and 2S storage proteins. The molecular packing is very compact. The intermolecular interactions are predominantly solvent mediated resulting in a high degree of mosaicity.

P15 CRYSTAL STRUCTURE FUNCTION RELATIONSHIP OF PHOSPHOLIPASE A2 FROM DABOIA RUSSELLI PULCHELLA AT 2.4S ,~

NEUROTOXIC

Vikas Chandra 1, Punit Kaur l, Ch. Betzel 2 and T.P. Singh l 1D . . . . . epartment of Btophystcs, All lndta lnstttute of Medical Sciences, New' Delhi 110 029, India ~Institlit fiir Physiologische Chemie, Geb.22A, c/o DESY, Notkestrafle 85, 22603 Hamburg, Germany

Phospholipase A2 enzymes catalyses specifically the hydrolysis of the C-2 ester bond of 3-sn- phosphoglycerides to produce lysophosphatidylcholine and fatty acids. The PLA2 from Daboia russelli pulcheUa is a basic secretory and potent presynaptic neurotoxin. It consists of a single polypeptide chain of 122 amino acids, cross linked by seven disulphide bridges and belongs to the class II category of PLA2s. The enzyme was crystallized by vapour diffusion method using ammonium sulphate as a precipitating agent in orthorhombic space group C222t with a=77.048 b=92.347 ~77.059A with two molecules in the asymmetric unit. The intensity data were collected to 2.45/~ with a completeness of 98%. The structure determined by molecular replacement method has been refined to an R=18.8%, The side chains of His48, Asp99 and Tyr52 t'orm an active site, which are conserved in all phospholipases. The calcium binding loop is formed but contains no calcium.

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P16 The structural view of Nature's another 'Swiss Army Knife', Catol~opin DII, a thiol protease at 2.1 A resolution S. Ghosh, A. K. Bera, S. Bhattacharya, S. Chakraborty, A. K. Pal, B. P. Mukhopadhyuy, I. Dey, U. Haldar, and As.ok. Baneriee Biophysics Department, Bose Institute, ~ a 700 054,INDIA

Calotropin DII isolated from latex of Calotropis gigantea, a thiol protease, 215 amino acid residues, crystallized in P21, a=48.7A, b=32.0A, ~131.6A and l~=97.7" with two molecules per asymmetric unit is solved at 2.1A resolution by X-ray .The initial model of Calotropin DII, obtained by molecular replacement method using Papain(1.6A), refined by molecular simulation and leest-square minimization (X-PLOR). A non-crystallographic two fold symmetry neady parallel to b-axis exists between the two molecules in the asymmetric unit(x=1/4 and z=1/4). Positional parameters and isotropic temperature factors for non hydrogen atoms and 120 water molecules refined with 11219 Fo between 10.0 and 2.1A resolution(R=19%).The electron density map is well defined and superposed two molecules in the asymmetric unit shows r.m.s, deviation of 0.25)LThe deviation of bend length and bond angles are 0.015A and 2.1 ~ respectively. Molecules are ellipsoldal with two distinct domains, L and R separated with a deep cleft. A long a-helix at the cleft interface of the domains. This is a typical ((~,13) structure common to Papain family. The difference solvent accessibility(DA) reveals the difference in subsites and substrate specificity of this thiol protease.

P17 Structure of glucoamylase at 1.1 A and its complex with acarbose at 1.6

Jozef Sevcik and Adriana Solovicova Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic

The crystal sm_,cture of recombinant non-glycosylated glucoamylase from Saccharomycopsis fibuligera was solved by molecular replacement, using the catalytic domain of Aspergillus awamori glucoamylase as search model (coordinate set 3GLY). The enzyme crystallizes in space group P212121 with unit cell dimensions a = 56.90, b = 85.70, c = 98.24 A. The data were collected on EMBL beam lines at DESY, Hamburg: at cryogenic temperature from glucoamylase crystal to 1.1/3, resolution and at room temperature from the complex crystal to 1.6 /~. The refinements with the maximum likelihood program REFMAC in combination with automated refinement procedure ARP converged with R factors 15.9 and 13.6 %, respectively. The enzyme consists o f 492 amino acid residues and has 14 a-helices, 12 of which form an (cHa)6 barrel. The core at the center o f the barrel is conical and is filled with hydrophobic side chains. Both ends of this core are open to solvent. The active site is in a pocket formed by long loops at the narrower end of the core. Acarbose fits tightly into the pocket and this readily explains the exoglucanase activity of glucoamylases. Overall coordinate error of the structure at 1.1 A based on R given by REFMAC is 0.03 A and that of the complex is 0.13 A. The slructure of glucoamylase at 1.1 A is one of a few protein structures refined with truly atomic resolution.

P 1 8 1.5 A Resolution reffmemnet of Ortho~ombir Bovine Pancreatic Phospholipnse A2

K. Sckar & M. Sundarn_iingam ~ Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012 and

' Biological Macmmolecular Structure Center, Departments of Chemistry and Biocbemistry, 100W 18 ~ Avenue, The Ohio State University Columbus, OH 43210, USA

The X-ray crystal structure of recombinant bovine pancreatic Phospholipase A2 (PLA2) which specifically cleaves the sn -2 acylester bond of phospholipids, has been refined at I.SA resolution. The crystal belongs to the space group P2~2~2~ with unit cell parameters a=47.12, b=64.59 and c=38.14A similar to the native enzyme. The refinement converged to an R-value of 18.4% (Rfr~ = 22.8%) for 16,374 reflections between 10.0 and 1.5A resolution. The surface lo0p residues (60-70) are ordered in the present orthorhombic recombinant enzyme, but disordered in the Irigonal recombinant enzyme. The active site residues His 48, Asp 99 and the catalytic water superimpose well with the trigonal form. Besides the catalytic water, which is hydrogen bonded to His 48, it is often seen that there is a second water attached to the same Nitrogen atom of His 48. The catalytic water is also hydrogen bonded to the equatorial water coordinated to the calcium ion. In addition, to the equatorial water, there is also an axial calcium water and the additional slrucun~ water. These five common water molecules are hydrogen bonded to the additional 16 water molecules in the present orthorhombic structure which may further enhance the structural integrity of the active site. Details will be presented.

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P19 From Snake to Venom towards Drug Ch. Betzel ], N. Genov 2 & T.P. Singh ~

]Institute of Physiological ChemisUy, LIKE, C/o DESY, Notkestal~e 85, 22603-Han~urg, Germany 2 Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria

3 Department of Biophysics, All India Institute of Medical Sciences, 110 029 New Delhi, India

Phospholipases A2 (PLA2s) (EC 3.1.14) are widespread in the living organisms as both intracellular and extracellular enzymes. They are among the smallest enzymes known which perform various vital physiological roles and were isolated from a number of sources: snake venom, nmmmaliar l~increas, lung, gastric mucosa, fiver spleen, alveolar macrophages, intestine, membranes, heart, placenta and brain. The ~ake venom enzymes, although they share structural and catalytical similarities with the crude PLA2 enzymes cause in addition a enormous destruction by interfering in the normal physiological process of the victim including a variety of pharmacological effects. Using as target structure the PLA2 complex Vipoxin, isolated from the venom of the most toxic snake known to day. Vipera ammodytes meriodinalis, we will present detailed results for the heterogeneous strucULre-function-relationship p . ~ l e of this protein family. Furtheron we. will s-mmarise pharmaceutical effects which can be explained by the three-dimensional structure of Vipoxin; we have recently analysed with synchrolron radiation to high resolution. P 2 0 Structure and function of chromophores in R-Phyeoerythr in Dong-cai Liang, Tao Jiang, Ji-ping Zhang, Wen-rui Chang ;Institute of Biophysics, Chinese Academy of Sciences, Beijing 10010, China The crystal structure of R-Phycoerythrin (R-PE) from Polysiphonia urceolata has been refmed to a resolution of 1.9A, Crystallographic R-factor of the refined model is 0.195 (R~ee=28.2)from 8-1.9A.

resolution structure of R-PE showed precise interactions between the chromophores and protein H i g h ~ "

residues, which explained the spectrum characteristic and function of chromophores. Four chiral atoms of phycourobilin (PUB) were identified as C(4)-S,C(16)-S, C(21)-S and C(20)-R. The coupling distances o f 19A to 45A between the chromophores were calculated and the function of the chromophores in the energy transfer pathway was discussed. High resolution structure of R-PE suggested other pathways o f energy transfer, such as the ultrashort distance between a l 4 0 a and 13155 It has been proposed that aromatic residues in linker proteins not only influence the conformation of chromophore, but may also bridge chromophores to improve the energy transfer efficiency.

P21 Transferred Cross-Correlated Relaxation Complements Transferred NOE: NMR Structure

of an IL-4R-Derived Peptide Bound to STAT-6

Wolf~an~ Jahnke, Marcel Blommers

Novartis Pharma AG, Preclinical Research, CH-4002 Basel, Switzerland

NMR spectroscopy is a powerful method to determine structures of ligands such as protein inhibitors when bound to their molecular receptor. In the case of weak binding (KD = mM to high gM), where only a small fraction of inhibitor is bound at any time, this had to be achieved solely by analysis of transferred NOEs, since coupling constants in this case do not give information on the bound state. Structure calculations of bound figands then had to rely on distance restraints only, rather than on additional dihedral angle restraints. We have developed a technique to obtain angular restraints for the case of weak binding. The method is based on cross-correlated relaxation (cross-correlated HN-N/I-F '-C' dipol-dipol relaxation or cross-correlated I-F'-C' dipoFCO CSA relaxation) and exploits its correlation time dependence. The method is illustrated by determination of a partially 13C,]SN-labeled IL-4R-derived peptide bound to STAT-6. M. Blommers, W. Stark, C.E Jones, D. Head, C.E. Owen, W. Jahnke, JACS 121, 1949-1953 (1999).

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P 2 2 Multidimensional NMR Studies on the Complex of Pepstatin with HIV-I Protease Tethered Dimer

S. C. Panchal, R. V. Hosur, Bindu Pillay ~ and M. V. Hosur t Department of Chemical Sciences, Tam Institute of Fundamental Research, Homi Bhabha Road,

Mumbai 400 005, India Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 005; India.

Maturation of infectious HIV, the causative agent to AIDS, requires a viral-encoded protease. Genetic and inhibition studies have shown that the spread of viral infection can be arrested ff the protease activity can be blocked. Hence, this enzyme has become a primary target for rational drug design, based upon structures of the free protease and the protease complexed with a variety of inhibitors. One of the potent inhibitors is Pepstatin ~vhich has a rare amino acid statine. The hydroxyl group of statine is responsible for inhibition.

We report here the NMR spectral assignments of the HIV-I protease tethered dimer complexed to Pepstatin obtained from a battery of triple resonance experiments. The spectral features of this complex are compared with those of the free protein and also with those HIV-I protease- inhibitor complexes reported in the literature. We observed that pepstatin provides substantial stability to the folded protein and the comparisons indicate that the global fold of the protein is not affected significantly on inhibitor binding.

P23 Dehydrophenylalanine containing analogs of Tritrpticin show increased biological activity Puniti Mathurl., S. Srivatsun 2, Ratan Mani Joshi 3, N. R. Jaganathan I and V. S. Chauhan 3.

~Department of NMR, All India Institute of Medical Sciences, New Delhi 2 Indian Institute of Technology, Khamgpur, 3 International Centre for Genetic Engineering and Biotechnology, New Delhi.

Tritrpticin, a thirteen residue peptide antibiotic, was isolated by Pungercar et al from a putative precursor protein for porcine cathelin. The peptide has similarities with autolysin, a bacterial suicide enzyme, o~,j3--dehydroamino acids have been found to be effective stereochcmical directors of pcptide folding. Two peptide analogs of Tritrpticin containing dehydrophenylalanine (APhe) were synthesised on solid support, in addition to the original pcptide. The peptidcs were HPLC purified and then assayed for biological activity. The sub~'tution of APhe residues have been shown to greatly enhance the biological activity of the pcptides. The substituted peptide was found to be a more effective antibiotic. The haemolytic activity was also found to increase when more residues were substituted with APhe. Preliminary structural studies have bccn performed by circular dichroism spcctropolarimetry and nuclear magnetic resonance spcctroscopy. The results indicate that the APhe residues increase the number of helical ordered conformations in solution. Thus. dehydrophenylalanine-substituted synthetic pcptide analogs exhibit more ordered conformations and hence, increased biological activity. Such synthetic pcptides could prove to be effective drug candidales. P 2 4

An au tomated approach for resonance assignments in proteins H.S. Atreya, S.C. Sahu, K.V.R. Chary and Girjesh Govil,

Tara Institute of Fundamental Research, Mumbai-400005, INDIA

The algorithm TATA (Tracked AutomaTed Assignments) for the assignment o f IH, 13Ca, 13C~, ]3C' and l~q spins in proteins uses peak lists from CBCANH, CBCA(CO)NH, FIN(CA)CO and HNCO spectra and the protein primary sequence. The program searches for neighboring partners of a given residue on its either side in the primary sequence and identifies a maximum possible stretch o f amino acid residues. While doing so, it tracks Ala, Ser, Thr and Gly residues on the basis of their characteristic ]3C~ and 13C~ chemical shidfls. Such a tracked stretch o f amino acid residues is then mapped onto the primary sequence for sequence specific assignment. The program has been tested, using experimental data, for a calcium binding protein from Entamoeba histolytica (15 kDa) and also in the case of three other proteins, namely, fragment o f Fibroblast Collagenase (18kDa), Metailo-beta-Lactamase (25.5 kDa) and Borrelia-Burgdoferi OspA (28 kDa), for which peak lists were simulated using published assignments. In all these cases, nearly complete (> 95 %) assignments could be attained.

42


P 2 5 Characterization of the Internal Motions in wild type and C37A-C47A mutant of spo-NCS

by a Combination of nC-NMR Relaxation Analysis and Molecular Dynamics Simulation. Elisabeth ADJADJ ', Eric QU1NIOU ', Nadia IZADI-PRUNEYRE ', Yves BLOU~U1T ', Jolg MISPELTER ',

Bernadette HEYD ~, Guilhem LERAT ~, phil#Tpe MINARD ~ and Michel DESMADRIL 2, 1-1nstitut Curie, 1NSERM U350, Centre Universitaire Bat.l l 2, 91405 Orsay-Cedex France

2-Laboratoire de Moddlisation et d'IngSnierie des Protdines, Centre Universitaire Bdt. 430, 91405 Orsay-Cedex France.

It is now well established that the functional diversity of binlogical molecules closely depends upon the static and dynamic properties of their three-dimensional smtcttae. "C relaxation parameters, obtained from NMR, provide an indirect means to measure the internal mobility in a protein, because the relaxation processes are due to variable local magnetic fields which are created by the ps-ns fluctuations of the C-H bond. These rapid motions are of low amplitude, and are related to the existence of very strong local interactions. However; although they are not directly related to the biological function of the protein, they nevertheless play a fundamental role in its expressio~ This is because they reveal the factors which stabilize the protein structure. The collective motions of larger fragments of the protein on a p.s-ms time scale are more directly implied in the biological function. In some cases, they can also be appreciated qualitatively by relaxation methods. To gain a better insight of protein flexibility, the complete set of experimental results need to be confronted with dynamics "simulation, which makes it possible to visualize the trajectory of the movements. We present here a summazy of our results and discuss their interests in the context of protein design.

P 2 6 Characterizadon of Active-site Conformation of E. coli Adenylate Kinase by High-Resolntion NMR

Methods Y. Lin* & B. D. Nageswara Rao

Dept. of Physics, Indiana University Purdue University Indianapolis, Indianapolis, Indiana 46202, USA.

Adenylate kinase (AK, MW 20-24 kDa) catalyzes the reaction: AMP+M(H)ATPC~ADP+M(II)ADP, where M(II) is an obligatory divalent cation, Mg(II) in vivo, and may be substituted by Mn(H) and Co(H) in vitro. The AMP-site on this enzyme specifically binds cation-free AMP and ADP, and the ATP-site binds ATP(ADP) as well as GTP(GDP) with or without the cation. The nucleotide conformations in M(II)ATP.AKeco and AMP.M(IDGDP.AKeco complexes have been determined by two types of NMR measurements: (i) 2D TRNOESY, which yield interproton distances allowing the determination of the conformation of the adenosine moiety, and (ii) 31p and 13C paramagnetic relaxation measurements, which yield Co(II)-31P distances as well as Mn(II)J3C distances (by using [u]-I3C] nucleotides). The active-site conformation was established by combining these results using molecular modeling methods. Theses conformations are found to differ significantly from those obtained by x=ray crystallography. Attempts to dock the NMR-determined enzyme-bound nucleotide structures with the protein structures obtained by the x-ray crystallography yield some insight into the roles of amino-acid environment, and the role of the cation at the active site. This research was supported by the National Institutes of Health, U.S.A (GM43966) and by IUPUI. * Address after July 1, 1999: Dept of Biocbem & Biophys, University of Pennsylvinia, Philadelphia, PA 19104-6059.

P 2 7 Conformations of MnATP and MnADP Bound to Yeast 3-Pho.sphoglycerate Kinase Determined by UC

Relaxation Measurements Using [ul-"ClNuclentides Vidya Ra~hunathan*, Mei H. Chau, and B. D. Nageswara Rao

Department of Physics, Indiana UniVersity Purdue University Indianapolis, Indianapolis, IN 46202, USA.

A complete characterization of the conformations of MnATP and MnADP bound to the active site of yeast 3- phospboglycerate kinase is obtained on the basis of "C relaxation measurements, performed on exclusively enzyme- bound complexes, E.Mn[ulJ3C]ATP and E-Mn[ulJ3C]ADP, at three distinct 13C-NMR frequencies, 75 MHz, 125 MHz, and 180 MHz. The frequency dependence of the relaxation data has been analyzed to evaluate distances from the cation for all the ten 13C nuclei in the adenosine moieties of E.MnATP and E.MnADP. These distance data, taken along with previously published cation-3~P distances have been used as constraints in the molecular modeling program, Quanta, in which molecular dynamics simulations and energy minimization have been performed to determine the conformations compatible with the distance data. The details of the enzyme-bound MnA'rP and MnADP conformations are distinguishably different from each other. The position of the cation is displaced by 2.4 ,~ when the adenosine moieties of the two structures are superposed. Furthermore, the NMR-determined structures of enzyme-bound MnATP and MnADP are significantly different from those in published x-ray crystal structures of the enzyme-nucleotide complexes. This research was supported by the National Institutes of Health, USA (GM43966) and IUPUI. * National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

43


P28 NMR and MD studies on fragment $79-601 of gp41 domain of HIV

pgssbant Den i s, Sudba Srivastava*, Evans Coutinbo s and Anti Saran*, *Tara lnJlu~ o f ~ Research, l~nnl~i 400005

couc of 400098

The fragment 579-601 o f t h e gp41 domain o f HIV is now seen as a major epitope recognized by amibodies o f HIV patients. The conformation of th is peptide been investigated by 2D-NMR at 600 MHz in DMSO-d6. Sequence-specific resonance assignments have been made from DQF-COSY, TOCSY and NOESY spe~-L The temperature coefficient o f N H chemical shifts are above 4.0 ppm/~ and hence are not involved in iuh-amolecular H-bonding. The 3 j ~ coupling constants measured from E-COSY spectrum are in the range 10-13 Hz. This data along with the observation o f strong d~r and d ~ h o e ' s throughout the length o f the peptide indicate that the peptide has a I ~ - p l ~ structure in DMSO-d6. The hOe's in the linear portion o f the build-up curves were converted into distances and used as constraints in a MD simulation to generate the solution structure in presence o f explicit solvent molecules.

P29 Solution studies of lnterleukin-15 reveal a tertiary fold different than IL-2. Leizl F. Sapico, Jayson Gesme, Herbert lijima, Raymond Paxton~ & Thamarapu Srikrishnan, Department of Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263 and t Department of Protein Chemistry, Immunex Corporation, Seattle, WA 98101. lnterleukin 15 (IL-15) is a novel cytokinr that has been recently cloned from the simian kidney epithelial cell line. Although I!-15 has no sequence homology with IL-2, it uses the components of the IL-2R for binding and signal transduction. IL-15 is a ligand that activates human NK cells through components of the IL-2R receptor in a pattern that is similar to but not identical to that of II,-2. We present here a detailed report on the secondary structure of the human and simian I1.-15 using circular dichroism (CD) and FTIR spectroscopy. At room temperature in PBF buffer (pH 7.0) human IL-15 has 18% a-helix, 38% 13 and 42% random coil and the simian IL-15 has 33% a-helix, 32% 13 and 35% random coil. The simian IL-15 is very stable at higher temperatures, retaining as much as 19% helical structure at 80"C with 43% 13 aad 38% random coil. At lower and at higher pHs, both human and the simian IL-15 have much less helical content than at physiological pH. At pH 9.0, the human has 5.8% helical content as against 12.0% for the simian. At pH of 2.8 the human is almost non-helical ( 2.8% a-helix) and.the simian has 16.1% helical content. From an analysis of the FTIR spectrum, human 11-15 has 10",4 alpha helix with 43% beta and 47% random coil. These values indicate that IL-15 has a different tertiary structure than IL-2 which has a 4a-213 fold with a 59% helix and a 41% beta structure.

P 3 0 Solution Conformation of the Nonmammalian Tachykinin Eledoisin

C.R. Graco 2, G.Nagenagowda 2, A.M.Lynn I and Sudha M.Cowsik l 1School of Life Sciences, Jawaharlal Nehru University, New Delhi

2SIF, Indian Institute of Sciences, Bangalore

The tachykinin family is characterised by the C-terminal pentapeptide sequence Phe-X- Gly-Leu-Met-NH2 (X=an aromatic or aliphatic residue). These peptides show a wide spectrum of biological actions in the central and peripheral nervous system such as these excite neurons, evoke behavioral responses, are potent vasodilators and contract many smooth muscles. The possibility that tachykinins may act as growth factors or as messengers between the nervous and immune system has generated much interest and excitement. To understand the structural basis of biological activity, the nonmammalian tachykinin eledoisin was investigated by use of CD, two dimensional NMR and distance geometry techniques. In aqueous solution eledoisin is conformationally averaged, but addition of membrane mimetic solvents induces helical structure. Our data indicates that the helical core of eledoisin is better defined in the miceller environment than TFE.

44


P31 Solution S t ruc ture of a Calc ium Bind ing Prote in f rom Entamoeba histolytica Sarata. C. Sahu, 1 S. Chauhan, 2 A. Bhattacharya,2 K. V. R. Chary, 1 and G. Govil 1

Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India, 400005 2 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, 110067.

In order to understand the mechanisms of Ca 2+ signaling in E. histols a calcium binding protein (Eh CaBP; Mr-I 5 kDa) was over-expressed in E. coli. Nearly complete sequence specific resonance assignments for IH, 13C and 15N spins in Eh CaBP were done using triple resonance NMR experiments. Qualitative interpretation of the nOe's, chemical shift indices and of hydrogen exchange rates threw valuable light upon the secondary structiare of this protein. CaBP is found to have two globular domains each of which consists of two pairs of helix-loop-helix motifs, like in other homologous proteins. The eight a-helical segments are 4-11, 19-28, 35-47, 55-63, 76-87, 95-103 and 107-115. The protein also consists of two short anti-parallel [3-sheet structures. The two domains are connected by an extended conformation. Intricate details of 3D structure of Eh CaBP will be presented. P 3 2 STUDY O F C R O S S - C O R R E L A T I O N S IN N M R AS S O U R C E O F S T R U C T U R A L I N F O R M A T I O N O F

P R O T E I N S . M E A S U R E M E N T O F CSA/CSA C R O S S - C O R R E L A T I O N S .

Anii K u m a r ~, Maur iz io Pellecchia ' and E r i k R.P. Zuiderweg z J Depar tment o f Physics and Sophist icated Ins t rumen t s Facility, Ind ian Inst i tute o f SciencepBangalore 560

012, India, ZBiophysics Research Division, The Universi ty o f Michigan, Ann Arbor , M I 48109, USA.

Cross- terms between different pa thways o f relaxation o f a spin, can lead to detailed in format ion on s t ructure and dynamics o f biomolecules. We have carr ied out detailed calculations to investigate situations where such cross-correlat ions show up in longitudinal as well as t ransverse relaxation o f coupled spins. In t ransverse relaxat ion a p rominent s ignature o f cross-correlat ions is the differential line b roadening /nar rowing . I t is found that the chemical shift an iso t ropy (CSA) contr ibutes significantly to the relaxation of J3C and tSN spins at high magnet ic fields and that the cross- terms between the two C S A ' s contributes differentially to the double and zero q u a n t u m coherences of such spin pairs. We have carr ied out measurment o f the differential relaxation rates o f such double and zero q u a n t u m coherences of JSN- J3CO along the back bone of protein Binase (12.3 KDa). The results o f these measurements will be presented.

P33 Solution structure of an insect growth factor, growth-blocking peptide and its

structural and functional similarity with epidermal growth factor

Keiichi Kawano. Tomoyasu Aizawa, Naoki Fujitani, Yoichi Hayakawa, Atsushi Oimishi, Tadayasu Ohkubo, Yasuhko Kumaki, Kunio Hikichi, and Katsutoshi Nitta Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Division of Biological Sciences, Graduate School of Science, Hokkaldo University, Institute of Low Temperature Science, Hokkaido University, Japan Adveaced Institute of Science and Technology

Growth-blocking peptide (GBP) is an insect growth factor consisting of 25 amino acid residues that retards the development of lepidopteran larvae at high concentration while it Stimulates larval growth at low conceniration. In this study, we determined the solution structure of GBP by two-dimensional ~ NMR spectroscopy. The structure contains a short segment of double-stranded 15-sheet involving residues 11-13 and 19-21 and a type-II ~turn in the loop region (residues 8-11), whereas the N and C termini are disordered. This is the f'wst report of the three-dimensional structure of the peptiderigic insect growth fctor, and the structure of the well defated region of GBP was found to sha~ similarity with that of the C-terminal domain of the epidermal growth factor (EGF). Because GBP has been reported to stimulate DNA synthesis of not only insect cells but also human keratinocyte cells at the same level with EGF, the structural similarity between GBP and EGF may lead to the interaction of GBP to EGF receptor.

4 5

A1 : Pro~ns: Stn /Ct~ and Dynamics

P34 A novel s tructural fold o f a neurotoxin from Bungarus candidus: N M R characterizat ion

Rani Parvathy V, t, K. V. R. Chary t, R. M . K i n f , (7. Govil t 1Department of Chemical Sciences, Homi Bhabha Road, TIFR, Mumbai 400 005

2 Bioscience Centre, Faculty of Science, National University of Singapore, Singapore 119260

A neurotoxin has, been isolated and purified from Malayan krait. The toxin consists of sixty-six amino acid residues and is devoid of glutamines and histidines. Three dimensional structure of this cysteine rich (10/66) protein has been studied. Nearly complete sequence specific IH resonance assignments have been done using standard 2D NMR experiments. Qualitative interpretation of the nOe's and hydrogen exchange rates threw valuable light upon the secondary structure of the protein. The toxin is predominantly in anti-parallel ~sheet structure. The protein consists of four stranded antiparallel sheet. The individual ~strands are MI-K4, K24-R32, T37-A45, L55-C60 and D63-C65. On the whole the toxin has a novel structural fold. This is the first neurotoxin with a disulfide bridge in the first loop instead of second loop as observed in case of long chain neurotoxins and K-bungarotoxin. Intricate structural details of the protein will be presented.

P35 Structural and thermodynamic responses of a protein with an exraordinary stability of molten g lobule state Takumj" Koshiba, Yoshihiro Kobashigawa, Min Yao, Makoto Demura, Astushi Nakagawa, Isao Tanaka, Kunihiro Kuwajima ~ and Katsutoshi Nitta Division of Biological Sciences, Graduate School of Science; Hokkaido University, Sapporo, Japan, and 1Department of Physics, School of Science, The University of Tokyo, Tokyo, Japan

To understand protein folding mechanism, the concept of molten globule state is considerably important. Here we show that the molten globule state of canine milk lysozyme, which belongs to the family ofcalciumdainding lysozymcs, as a useful model state far elucidating the molecular mechanisms by which the molten glubule state is stabilized. In order to investigate the stability of the molten globule state of this protein, we measured hr- and ncar-UV CD spectra and the thermal transition ofthe protein using d i f f r scanning calorimetry (DSC), andwr interpreted with the structure revealed with X-ray crystallography and NMR spectroscopy. The X-ray crystal structure of this protein has been determined by m o l ~ l a r replacement and refinedat the high-resolution ~1.85 A to R-lacier of 17.8 % (fir162 R-factor = 21.5 %). From these results, it has been revealed that the molten globule state of canine milk lysozymr was extraordinarily stable because oflbe specific packing interactions ofct.domain, and structural and thermodynamic responses has been discussed.

P36 Implementation of a new relaxation matrix approach in an iterative assignment strategy with ambiguous d i s t a n c e r e s t r a i n t s Jens Linge, ScAn O I)onoghue, Michael Nilges European Mole('ular Biology Laboratory, D-69012 Heidelberg, Germany The importance of ambiguous distance restraints for automated N e E assignment and NMR structure determination ha.s been demonstrated in several recent projects 9 Automated N()E assignment can avoid errors in data interpretation and leads to a fa.ster stru('ture determination process. Starting with an almost ('omplete list of chemical shifts, s()me few assigned NOEs and otherwise uninterpreted N e E peak lists, the program ARIA (Ambiguous Restraints in Iterative Assignment) calibrates NOEs, merges the obtained ambiguous (tistanc(' restraints from different N e E spectra and assigns the N e E peaks in an iterative manner. Spin diffusi.n is one of the main factors which allow to derive only approximate ranges of interproton distances. In order t~ impr,~ve the accuracy of these distance restraints, the integrated N e E volumes are corrected'for spin diffusion by a new relaxation matrix algorithm. After each iteration, the distance restraints can be recalibrated by comparing simulated and mea.sured NOEs. ARIA ha, s an user fri,,ndly browser driven interface for editing of all the parameters for calibration, assignment, relaxation matrix refinement and structure calculation. It ha.s an interface to interactive assignment programs, which makes it possible t.o inspect the ~ssignments together with the original data. .lens Linge is supported by a Boehringer Ingelheim pr(,doctoral fellowship.

46


P37 Native state hydrogen exchange studies of a fragment complex can provide structural information about

the isolated fragments G Chakshusmathi, Girish S. Ratnaparkhi, P.K.Madhu and R.Varadarajan

Molecular Biophysics Unit, Indian Institute o f Science, Bangalore-560012

Ordered protein complexes are often formed from partially ordered fragments that are difficult to structurally characterize by conventional NMR and crystallographic techniques. We show that concentration dependent hydrogen exchange studies o f a fragment complex can provide structural information about the solution structures o f the isolated fragments. This general methodology can be applied to any bimolecular or multimeric system. The experimental system used here consists o f Ribonuclease S, a complex of two fragments o f Ribonuclease A. Ribonuclease S and Ribonuclease A have identical three dimensional structures but exhibit significant differences in their dynamics and stability. We show that the apparent, large dynamic differences between Ribonuclease A and Ribonuclease S are due to small amounts o f free fragments in equilibrium with the folded complex and that amide exchange rates in Ribonuclease S can be used to determine corresponding rates in the isolated fragments. The studies suggest that folded RNase A and the RNase S complex exhibit very similar dynamic behavior. Thus cleavage of a protein chain at a single site need not be accompanied by a large increase in flexibility o f the complex relative to that o f the uncleaved protein.

P38 LIGAND BINDING AND PROTEIN CONFORMATIONAL RELAXATION IN CYTOCHROME P450CAM

C. Tetreau , M. Tourbez & D. Lavalette. lnstitut Curie (INSERM U350), Orsay, France Many experiments indicate that following laser flash photolysis of their iigated-complexes, hemoproteins undergo

structural relaxation towards the unlighted structure. This relaxation is rapid at physiological temperatures and greatly affects the kinetics of ligand rebinding. Although the numerous studies devoted to CO-Myoglobin have shown that a rearrangement of the heine pocket is involved, the proximal and (or) distal structural origin still remain an open question.

Here, further insight is obtained from the study ol ~ Cytochrome P450cam. The kinetics of CO geminate rebinding with the substrate-bound protein measured in a highly viscous medium between 220 and 140K exhibit two well resolved processes respectively attributable to rebinding with the constrained and the relaxed forms of the protein.

To analyze these observations, we use a kinetic model which takes the fact into account that the rates of geminate rebinding and relaxation are distributed because conformational substates do not interconvert on the time scale of the experiments. We found that the relaxation rate obeys an Arrhenius relationship even below the glass transition of the solvent and is faster than solvent relaxation by several orders of magnitude, suggesting that the structural rearrangement is not coupled with the surface and remains confined within the internal part of the protein.

Examination of the kinetics in the absence of substrate and in the presence of several camphor analogs is in progress in order to clarify the proximal and distal contributions to the relaxation process.

P39 PROTEIN COAGULATION DUE TO INTERACTING PROCESSES

M.Manno (I,2.3) P.L. San Biagio t~.3), V. Martorana t~.3), A. Emanuele (~.2~ S.M. Vaiana ~.2), D. Bulone (~.3) M.B. Palma-Vittorelli t].2) M.U. Palma ~.2).

t~) INFM - Uniter di Palermo and (2) Dept.of Phys. and Astronom. Sciences, Univ. of Palermo, via Archirafi 36, 1-90123 Palermo, Ita!y. (3~ CNR - IAIF, via U. La Malfa 153, 1-90146 Palermo, Italy.

Processes participating in protein self-association (or coagulation) and deposit are currently stirring a very high interest. Deposit usually involves protein conformational changes, that is formation of an "intermediate", and can occur even at low concentrations. It is responsible for pathologies such as systemic amyloidosis, Priori and neurodegenerative diseases (Alzheimer, Creutzfeld-Jacob diseases, BSE). Readily available proteins, exhibiting at low concentration self-association properties related to conformational changes, offer very convenient model systems capable of providing insight into this class of problems. Experiments on Bovine Serum Albumin reported here show that supramolecular association is due to the interaction of many processes: molecular crosslinking, thermodynamic phase-separation and a conformational change towards the intermediate required for coagulation. This three-process interaction allows coagulation even at very low concentrations. An analogous pattern has been recently observed in the case (that will be briefly recalled here) of a non- peptidic polymer. This suggests that interaction of these three processes can be a fairly common feature in protein coagulation as well as, more in general, in biopolymer association/gelation.

47


P40 Structure and conformational flexibility of the amino-terminal region of gp41 of HIV-1 and its implications

on viral fusion mechanism: A Biophysical Study ~ , S. F. Chen& W. J. Chicn, S. 14. Yang. S. Francis and D. IC Chang

Institute of Chemistry, Academia Sinica, Taipei, ROC 115. Taiwan The fusion process betwcen enveloped virus and host cell is an essential step for Viral infection. The fusion domain within the tran.~uembranc proteins has been known to be responsible for viral-cell fusion. Various studies in recent past have pointed out the oligomedzation phenomenon of fusion proteins in their fusogcnic state. We have used the amino terminal sequence of giycoprotein, gp41, of human immunodeficiency virus type-I to study its conformation and assembly, in order to address its possible role in viral fusion mechanism. Earlier reports from our group with a shorter sequence of amino terminal fusion peptide of ~ 1 have shown that it that it inserts into micelle as a helix with the glyciue at position 16 at micelle-water interface (Chang et al, 1997, J. Virol, 71, 6593). We further observed that this extended region of amino-terminal sequence of leucine zipper like domain of gp41 induces into helix in the micellar solution as judged by circular dichroism and NMR spectroscopy. In addition, the fight scattering and other data showed the oligomerization status of this sequence. The biological significance of these findings on the conformatioro' flexibility and property of oligomcrization, in the form of a proposed model based on the NMR data will Ix: presented as a possible interaction model of ~ 1 with membrane during fusion process. (This work is supported by National Science Council and Academia Sinica, Taiwan)

P41 A dominant-negative myosin I1 mutant with myosini like properties- Role of the rigid relay loop containing Trp501 in setting the enzymatic activity of myosin Renu Batra*, Michael A. Geeves t, and Dietmar J. Manstein*

Max-Planck-lnstitut fiir Medizinische Forschung~ Jahnstr. 29, 13-69120 Heidelberg, Germany, t Department of Biosciences, University of Kent, Canterbury CT2 7NJ, Kent, UK

All members of the functionally diverse myosin family contain a conserved 80 kDa motor domain. The relay loop containing the ATP-seusitive Trp501 forms part of a mobile region in this motor domain and displays apparent class specific differences in primary sequence. We created chimacric myosin constructs by replacing the native Dictyostelium myosin H relay loop sequence with mammalian myosin I derived sequence. The purified mutant protein displayed a 20-fold reduced motile activity in an in vitro motility assay and reduced ATPase activity. Detailed transient kinetic .analysis revealed a 5-fold reduction in the rate of the ATP cleavage step and a 7-fold increase in ADP affinity, both in the absence and presence of actin. However, the most dramatic effects of the chimaeric replacement were observed when the functional competence of the protein was analyzed/n v/vo. Cells producing the chimaeric myosin were unable to perform processes that are myosin H-dependent in Dictyostelium, like fruiting body formation, capping of cell surface receptors and growth in suspension. Thus, this mutation is the first in nearly 100 characterized Oictyostelium myosin II motor domain mutations that leads to a dominant-negative phenotype.

P 4 2 PARAMETRIZATION OF AN APPROXIMATE VALENCE BOND (AVB) METHOD TO DESCRIBE POTENTIAL ENERGY SURFACE IN THE REACTION CATALYSED BY HIV-1 PROTEASE Joanna Trvlska l, Pawei Grochowski 2 and Maciej Geller t'2 lDepartment of Biophysics, Warsaw University, Zwirki i Wigury 93, 02-089 Warsaw, Poland; 2Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University, Pawifiskiego 5a, 02-106 Warsaw, Poland

HIV-1 Protease (HIV-1 PR), one of the three enzymes encoded by the human immanodeficiency virus, is indispensable for viral replication. The AVB method was parametrized to describe possible paths of the enzymatic reaction of HIV-1 PR, including proton transfers, nucleophilic attack of a hydroxyanion on th esubstrate's peptide bond, formation and breakage of the reaction intermediate. The parametrization was based on the results of density functional calculations performed for molecular structures modelling the reaction. Firstly, we studied optimal geometries, conformational deformations and electrostatic properties of the separate molecules. These results were used to fit intramolecular interaction parameters and atomic charges. Secondly, we analysed energy profiles for proton transfers, nucleophilic attack and peptide bond breakage to obtain the intermolecular interaction parameters. The AVB model of the enzymatic reaction region will be coupled with the conventional forcefield description of the rest of the protein and applied as a fast generator of the potential energy surface in classical or quantum-classical molecular dynamic simulations (QCMD) of the enzymatic reaction. d. T. acknowledges support by Warsaw University (BST 591/BF), P.G. and M.G. by KBN (8 T11 F 016 16).

48


P43 DFT-BASED PARAMETRIZATION OF AN APPROXIMATE VALENCE BOND (AVB) METHOD FOR

QCMD/AVB SIMULATIONS OF THE TRANSPHOSPHORYLATION PROCESS CATALYZED BY PKA

K. Ginalski i, p. Grochowski 2 and B. Lesyng 1.2 1 Department of Biophysics, Warsaw University, Zwirki i Wigury 93, 02-089 Warsaw, Poland

2 ICM, Warsaw University, Pawi~iskiego 5a, 02-106 Warsaw, Poland

Transfer of a phosphoryl group is a fundamental biological process involved in metabolism, gene regulation and signal transduction. We plan to apply Quantum-Classical Molecular Dynamics (QCMD), combined with the AVB method, to simulate the transphosphorylation reaction catalyzed by protein kinase A (PKA). The AVB method describes the possible path for ~,-phosphate transfer from ATP to serine, including formation and breakage of the pentacoordinate associative or dissociative transition state. The AVB method was parametrized with respect to precise DFT quantum-mechanical calculations. We have studied the optimal geometries, conformational deformations and electrostatic properties of molecular structures modelling the reaction, as well as the energy profiles for dissociation and association of the phosphate group and related proton transfers. The parametrized AVB method will be used as a fast generator of the potential energy surface for the PKA active site region, allowing precise QCMD simulations of the enzymatic process.

K.G. acknowledges support by Warsaw University (BST 591/BF), P.G. and B.L. by KBN (8 T1 IF O16 16).

P44 STOKES-EINSTEIN LAW REVISITED:

ROTATIONAL BROWNIAN MOTION IN A MACROMOLECULAR ENVIRONMENT ~ , C. Tetreau, M.Tourbez & Y. Blouquit. Institut Curie (INSERM U350), Orsay, France

We report on the failure of the Stokes-Einstein equation to describe the rotational brownian motion of proteins in a macromolecular environment. As a consequence, rotational correlation time measurements such as currently performed in vivo for the purpose of obtaining information on protein size or on local viscosity (e.g. in membranes or even whole cells) are likely to be biased and should be reinterpreted. In a macromolecular environment, the linear viscosity dependence in the Stokes-Einstein equation must be replaced by a power law of viscosity. The exponent of the power law expresses the fact that the probe protein experiences only a fraction of the hydrodynamic interactions of the macromolecular cosoivent. The exponent is smaller than unity and depends on the relative scale of cosolvent (macromolecular environment) and probe protein.

The modified Stokes-Einstein equation leads toan accurate definition of the microscopic viscosity. It is suggested that a similar effective microviscosity should be introduced in Kramers equation describing protein reaction rates.

These conclusions are based on a wide set of experimental data using the transient optical absorption anisotropy method to measure the rotational relaxation of proteins with MW in the range 66 to 4000 kD in presence of cosolvents. with MW between 1.5 kD and 2 000 kD at viscosities up to 200 cP.

P45 S t u d y o f a /~-ha i rp in f o r m i n g p e p t i d e in a c q u e o u s s o l u t i o n u s i n g M o l e c u l a r D y n a m i c s

s i m u l a t i o n s

D. Roccatano 1, A. Amadei 2, A. Di Nola 2 and H.J.C. Berendsen 1 i Dept. of Biophysical Chemistry, Univ. of Groningen Nijenborgh 4, 9747 AG Groningen, The Netherlands

2Dept. of Chemistry, University of Rome "La Sapienza", I taly

The structural and dynamical behavior of the 41-56/3-hairpin from the protein G B1 domain (GB1) has been studied using Molecular Dynamics simulations in aqueous environment. Different simulations of the wild type and mutan ts of the peptide have been performed. The purpose ef these simulations is to establish the stability of this hairpin in view of its possible role as a nucleation site for protein folding. The conformation of the peptide in the crystallographic structure of the protein GB1 was lost in all simulations. The new equilibrium conformations are stable for several nanoseconds at 300 (> 10 ns), 350 K (> 6.5 ns) and even at 450 K (up to 2.5 ns). The new structures have very similar hairpin-like conformations with properties in agreement with available experimental data. The role of the hydrophobic core region for the peptide stability was verified by simulations of different mutants. The results of these simulations provide a further evidence of the impor tance of th e hydrophobic interactions in the stability of this peptide in solution

49


P46 Twist and Shear in l~-Sheets Bosco Ho and P.M.G. Curmi

Initiative ifi Biomolecular Science (IBIS) University of New South Wales, Sydney, Australia

The right hand twist and shear are systematic assymetric properties of Is-sheets. Both properties arise Irom electrostatic backbone-backbone interactions. New measurements of interacting pairs of Is-sheets and Is-ribbons have provided a quantatitive test for the modelling of [3-sheets. A novel approach to model the Is-sheet has produced quantative predictions which agree very well with experiment. The Is-sheet can be modelled by considering the electrostatic interactions of generic sub-units of an extended Is-sheet. This approach articulates differences between Is-sheet and Is-ribbon sub-units. The shear is due to a dominant carbonyl oxygen-oxygen repulsion embedded in a periodically repeating motif. Given that intra-strand energies favour right hand twisted IS-strands, the twist is due mainly to the electrostatic interaction of the dipoles of the right hand twisted IS-strands and also to an interaction involving the Ca atom.

P47 Statistic conformation of fibronectin in physiological conditions

H. Berry l, D. Lairez 2, E. Pauthe t, J. Pelta 1 ] D6partement de Biologic, Universit~ de Cergy-Pontoise, France

-' Laboratoire D6on Brillouin, CEA-Saclay, France

Fibronectin is a muitifunctional glycoprotein (molecular mass M=500kg/mol) of the extra cellular matrix (ECM) having a major role in cell adhesion. In physiological conditions, the conformation of this protein still remains debated and controversial. Here we present a set of results obtained by scattering experiments. In physiological conditions, the radius of gyration (Rg= 150+_30A) was determined by static light scattering as well as small angle neutron scattering. The hydrodynamic radius (RH=122_+5,/~) was deduced from quasielastic light scattering measurements. These results imply a low internal concentration (M/Rg~, 3) as compared to the one of usual globular proteins. This is also confirmed by the ratio RH/Rg=0.8_-+0.2 consistent with a gaussian chain whereas RH/Rg=I.3 for spherical shaped molecules. However, adding a denaturing agent (Urea 8M) increases both Rg and Ru by a factor two. "i'his means that fibronectin native chain is not either completely unfolded. The average shape of fibronectin conformation was also probed by small angle neutron scattering performed for reverse scattering vector q-~ smaller than Rg (2A<q-~<ISOA). The measured form factor is in complete agreement with the form factor of a random string of 50 beads of 45,~ diameter, it rules out the possibility of unfolded chain as well as globular structures. These results have structural and biological implications as far as ECM organisation is concerned.

P 4 8 Molecular dynamics study of ligand induced perturbative changes in cyclooxygenuse I and 2. V.Kothekar, Shakti Sahi & M. Srinivasan. Dept. of Biophysics, A.I.I.M.S, New Delhi-110 029.

Cyclooxygenase(COX) is a primary target for interaction of Non-Steroidal Anti Inflammatory Drugs(NSAIDS). Two isoforms of COX ate known namely COX-I and COX-2. While COX-I inhibition leads to gastrointestinal disorders, COX-2 inhibition is important for anti-inflammatory activity. Some of the currently known NSAIDs show select inhibition of COX-2, compared to COX-I while others do not. The mechanistic cause of differential activity of NSAIDs is important for the design of painkillers without any side effects. The main problem in aesigning a drug which would specifically bind to COX-2 is that both overall structure and the drug binding sites of both COX-I and COX-2 are, quite similar with no difference in the amino acid side chains in the channel except that Ile-523 in COX-1 is replaced by Val in COX-2. We have applied computer simulation technique to study interaction of two NSAIDs indoprofen and NS398 with COX-1 and COX-2 enzymes. Both the drugs were docked in the cyclooxygenase channel using in house docking program IMFI. The coordinate output from MD trajectories is analysed using analysis package of AMBER 5.0, MOLMOL, P-CURVES 3.0 and in house packages: ANALMD, ANALPI. We have observed perturbafive changes in COX-1 and COX-2 structures due to indoprofen and NS398. In case of indoprofon specific changes between COX-1 and COX-2 were noted in helix D, H6,S6 and helix 1t8 in the cyclooxygenase cavity. In case of NS398 these were in helix B in membraue binding domain helix 1t6, S8 and SlO in cyclooxygonase cavity and helioes Hl4-H16-in small lobe close to haem binding region. Imvlications of these results in enzyme selectivity by NSAIDs is discussed here.

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P49 PHOTOCONTROL OF THE STRUCTURE AND FUNCTIONS OF a-CHYMOTRYPSLN

Aail K. Sineh* and Kartha S. Madhusudnan, Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai - 400 076, India

ct-Chymotrypsin has been covalently linked to a photoisomerizable azobenzene unit and the hydrolyticc activities of the modified enzyme has been examined under photochemical conditions using p-nitrophenyl acetate as substrate. Both, E- and Z- forms of azo-~-chymotrypsins are found to retain their enzymatic activity, though slightly diminished as compared to the native enzyme. The hydrolytic activity of azo-~-chymotrypsin can be reversibly controlled by irradiation at two different wavelengths, viz. 314 and 430 nm. This study provides photochemical means of controll ing the structure and functions of biomolecules in general and gives a new direction for the development of photoswitchable biomaterials for applications in opto-electronic devices.

P50 Effect of chiral residues at terminal positions on the conformational structure of polydehydrophenylalanine peptides Fateh S. Nandel and ttarpreet Kaur* Department of Biophysics,Panjab University,Chandigarh- ! 60014,INDIA

a,15-dehydroamino acids are considered promising inducers even better than D-amino acids, the commonly appreoiated poptide modifiers of a I~ I1 turn which is a biologicall~ important structure. Because the presence of an sp 2 a-carbon atom and the electronic distribution caused by the C=C double bond, a dehydrophenylalamne (,~t~he) residue is characte~sed by peculiar structural lF, atures, so several works have been carried out in order to determine likely conformational censequonces of the presence of APhe residues m peptide sequences. In small peptides containing a single APhe, a bloc I113-bend structure was observed and in short peptides, the contbrmational behaviour of APhe is sensitive to the number, position and content of other usual amino acids in the poptide. In this study we report the conformational behaviour of peptides containing ix) ly -APhe viz. Ac-(APheh-NHMe,Ac-L/D-AIa-(,LPhe)~-NHMe and Ac-(APhe)o-L/D-AIa-NHMe. Due to the absence of chiral centre in APhe, the poptide Ac4~SJ~he~,-NttMe can adopt left as well as right handed 310 helical structure with ~=q~ =30~ presence of Ala residue, having a chiral centre alters the screw sense of the helix adopted by ~LPhe residues. In Ac-L-AIa-(APhe)6-NHMe the screw' sense of APhe residues are left handed with (IF q)=30 ~ and L-AIa residue adopts ~,q) value of-30~ ~ respectively. In this conlbnnation the carbonyl moiety of acetyl group lbrm a hydrogen bond with the NH moiety, of Phe residue at third position ~hereas in the peptide Ac-D-AIa4:~Phe)o-NHMe the screxv sense of the helix is right handed ~ith t~,q~ values for APhe residue equal to -300 each and D-AIa prefers ~,q~ values of 30~ ~

P51 Conformational Structure of N-terminal part of Gramicidin-A.

Fateh S Nandel*. Balwinder Singh and D.V.S. Jain* *Department of Biophx sics. *Department of ChemistD, Panjab University, C h a n d i g a r h - t60

014. INDIA.

Gramlcidin A is a hydrophobic uncharged pentadecapeptide containing alternating L- and D- amino acid residues A careful look at amino acid sequence of gramicidin A reveals that (i) only the amino acid residue: x~ith brancing at 13 or 7 positions are in D-form, (ii) N-terminal contains amino acid residues with smaller side chains, liii) C-terminal contains repeated sequence of D-Leu-Trp. Thus it is imperative to elucidate the role of D-amino acid residues having branching at 13 and Y positions. Therefore, the conformational bchax ~our of the x arious peptides has been studied viz: (a) tripeptide of the form Ac-X-D-Leu-NHMe where X=Gh.Ala .Abu .Va l .Leu . i l c and Ac-AIa-D-X NHMe with X=AIa,Val, l l e ,Leu ,Abu Gly and Aib. (c) pcnta.hcpta and octapeplide corresponding to the N-terminal of gramicidin A viz. HCO-VaI-GIy-AIa-D-Leu- AIa-N HMe. HCO-VaI-GIy- Ala-D-Lcu-Ala-D-VaI-Val-NHMe,HCO-CaI-GIy-AIa-D-Leu-AIa-D-VaI-VaI-D-Val- X~HMc and (d) dcsigned poptides by computational mutagenesis by substituting (i) Gly 2 by D-AIa (ii) Val b~ Ata tili) G h : bx D-ala and V a l b~ Ala (iv) Val by Ala, Gly 2 by D-Val and Val by Ala.

51


P52 Dynamical domain compositions of a single domain protein

K. Anton Feenstra and Herman J.C. Berendsen

BIOSON Re~earett Institute, Lab. of Biophysical Chemistry, University of Oroningen, Nijenborgh ~ Oroningen.

Recently we have shown that, using a novel method was developed to determine Dynamical Domains in proteins from from X-ray conformers and from Molecular Dynamics simulations, also in a typical single-domain protein of 85 residues characteristic dynamical domains can be filtered out, which can be translated into regions of increased and decreased rigidity [1]. Recent improvements on our simulation package, GROMACS, allow simulations of proteins in water to be performed at increased time step [2], thereby significantly extending the practical limit on the length of the large time-scale simulations that are necessary for these types of analysis, More detailed analyses will be performed, which will link regions of increased and decreased rigidity to o~ther local structural properties and dynamical properties of the protein. In this way we will attempt to identify internal elements of increased structural coherence, which will be correlated with the coherent motional behavior that is expected to exist in natural proteins. As an approach to study biologically relevant long-term protein behavior, these elements could subsequently be used to build simplified models of proteins.

P53 Study of Conformational Changes of Proteins by Normal Modes Analysis

F. Tamat'2~ Y.-H. Sanejouand I, N. Go 2 Laboratoire de Physique Quantique, IRSAMC, Universite Paul Sabatier (France), Department of Chemistry, Graduate

School of Science, Kyoto University

Conformational changes play an important role in the biological activity of macromolecules. It has been shown in a number of casesthat it is possible to obtain information about the nature of this conformational change by studying the largest- amplitude collective motions with theoretical methods: calculations of normal modes of low frequencies. The normal modes analysis can be carried Out in two different ways. A detailed potential energy or a simplified model (1) can be used in the calculation. Moreover, in the latter case, normal mod.e analysis by taking into account only Ccc atoms in the calculations are possible. Previous studies, with simplified model, on HIV protease and Adenylate Kinase have shown that the largest-amplitude collective motion obtained by normal mode analysis compare well with conformational change observed by crystallographers. However, in some rare cases, for example Enolase or Lactase Desydrogenase, with the simplified model, a poor agreement between low frequencies modes motion and conformational change is found. Normal modes analysis with detailed potential for these proteins should help us to understand the precedent results. 1- M.M Tirion, Phys. Rev. Letters, 77 (1996) 1905

P54 C o n f o r m a t i o n a n d A g g r e g a t i o n p r o p e r t i e s o f l o n g P o l y g l u t a m i n e p e p t i d e s

w i t h a n d w i t h o u t i n t e r r u p t i o n s Dbepak Sharma, Sunita Sharma, Santosh Pasha and Samir K Brahmachari* Functional Genomics Unit, Centre for Biochemical Technology, D.U Campus, Mall Road, Delhi-110007, INDIA.

Several neurodegenerative diseases are caused by expansion of polyglutamine repeats in the affected proteins. In most cases the proteins with expanded glutamine repeats are thought to precipitate and form toxic neuronal nuclear aggregates in the affected neurons. In SCA1, where the polyglutamine stretch is found to be interrupted by histidine residues, it has been reported that true indication of a mutant SCAI allele is the length of the longest polyglutamine tract coded for rather than the total size of the repeat region. Point mutation of CAT codon (His) to CAG codon (Gin) has been shown to induce repeat expansion and pathogenesis. Towards this end we investigated the conformational preferences of both the longest uninterrupted polyglutamine stretch as observed in various neurodegenerative diseases and those with histidine interruption(s) as seen in SCAI normals, using CD and second derivative FTIR (in KBr) spectroscopy. Our study suggests that substitution of histidines by glutamines induce a conformational change which results in decreased solubility and increased aggregation. Our findings also suggest that all the polyglutamine peptides with and/or without interruption(s) adopt [~ structure and not random coil as suggested earlier. Moreover the insolubility and aggregation phenomena in case of all the polyglutamine peptides are preceded by the formation of large intermolecular [3 sheet structures.

52


P55 PEPTIDE D E I G N USING O., I$-DEHYDRO-AMINO ACID RESIDUES ~ ~ l l E J l l . ] [ l l l b Jyofl Makker, Shanaisflm l)ey, S.Kmmw and T.P.Skagh Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110 029, India

The conformsfional preferences of amino acid side chains are of fundamental importance in determining the interactions that govern the preferred conformations of polypeptides and proteins. The eonformafional preferences are governed to a large extent by interactions of the atoms of a given side chain with atoms of the backbone as well as the atoms of tile twb neighbouring peptide units. In order, to develop an effective design tool, it is necessary to restrict the number of preferred conformations to the minim,m r amino acid residues have emerged as an effective tool in the design of precise secondary structures of peptides and proteins. In recent years, systematic investigations on dehydro-reaidue containing peptides have led to the deduction of several general rules of peptide design. APhe residues have been extensively studied and their cenformational preference is restricted to three ~ u angles, -60, -30 ; -60, 140 ; 80, 0.respectively. AAIa and AVal residues have also been studied. In view of such clear guidelines, the peptide design using r residues has become a useful tool. Thus, the prediction of sequences for various secondary structures is feasible. By varying dehydro-residues from the same class it is possible to modulate ~ u values, thereby obtaining a conformation of choice.

P56 P r o b i n g t h e d y n a m i ~ a t p r o t e i n - w a t e r i n t e r f a c e

G . S. L d ~ h m i l a p t h & G. Krishnamoerthy Department of Chemical Sciences, Tara Institute of Fundamental Research,

Homi Bhabha Road, Mumbai, 400 005, INDIA

Solvent interaction with proteins exerts its influence on the dynamics and functions of proteins. The nature of protein-water interface was probed by monitoring the rotational dynamics of solvent exposed side chains. The single U'yptophan (W) sidechains of subtilisin Carlsberg and myelin basic protein and covalently linked extrinsic fluorophores were used as probes. Solvent exposure of W was testified by fluorescence quenching by KI. The values of bimolecular quenching constant were very similar in native and denatured states. The motional dynamics of W was estimated by picosecond time-resolved fluorescence anisotropy measurements. The rotational correlation time associated with the internal motion of W scaled very poorly with the bulk solvent viscosity. In contrast, the scaling was linem as expected by Stokes-Einstein relationship when the pi'oteins were denatured. These reSults indicated the presence of bound water on the surface of native proteins. This water sVucture which insulates the protein surface from the bulk solvent gets disrupted on denaturation.

P57 The New Mechanism of Enzyme Activity Regulation by Ionic Strength

Mazhul V.M., Zaitseva E.M., Institute of Photobiology.Natl.Acad.Sci.Belarus, Akademicheskaya Str., 27, Minsk, BY220072, Belarus,

E-maih [email protected] The effect of ionic strength of the solution, created by Na § K', Mg 2§ Ca 2§ on internal dynamics and enzyme activity of aldolase from rabbit muscle (EC 4.1.2.13) has been studied. Changes of structural flexibility of the protein were monitored by the room temperature tryptophan phosphorescence (RTFP) technique. The RTTP lifetime and intensity of aldolase are known to report on the rigidity of the protein matrix around Trp 147, which is located in the hydrophobic core in the vicinity of the enzyme active site. It has been esteblished that rising of values of ionic strength of the solution (0.05-11a) results in dramatic increase of structural flexibility of the protein matrix (as reflected by the considerable decrease of both RTTP intensity and lifetime values) and reduction of the enzyme activity. The effect of ionic strength was more intensive and observed at lower Ix values in case of Ca 2" and Mg 2+ than of Na*, K § Good correlation between changes of internal dynamics and enzyme activity of aldolase shows functional significance of protein matrix structural fluctuations. We assume the decrease of enzyme activity to be associated with a modification of intramolecular interactions resulting in a greater flexibility of the active site structure. Based on the data obtained we introduce the new mechanism of enzyme activity regulation by ionic strength of the media by means of change of protein structural flexibility.

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P58 O N E - A N D T W O - P H O T O N I N D U C E D F L U O R E S C E N C E O F P R O T E I N S

Ktcrdamtk University o f Warsaw, Institute o f Experimental Physics, Department o f Biophysics, 93 Zwirki i Wigury St. 02089 Warsaw, Poland; E-mail: [email protected]

Two--photon excitation (TPE), or two-photon induced fluorescence (TPIF), o f proteins offers the potential advantages o f excitation to different excited states and the possibility o f new information content in the steady-state and time-resolved fluorescence spectra. TPE experiments require higher concentration o f fluorophores (sometimes in the m M range), and very high excitation power, usually obtained with lasers. Different transitions were observed for these modes o f excitation o f tyrosine and tryptophan, and higher selectivity for TPE was obtained. Such selective excitation was unequivocally confirmed from studies o f tyrosine-tryptophan mixtures and proteins containing tryptophan and tyrosine residues, where OPE resulted in dominant tyrosine emission, while in the case o f TPE, emission is dominantly from tryptophan. Results o f further studies will be presented, which led to characterization o f the effect o f environment and mode o f excitation on the fluorescence o f proteins.

P 5 9 FCS as a tool to monitor protonat ion kinetics and photodynamics of dyes - aspects of GFPs

and fluoresceins as pH sensors. Widengren, j?.4 Terry, ]3. 2 Mets, 0 3, Rigler R. 4 i. Max-Planck-Insitute f. Biophys. Chem., G6ttingen, Germany 2. Bieimage, Novo

Nordisk, Copenhagen, Denmark 3. Evotec Biosystems, Tallm, Estonia 4'Karolinska lmtlmtet, Stockholm, Sweden

Fluorescence correlation spectroscopy (FCS) has over the past decade emerged as a very versatile technique to study a wide range of dynamical processes on the molecular level with ultrahigh sensitivity. From the specific fluctuations in fluorescence photophysical processes, such as singlet-triplet state ~nteractions, photoinduced isomerization, electron transfer and photodegradation processes can be characterized. Exploiting the fluorescence fluctuations generated upon association and dissociation of hydrogen ions to a pH-sensitive dye, FCS can be used to monitor pH in microenvironments, yielding also temporal information of the binding process. Here, FCS is used to characterize the protonation kinetics and photodynamics specific for GFP. Distinct differences between FITC and GFP are seen indicative of a two-step protonation process of GFP, with an intermediate step following upon proton exchange at some external site of the proteitt and involving a conformational transition leading to fluorescence quenching. Several photophysical processes in the millisecond to microsecond time range are seen for GFP, compatible with a photo-induced isomerization process, triple state formation, as well as local conformational fluctuations in the microsecond time range close to the chromophore unit.

P 6 0 INTRINSIC QUENCHERS OF TRYPTOPHAN FLUORESCENCE IN PROTEINS: A STUDY BASED ON

CRYSTAL STRUCTURE DATA R. Swaminathan*, S. Thamotharan # and N. Yathindra #

# - Department of Crystallography and Biophysics, University of Madras, Chennai 600 025, India. * - Department of Chemistry, Indian Institute of Technology, Panbazar, Guwahati 781 001, India.

Investigations on the influence of intrins.ic quenchers on the fluorescence lifetime of tryptophan (Trp) in several single Trp containing proteins have been pursued using their crystal structure data. Proximities of quenchers such as tyrosine-OH, imidazole, -SH, -CONH2, -COOH, -NH3 + and the amide bond from the indole ring of Trp are estimated for the three rotamers, gauche +, trans and gauche around the Ca-Cp bond in glucagon, melittin, ribonuclease Tt and other single Trp proteins. Attempts are made to correlate the amplitude of the shortest fluorescence lifetime component in the fluorescence intensity decay with the Trp rotamer most vulnerable to fluorescence quenching. The results show that the peptide bond is a strong quencher of indole fluorescence in the case of melittin and glueagon. The rotamer populations are calculated based on the energies corresponding to the three conformational states and are compared with three different lifetimes and amplitudes. The rotamer corresponding to the quenched state appears to be different from that found in the crystal structure. Detailed results obtained for various proteins are described.

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P61 C o n f o r m a t i o n a l f l u c t u a t i o n o f p r o t e i n o b s e r v e d b y t i m e - r e s o l v e d h o l e - b u r n i n g

Ay. Shibata, AH. Chosrowjan, AN. Mataga, BI. Morisima, Inst. for Laser Tech, A, Kyoto Univ. B

In a protein, various types of molecular motions occur in different time scales ranging from sub-picosecond to milliaecond region. To elucidate the molecular medmnisms of protein functioning, it is essential to make a time-domain observation of these conformational dynamics over a whole temporal range by a single observation technique. Time-resolved transient hole-burning (TKFHB) spectroscopy is one of the most powerful tools to do such an observation. In TRTHB technique, conformational fluctuation of chromoprotein is studied by detecting the fluctuation-induced temporal variation of the surrounding environment felt by the chromophore This is done by observing the temporal variation of the transient hole-burning (THB) spectrum burned by the irradiation of laser pulse. So far, we have observed the conformational dynamics of some heine-protein samples in a ns-ms time region. These observations have revealed the importance of the solvent flexibility to the protein dynamics. In the premmtation, we will review our recent results. Then, in order to get a more detailed insight of the nature of the observed dynamics, we will discuss our recent investigations of the effect of point mutation on the proton d3rnamics, which is now in progress.

P62 The structure of the Myosin dimer: measuring light-chain to light-chain distances using fluorescence- and

luminescence resonance energy transfer ~ l l ~ L ( ~ g g I g ~ , Ming Xiao, Roger Cooke ", Paul Selvin. Physics & Ctr. for Biophys. & Comp. Biol, UIUC, Urbana,

IL 61801 & * Biochem/Biophys. UCSF, San. Francisco, CA 94143.

The dimeric protein myosin binds to actin and hydrolyzes ATP to generate force and movement which leads to muscle contraction. Why myosin is a dimer, what is the spatial arrangement between the two halves, how these distances fluctuate, and how the dimer interacts with actin are poorly understood. As a fast step towards understanding these issues, we have measured distances between the two regulatory fight chains within each dimer. Distances were measured using fluorescence and luminescence resonance energy ~rausfer (FRET & LRET). Both techniques measure distances between a site-specifically placed donor and acceptor, but are sensitive to different motionsl time-scales. Specifically, we use single-cyste/ne mutants of skeletal RLC that are labeled with a donor (a luminescent lanthanide or a fluorescent organic dye) or an accvptor such as tetramethylrhodamine, and exchange into purified skeletal heavy me romyosin. The donor transfers energy in a distance-dq~*ndent fashion to the acceptor. We find that probes placed near the intersection of the two dimeric hafts are in close-proximity by LRET, but significantly farther as measured by FRET, indicating that they move on the sub-millinecond time-scale. Surprising!y, the distances are not affected by binding of myosin to actin. This work was supported by NIH grant AR444220 and by the Research Board, University of Illinois-Urbana-Champalgn.

P63 Study of Bioprotective Effectiveness of Disaccharides by Light and Neutron Scattering

C. Branca, A. Faraone, S. Magazd, G. Maisano, P. Migliardo, V. Villari Dipartimento di Fisica and INFM, Universit~ di Messina, P.O. Box 55, Papardo, 98166, Italy

Water is usually thought to be required for the living state but many organisms can withstand anhydrobiosis when essentially all of their body water has been removed. Indeed, numerous restrictions have led many organisms, as some species of soil-dwelling organisms, to some adaptations to survive. The mechanisms for survival to this kind of stress is connected with the accumulation of trehalose. The present work shows that light and neutron scattering allow to get information on the effects of these disaccharides on the hydrogen bond network of water. The analysis of the results nnmnbiguously show that trehalose induces the most extensive destructuring effects on the adjacent water molecules, playing the role of structure-breaker. These results make it plausible the hypothesis that the crystallization process, in the presence of trehalose, is more obstructed with respect to maltose and sucrose, by a relatively high reduction of the amount of freezable water.

55


P 6 4 Steady State Fluorescence and Quenching Studies on Lactoperoxidase

and M. Sharique Zahida Wahecd ~ m e n t of Chemical Sciemes, Tara Institute of Fun6amental ~ c h ; Homi Bhabha ~ Navy

Nagar, Colaba, Mumbai 400 005, Irma Intrinsic steady-state fluoresome, of lactoperoxidase (LPO) and its llgand bound complexes have been characterised as a structural probe of its stmcttae in sohfion. The Quaatum yietd of 0.0185s for LPO and the emimon ~ observed at -338 am, for LPO aad the complexes, indicate the Trp--~heme energy tinnier. Trps are located away from the heine. They are appropriately equally distn'buted in the hydrophobic and hydrophilic regions of the protein. The "average" distance between Trps and the heine within the tmzyme is 25.1!'0.2A. These fluormcet~ properties are comistent with the recmt theoretical threo-dimc~tional model for LPO and reveal that Trp337 and Trp404 dominate the ~ c fluorescence, and together contn'bute -64% of the observed intensity. Quenching studies using I ' , Cs + and polar neutral acrylamide are consistent with this picture. Actyiamide penetrates the protein matrix. It is an efficient quencher and the quenching proce, s is essentially homogeneous with all the Trps being mr.essible. C + ion is a very ineffident, quencher but the iodide ion shows the qutmching process to be predominantly heterogeneous with widely differing Trp accessibility. The Stem-Vohner constants deduced are K,, =8.4+1.4 M'Zand/~ =4.05s M "n for aczy]amide and iodide quanchin 8, respectively. The fi'actional accesstWdity, f~, = 0.52i-0.03, was deduced foriodide quenching.

P 6 5 DOES PICOSECOND PROTEIN DYNAMICS HAVE SURVIVAL VALUE? M. Brunori~, F. CutruzzolU, Q.H. Gibson 2, C. Savino', C. Travaglini-Allocatelli' and B. Vallone t. ~Dept. Biochemical Sciences and CNR Centre of Molecular Biology, Univ. Rome "La Sapienza", Rome (IT) and 2Dept. Biochemistry and Cell Biology, Rice Univ., Houston, TX (USA).

The control of oxygen delivery to tissues is of great significance to the fitness of an organism. Different oxygen carriers vary in the kinetics of oxygen binding and release. The structural dynamics of oxygen dissociation in non- cooperative haemoproteins allows rationalization of experimental data obtained by overall reaction kinetics, single crystal x-ray diffraction, laser photolysis and molecular dynamins simulations. An accurate 3D structure of the reactant and the product (in our case deoxy and oxy Mb-YQR, a new triple mutant of sperm whale Mb) is essential but insufficient to understand the biochemically relevant oxygen rates and affinities. We present the hypothesis that the overall oxygen kinetics of a carrier haemoprotein is controlled not only by the iron-ligand coordination bond and the local H-bonding by distal site residues, but also by fast fluctuations of internal side chains which may either permit or obstruct access of the ligand to cavities within the protein before escape to the bulk. Since these motions occur on a picosecond time scale t "L'heir significance in controlling the rate of oxygen delivery dictates the physiological competence of the carrier. Thus fast dynamic fluctuations acquire a "survival value" in haemoproteins and possibly in all proteins.

P 6 6 STEADY-STATE AND PICOSECOND-TIME Pseudomonas/mt/da CYTOCHROME P450cam

RESOLVED ~'LUORESCENCE STUDIES ON

Swati Pr,ar,.qd, Shyamalava Mazumdar and $amaresh Milra Department of Chemical Science& Tara Institute of Fundamental Research, Homi Bhabha Road, Colab~ Atumbai-400005, India

Steady state and time resolved fluorescence has been used to probe the local mvimmmm of cytochrome P450cam and the effect of the binding ofthe atbsWae (camphor)with the eazyme, l~e steady-state fluorescence quenching expetime~ show that all the five-tryptophan residues in the ~ - f r e e and camphor-bound mzyn~ are accessible to the neutral quencher, agIylamide. Thc D nmh~ Of ~ residu~ ~ to iolliC quaghef (KI) increases ou mlmtrate binding to the enzyme. This indictmm oa removM of,'~mphor frmn the ~ .mp~r_ -bound enzyme, the tq0?tophan residaes move towards its core. The time remlv~ flumtscence m~i~s shows that the f l ~ decay follows a three-exponential model, giving three lifetime mmpmm~ in the range from 100136 to 4as. The binmn~ of ~ does not ,-~_ ,~- any significant clnan~ in the lifaime comimamm ef the mzyme, ~l~ng m~ the ~o~n rescues are Ixmm~ly far mmved flora the sMmme ~ ~ ~ ~ ~ of c~0r does ~ ~onifl~y affect the ~ congxinin~ ~ (fflbe ~ . ~ d~ay-gm~q~xl emission spectrum was utilized to ~ g n the diffetem lifetime compenems a ' ~ residues.

56


P67 Electrostatic Behavior of synthetic peptides in polar solvents

P. Soto and R. Fayad Departmento de Fisica, Universidad de los Andes, A.A. 4976 Bogo~, Colombia

Our aim is to describe solubility of synthetic pcptides of biological interest immersed in polar solvent. We start with a careful analysis of molecular dynamics simulation of peptides in vacuum and in polar solvent. As an important tool to investigate and compare peptide activity. We calculate electrostatic potential and SAS. We use the above results to predict free energy of solvation and conclude about those synthetic pcplidc activity.

P68 K1NETYCS PROPERTIES OF BACTERIAL L UCIFERASES IN WATER-ORGANIC SOLVENTS

I. E. Sukovataya. N. A. Tyulkova N.A. Institute of Biophysics Russian Academy of Science, S]~oerian Branch, Krasnoyarsk, 660036

The influence of acetone, methanol and ethanol on kinetic parameters (the emission intensity, the quantum yield and the rate constant of bioluminescence decay) ofbioluminescent reaction catalyzed by luciferases from Photobacterium leiognathi and Vibrio harveyi was studied. The reaction was initiated by the injection of the photorecovered FMNH2. The long-chain aliphatic aldehydes, e.g. decanal, dodccanal, tctradecanal were used as substratcs. The series of changes in parameters of bioluminescence reactions depends on the type and concentration of the organic solvents added. The dcnaturating concentration of solvents has good correlation with hydrophobicity of those. The n0ndenaturating (of less then 10%) concentrations of acetone, ethanol, methanol increase the rate of bioluminescence reaction catalyzed by P. leiognathi, with dccanal and tetradecanal used as substrates, 1.5-2 times as much. The increasing of the reaction intensity for luciferase from V. harveyi by a factor of 2-3,5 occurs in water-organic mixtures with all aldehydes. The presence of organic solvents essentially changes the value of the rate constant of bioluminescence decay with dccanal and tctradecanal used as substrates. The rate constant value for biolnminescence decay, as the organic solvent concentration increases, is determined by the aldehydes chain length used. In case of tetradecanal the rate constant of bioluminescence decay degreases, with dodecanal - it remaing constant, in the dec.anal case its value grows for both lueifemse~. The factors affecting the binding of substrate andthe xeleasing of the product by bacterial luciferascs arc discus~:d.

P69 THE ELECTRON SPIN RESONANCE PROPERTIES IN WOOL

KERATIN AND THE EFFECTS OF PIGMENT AND y AND UV IRRADIATION ON THESE PROPERTIES

Sh.V.MAMEDOV t~, B.AKTAS 3, M.CANTURKI, B.AKSAKAL j and R.YILGIN J l.Yddtz Technical University, Department of Physics, 80270 Sisli - lstanbul, TURKEY

2.Institute of Physics of the Azerbaijan Academy of Scien., Bake, H.hvid St33, AZERBAIJAN 3. Gebze High Technological Institute, Department of Physics, 41410 Gebze- Kocaeli, TURKEY

Different from the studies in literature, the e|ectron spin resonance (ESR) spectrum of wool keratin was examined in a wide magnetic field range and a more complex spectrum was observed: !) A central signal with AHm=I.lmT and g=2.0075 approprite to the doublet in literature; 2) The sulphur signal with AHm=2.2 mT and g=2.0218; 3) A signal with AHm=5.0 mT and g = 2.1911;4) A very wide signal with AHm=66.0 mT and g=2.1575 ; 5) A very intense signal with AHm=0.6 mT and g~2.0065, the intensity of which depends on the degree of pigmentation in wool keratin with pigment. In this study, all these signals are observed for the first time at room temperature without any effect. After samples are irradiated by 7 and UV, the intensity of the signal in wool keratin increases definitely. This increment continues till the light brown wool keratin, however, there is not any increment at all in this sample (light brown wool keratin ) and also a decrease by 1.7 or more times in black wool keratin is observed. These results, as mentioned in our publications prove that pigment (melanins) plays a capturing role for the free electrons appearing in the irradiation process and for free radicals occurring and it plays a protector (defence) role- for keratin against the irradiation.

57


P70 Effect of metals ions on ATPase activity and superprecipitation of cardiac muscle actomyosin

Bogutska K.I., Miroshnichenko N.S. Shevchenko Kyiv University, Department of Biophysics

The inhibiting effect of Zn 2+, Cd 2§ Sr 2§ on Mg2§ superprecipitation and ATPase activity of cardiac muscle actomyosin has been investigated in order to elucidate possible role of such catiofas in the contractile process. Cations of Zn 2§ Cd 2+, Sr 2+ (1-5 mM) can influence on superprecipitation of actomyosin but the level of the process decreases respectively as compared to control (in presence of Mg2+). Mg 2+- ATPase activity of cardiac act0myosin is decreasing in presence of these ions and depending on their concentration. It was settled that ions can influence on actomyosin ATPase and structural changes of cardiac muscle actomyosin complex.

It is probable that such cations can substitute magnesium ions in the active centre of myosin as well as in the section of significance for the process of superprecipitation of actomyosin but the substitution is less efficient for realization of the superprecipitation reaction and ATP-hydrolyze process than when magnesium ions are available in the incubation medium. It is possible that these results are evidence of the direct effect of heavy metals ions on actin-myosin complex. The results obtained prove that the ions of heavy metals can effect the cardiac muscle at the stage ofactin-myosin interaction.

P71 ALTERATIONS IN THE EXPRESSION OF SMOOTH MUSCLE MYOSIN ISOFORMS IN HYPERTROPHY OF THE SMOOTH MUSCLE IN THE URINARY BLADDER WALL. S. Chacko, M. DiSanto, J.A. Hypolite, Y- M. Zheng, and A.J. Wein from the Division of Urology and Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104.

Myosin is the molecular motor for muscle contraction. Although them is only one smooth muscle myosin heavy chain (MHC) gene, MHC mRNAs that encode tissue-specific isoforms which differ at the N-terminal (SMA and SMB) and C-terminal (SM 1 and SM2) regions are formed by alternative splicing of the pre-mRNA. The presence of SM-B isoform has been shown to affect the ATPase activity and shortening velocity of smooth muscle, and the SM-1 isoform is shown to form more stable myosin filaments compared to that of SM2. Partial obstruction of urinary outflow leads to Compensatory hypertrophy of the bladder smooth muscle. The objective of this study was to examine whether the expression of the alternatively spliced MHC isoformsis altered in response to the remodeling that follows partial outlet obstruction. The SM-A isoform, typical of tonic smooth muscle, increased from <1% to 60% in hypertrophied muscle. Both SM 1 and SM-A isoforms returned to near normal levels upon reversal of the hypertrophy. Our results indicate

that the increased level of SMI isoform is likely to form stable myosin filaments, and theoverexpression of SM-A isoform makes the bladder muscle more tonic in the remodeling of smooth muscle following outlet obstruction. Supported by NIH Grants. P50 DK52620 and DK39740.

P72 ELECTROSTATIC AND TITRATION PROPERTIES OF PHOSPHORYLATED

MODEL COMPOUNDS AND IrEPTIDES M. Wojciechowski l'*, T. Grycuk 1, J. Antosiewicz 1, B. Lesyng 1'2

t Department of BiophyMcs, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland 2ICJ~I, University of Warsaw, Pawinskieg.o 5,4, 02-106 Warsaw, Poland; * e-mail: [email protected]

Phosphates play a key role in living cells. The biological role of phosphates can be explained referring to their electrostatic properties. This problem requires, however, a more detailed study. The first pKa of phosphoric acid and of phosphate mono- and diesters is about 2, which results in at least their monoanionic state at physiologica! pH. However, their secondary pKa can result in change of the charge, depending on the molecular environment. The goal of the present study is to elaborate a theoretical approach of predicting ionization states of the phosphate groups in model molecular systems and in peptides. We computed ESP charges for several monosubstituted phosphates. Using the Poisson-Boltzmann approach and the computed ESP charges, protonation energies of the investigated compounds were predicted. Results of these calculations were used to propose a model pKa of the phosphate group. It can be used in predictions of titration properties of the phosphate groups in proteins by means of the continuum molecular electrostatics methods. The refined PB model was validated using a short phosphorylated peptide Gly-Gly-Tyr(P)-Ala.

M. IZ. acknowledges support by Warsaw University (BST 591/BF), T. G., d.A. and B.L. by KBN (8 TI IF 016 16)

58


P 7 3 How do Packing and Chain Topology Affect the Structural Dynamics of Proteins?

Marc A. Ceruso and Alfredo Di Nola Department of Chemistry-University of Rome "La Sapienza"-P.le Aldo Moro, 5-00185 Rome Italy

A remarkable feature of protein structure is the tight and regular packing of core residues. Specific side chain/side chain packing interactions are recurrent throughout protein folds. The density and specificity of these interactions is thought to control not only the folding topology but the folding pathway as well. The purpose of this work was to assess the relative importance of packing and chain topology in determining protein dynamics.

The effects of packing and chain topology on the structural dynamics of proteins have been investigated in a systematic manner using molecular dynamics simulations. A total of 12 proteins systems with 4 different topologies were simulated for at least 2ns each in the presence of explicit water molecules. D.vnamic properties derived from principal component analyses of the trajectories were quantitatively compared by using groups of proteins that belong to the same fold family such as (1) protein G, protein L. Ubiquitin, and (2) Cytochrome C551, C552 and C. Alternatively, proteins that do not belong to the same fold family but present a significant homology of packing and chain topology were comparatively analyzed: (3) Initiation factor 1, r SH3 domain and Interleukin 113. Finally, (4) core-repacked variants were compared to the wild-type protein in the ease of the four-helix-bundle Rop.

P 7 4 PH NMR study on the binding mechanism of Me-tx-D-GalNAc to Artocarpus lakoocha lectin

Subhasis Bandu and Bistmu P. Chatterjee Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032

The dynamics of the binding mechanism between Artocarpus lakooeha lectin and Me-r have been studied using nH NMR spectroscopy. Various thermodynamic and kinetic parameters have been calculated with the help of temperature dependence line broadening of the methoxy group resonance of Me-r No change in chemical shift has been observed while linewidth at half height of the sugar protons found to have increased with increasing temperature indicating that binding ligand experiences a fast exchange and a similar magnetic field between bound and free environments. The activation parameters obtained from the association and dissociation rate constants suggest that the association process is controlled by high activation entropy which is due to the specific orientation of both lectin and sugar whereas the contribution of activation enthalpy is small. On the other hand, the dissociation reaction is controlled by high activation enthalpy due to breaking the interaction between the Sugar and lectin. From the NMR data a two-step binding mechanism has been proposed. The associated complex is stabilized mainly by hydrogen bonding and van der Waals attractions since AH ~ and AS ~ have been found to be negative while hydrophobic interaction is not significant, if present at all, otherwise a positive AH ~ and AS ~ values would have been obtained.

P 7 4 A Protein Prosthetics: X-ray crystal structure of FimC-FimH chaperone-adhesin complex. _Devapriya ChoudhutT x,Andrew Thompson 2, Vivian Stojanoff 3, Jerome Pinkner 4, Scott Hultgren 4 and Stefan Khight n. nDepartment of Molecular Biology, Swedish Iniversity of Agricultural Sciences, Uppsala, Sweden, 2 3 EMBL Grenoble Outstation, Grenoble,France, ESRF Grenoble France, 4Department of Molecular Microbiology, Washington University School of Medicine, St.Louis, USA.

Molecular interactions that occur between the pathogen and the host cell surface receptors are among the earliest events in pathogenesis. In gram-negative bacteria these interactions are often mediated by hair like appendages called pili on the surface of the bacteria. Specific proteins called adhesins are situated at the pilus tip which are responsible for binding to cell surface receptors, thus initiating pathogenesis. We have solved the X-ray crystal structure of the adhesin protein FimH in complex with the bacterial chaperone FimC at 2.5/~ resolution. FimH is present at the tip of the so called Type I pill in uropathogenic E.coli and is responsible for urinary tract infections in humans. FimH consists of a two domain structure, one of which, called the lectin domain, binds to mannose containg receptors in the host cells. The other domain called the pilin domain has a unique mode of interaction with the chaperone FimC, in which a beta strand fom FimC is inserted into the protein. This phenomenon termed 'Donor strand Complementation' appears to have important ramifications not only for the stabilization of FimH tertiary structure but also for the assembly of the entire pilus.

59

A2 : Proteins : Folding and Stability

P75 Stability and Unfolding of p53 Human Core Domain

Delphine Flatters and Julia ~ e l l o w , Department of Crystallography, Birkbeck College, Malet Street, London

The protein p53 is a key element in maintaining genomic stability being involved in several different aspect of control of genome integrity. Most importantly, more than 50% of human cancers contain mutations involving 1)53. These alterations lead to complete loss of wild-type functions reducing DNA binding and transcription. The majority of these mutations map to p53 core domain, the 3D structure of which is known from X-ray Crystallography. The mutations seem to fall into those that directly affect the binding of DNA to the protein and those that destabilise the conformation. We are using molecular dynamics simulations to study the stability and unfolding of both the wild-type and mutant p53 core domain. One mutant, R175H, is thought to deatabilise the native structure. At high temperature, our trajectories show several common features of wild-type and mutant unfolding in agreement with experimental data. But, in the case of the mutant, the LSH region which interacts with DNA seems to be more destabilised and, one of the zinc ligand move away from the zinc ion earlier in the trajectory than for the wild-type. We propose an unfolding mechanism Which effects the conformation of the N-terminns, the $6-$7 epitope region and the LSH domain.

P76 S e l f - c o n s i s t e n t p o t e n t i a l s f o r p r o t e i n f o l d i n g s i m u l a t i o n s

F ~ . ~ Z ~ I " ~ , Minoru Kanehisa1" and Masaki Sasai

Institute for Chemical Research, Kyoto Universityt Graduate School ofHurnan Informatics Nagoya University~

We present a new idea to construct self-consistent statistical potentials which are suitable for protein folding

simulations. In order to determine the appropriate parameters, we take unfolded and misfolded structures into

consideration. For compensating the lack of knowledge of sU'uctures that appear during the folding processes, a series of

conformations were generated by molecular dynamics simulations using an idealized potential constructed from a

dataset that contains the native structure of the target protein with high weight. Then, the statistical potential is adjusted

so that energy spectrum of generated conformations is consistent with the sufficiently smooth potential surface. The

adjusted self-consistent potentials are applied to folding simulations of several proteins.

P77 Energy landscape analysis of kinetic partitioning phenomena in a lattice model

l ~ l ~ i . ~ a n ~ l ~ , Masaki Sasai

Graduate School of Human Informatics, Nagoya University The mechanism of kinetic partitioning phenomena in protein folding is studied with a simple lattice model. The chain contains two kinds of residues, H (Hydrophobic) and P (Polar). Chain with the length of 16 residues is put on the 2- dimensional square lattice and is moved with the Monte Carlo (MC) method. In order to fully understand the equilibration process, the chain motion is restricted in the space containing 4911 conformations. The sequence is designed so as to have two competitive energy minima (Ml and M2) in this restricted conformational space. Kinetic change of the residence probability of the MC trajectories at each conformation is monitored and the way the residence probability approaches the Boltzmann distribution is analyzed. For the certain time ranges the probability to visit Ml is larger than the one to visit M2 when a lager number of the kinetic paths are linked to Ml than M2. The residence probability at M2 increases through two kinetic phases. Relevance to the a - fl transitions in prion or in f l- lactoglobulin is discussed.

60


P78 A un i f i ed des ign a p p r o a c h fo r t h e i nve r se fo ld ing and d i r e c t folding of protein

Wang I~ao Han Yuan Zheng Wang Zhi Xin and Pan xin Min Institute of Biophysics, Academia Siniea, Beijing, 100101, China

The protein design or inverse folding problem, which maybe have improtant significance for biotechnology, have been intensively studied on the methodology. The recent progress is mainly concentrated on the Monte Carlo method with regard to the some poteintial function H(s, r) of protein. Here we suggest a algorithm, which, in theoretical principle, could apply both to the inverse folding and to the direct folding. The general form of algorithm for the inverse folding is

in which Si denotes what amino acid residue locates at the i - th site along the protein chain. The convergence of the algorithm is proved mathematically and applied to 21) and 3D lattice models of protein respectively so as to test the validity of the algorithm. The numerical simulations show that the better results are obtained. Espe- cially, for a example of 2D lettice HP model, i .e. there are just two types of residues, H(hydrophobie) and P (hydrophilic) residues, the result is identical to one of dual Monte Carlo method.

P79 Pm~dally unfolded intermediates of muRimeric proteins: Studies on guanidine

hydrochloride stabilized intermediates.

VimMI Rhakunl*, Sangeeta Knlkami, Atta Ahmad~ goodathingal Prakash and Shashi Prajapati

Dive'on of Membrane Biology, Central Drug Research Institute, Lucknow 226 001, India.

Multimeric enzymes provide an attractive system for investigating spontaneous self-assembly of protein stmchlres and for examining regulatory interactions between subnnits. Despite the preponderance of multimeric proteins in biological systems, quantitative equilibrium experiments on large polymeric structures are still scare because of their complexity and low stability. Understanding the folding/unfolding and self assembly processes of such macromolecules remains a major problem in protein chemistry. We have observed that in case of multimeric proteins presence of low concenlrations of GdmCI leads to formation of stable intermediates of proteins with significant folded structure and on intact suburut composition. The results of our studies on bovine liver catala.se and Glucose oxidase gill be discussed.

P80 Protein folding at subzero temperatures

Alexey Surin ''~', Tomoharu Matsumoto '), Li Yang'), Yuki Nakagawa", Kazumoto Kimura ~), Yoshiyuki Amemiya '), Gennady V. Semisotnov 2' and Hiroshi Kihara ')

1) Kansai Medical University, Japan, 2) Institute of Protein Research, Russia, 3) Dokkyo University School of Medicine, Japan 4) University of Tokyo, Japan

Proteins fold very fast, faster than ms. It prevents from monitoring directly the early events of the folding process by circular dichroism or x-ray scattering. We have been developing stopped-flow appratuses working at subzero temperatures for very viscous solutions. To monitor folding at subzero temperatures, we have to add anti-freeze. We have tested mixtures of ethylene glycol, methanol and water, which gives stable intermediates in case of myoglobin denaturation. Folding process was found drastically slow in comparison with that at room temperatures, when monitored by optical absorption. This enables us to monitor early events of folding process by stopped-flow combined by fluorescence, optical absorption, x-ray scattering and circular dichroism.

61


P81 Anion-induced Foldinl~ of Human Serum Albumin under Low pH Conditions

Sasd Tayyab. Salman Muztmmil and Yogesh Kumar

Interdisciplinary Bioteclmology Unit, Aligarh Muslim University, Aiigarh 202 002 Acid denatured human serum albumin (lISA) at low pll (2.0) exists in s molten globule like oonfmmatlou

characteriTed by. the presence of abundant secondary structure, high ANS binding, loss of tertiary structure and mm- cooperati~: themud amsitim. We studied the effect of vtr/ous anions (of acids and salts) on the acid denatured state of ! ISA by near-UV CD. far-UV CD. ANS binding, tryptophen flumcsceuee and thermal transition. Addition of different acids and salts caused an induction of a-helical structure as evident from the inow.ase in the MItE value at 222 nm and loss Of ANS binding sites e.~ibited by the decrease inthe ANS fluore~ence intensity. However, the concentration range of acids / salts required to brin 8 about the transition varied greatly among different acids and salts. Among various acids / salts tested, K3Fe(CN), was most effective in inducing the preperties clo~ to native .~na,~ure. It followed the electroseleotivity .~-ries. The near-UV CD .,q~ectra showed an increase in MRE iowards the na~'e a te . whereas the .tWptophan flumescenee emission spectra produced a red shift of about 6 um on addition of KCI(h. "lhe temperatta~induced transition in the presence of 40 mM KCIO, monitored by eliipticity measurements at 222 nm was chmcterized by the presence of an inlemtediate slate having abundant mxxmdmy stnglure and existing in between temperature range 30~ - 50~ These results suggest that lISA at low ptl and in the Wesem~ of acids or salts, exists in a partially foMed state characterized by native like s e ~ stmotme and tertia~ folds.

P82 Partially folded tetrameric intermediates o f bovine liver cata lan: Guanidlne

h y d r o c h l o r i d e a n d u r e a d e n a t u r a t i o n . Sangeeta Kuikarnl, Shashi Prajapa~ Koodathingal Prakash, Atta Abroad artd Vinod Bhakuni.

Division of Membrane Biology, Central Drag Researc~ Institute, Lucknow 226016, India.

Equilibrium guamdine hy&ochloride and urea unfolding studies on tetuuneric enzyme, bovin~ liver catala_~e showed that in presence of low (0-0.5M) guanidine hydrochloride (C-dmCl), it undergoes a temperature dependent change in enzymic activity. S ~ studies showed that upto 0.8M GdmCl the enzyme exists as a tetramer. However, the fimctionai, stn]cttlml and ~lubility properties o f these tetmmer/o species were found to be significantly different as compared to native tetramer, sugsesting the stabilize'on of partially unfolded intermediate of enzyme under these conditions. For urea denatmalion, no significant effect on enzyme activity or subunit assembly ofprotein was observed upto 2M ere& However, at higher urea concentration ( > 5M) the enzyme was e. letely dissociated into monomers.The study demonstrates that GdmC1 induced d~mturation of bovine liv~ catalAcc is a multiphasic process whereas, that of urea is a simple two-state process of dissociation ofteUameric enzyme into monomers without the ~ o n of any intermediate.

P83 Physicochemical characterization of ervatamin B. Occurrence of intermediates at acidic pH

and in presence of denaturants.

Monies Stmdd~ Suman Kundu and M V Jagalmadham*

Molecular Biology umt, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005

Solution conformation of ervatamin B, a plant cysteine protease from Eravatamla coronaria, belonging to the papain supeffamily, was studied by spectroscopy (CD, fluorescence and absorbance) at 'different pH, tempemtme and in presence of denaturants. The enzyme retains stu~cture as weU as activity over a wide range of pH (3.0-11.0) even after a prolonged exposure. At neutral pH, the enzyme is stable upto 60 ~ and in the presence of 8 M urea and 2.3 M GuHC1. Thermal and chemical induced unfolding of the enzyme both at neutral and low pH was cooperative. At pH 2.0, ervatamin B shows a strong binding to ANS, the hydrophobic dye, with reduced tertiary stxucture along with substantial secondary structure and no proteolytic activity. These features show that the e ~ y m e exists in molten globule state at pH 2.0. However, the enzyme is stable in pH 3.0 and exhibits the characterstics of molten globule state in the presence of low concentration of denaturants like urea and GuHC1. The transition curves at pH 3.0 are non-coincidental indicating the presence of intermediate states.

62


P 8 4 Structural characterization of a highly stable eysteine prote.ase ervatamin C: Identification of a

Partially structured intermediate state at low pEL

Suman Kundu, Monica Sundd and Medicherla V. Jagarmadham

Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India

The structural aspects of ervatsmin C, a higldy stable cystcine protease from the latex of Ervatamia eoronarla, has been studied in relation to its activity and stability using circular dichroism (CD), fluorescence and absorbance as a funchon ofpH, guanidine hydrochloride (GuHC1) and temperature. Ervatamin C belongs to ~z +[3 class of proteins with mixed ~x-helix and 13-sheet rich regions and retains both secondary and tertiary muetures along with biological activity over a wide range of pH and at high concentration of other denattmmts. Its unfolding by add, GuHC1 and temperature was cooperative with high transition midpoints reflecting a remarhable stab'flity. Besides, in presence of ~ea at neutral conditions, ervatamin C does not undergo any s~ructural changes and exhibits fxdl activity even after prolonged exposure. At pH 1.0 the enzyme partly unfolds losing its tertiary sCuctme and proteolytic activity but retains a predominant secondary sWaotufe with an absorbance spectra different from those under native and denaturing conditions. In this state the enzyme binds the fluorescent dye 8-Anilmc~l-napthalene-sulfomc acid (ANS) strongly and differs from the native or denatured state suggesting the presence of an intermediate in the folding pathway.

P 8 5 INTERACTION OF FROTEIN STABHJZgRS V~TrH PROTEINS : A

PROTEIN - REFOLDING STUDY Bi.na Chanda_n_i, Ruby Dhar, Latankumar Sinha, Deepti Warrier and Sonata Mehrotra

Department of Microbiology and Biotechnology Centre, M.S. University of Baroda, Baroda - 390 002, India

Osmolytes are known to stabilize protein structure. These small stabilizers generally have little direct interaction with proteins but affect the bulk solution properties of water. In the presence of polyethylene glycol (a polymeric stabilizer) also, globular proteins are preferentially hydrated, leading to stabilization of the native compact state of the protein. Renaturation of reduced and denatured lysozyme in the presence of low concentration of osmolytes and PEG was studied. The renaturation yield was obtained by monitoring the lytic activity of refolded lysozyme. Depending upon the type and concentration of the additive used either an increase or a decrease or no change in renaturation yield was observed.

P 8 6 CD and NMR Investigations on Trifluoroethanol Induced Stepwise Folding of Helical Segment from

Scorpion Neurotoxin

Purnima Khandeiwal, Subhendu Seth and iL V. Hosur Department of Chemical Sciences, Tara Institute of Fundamental Research,

Homi Bhabha Road, Mumhai 400 005, India.

A 14 amino acid residues peptide from the helical region of Scorpion Neurotoxin ms been structurally characterised using CD and NMR spectroscopy in different solvent conditions. TFE titration has been carded out in 11 steps from 0 to 90% concentration of TFE and the gradual stabilisation of the conformation to form predominantly tz-helix covering all the 14 residues has been studied by tH and L3C NMR spectroscopy. Detailed information like coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each concentration of TFE has been extracted and analysed to derive the stepwise preferential stabilisation of the helical segraents along the length of the peptide. It has been found that there is a finite population of the helical conformation in the midd/e residues from A5 to AI 1 even at low percentages of TFE. We also observed that greater than 75% TFE v/v is required for the propagation of the helix to the N and C termini and for proper packing of the side chains of all the residues. These observations would be significant from the point of view of understanding folding of this segment in the protein and would also throw light on the inherent preferences and side chain interactions in the formation of the helix in the pentide.

63


P87 Conformational Features Of Certain Peptides From Lysozome And Their Possible Role In The Folding

Of Lysozome

Y.U. Sasidhar', C. Rama Prabha and Anm Gidwani, Department of Chemistry, Indian Institute of Technology, Mumbai 400 076

In a peptide model a small part of the protein is present and the rest of the protein is not present and the peptide will serve as a model for protein folding initiation site. In the present work the overlapping pentapeptides CELAA, ELAAA and LAAAM have been selected from A-helix of lysozyme, spanning residues 6-12 and their conformational features are studied using quantum mechanical PCILO method. The peptides CELAA, ELAAA and LAAAM exhibit turn propensities in most of the minimum energy conformations. Interestingly CELAA displays helical conformation as one of its low energy conformations. Since turns are expected to play a role in helix folding, these results assume significance for helix folding in lysozyme considering that a-domain of lysozyme is known to have folded early ( within - 80 ms ) in a refolding experiment. * Corresponding author; Email: [email protected]

P 8 8 Trlchloroacetic acid Induced unfolding o f cytoclwome c results in stabilization o f

molten globule intermediate. Atta Abroad, Sangeeta Kulkami, K.P. Madhusudan and Vinod Bhaktmi.

Division o f Membrane Biology, Central Drug Research Institute, Lucknow 226016, India.

A sTntzmatir invesligation of triohloroac~'ti 9 avid (TCA) and trifluoroaeetiv avid ('ITA) induced denaturation o f native horse cytochrome c has been carried out using a combination o f optical spectroscopy and electro spray ionization mass spectroscopy (ESI MS). Huorascencv, circular dichroism and absorption Sl'metroscOlD, showed that addition o f TCA to native cytoehrome c does not remllt in significant unfolding of protein but stabilirntion o f a molten globule intermediate. In contrast to this, for TFA, an initial extensive unfolding at low concentrations (TraM) chataetexised by a significant breakdown of secondary and tertiary structure was ob~an~ . This was fonowed by partial folding o f the acid unfolded protein in presence o f higher TFA conc~mtrafions.R~ed on RSl MS stluti~, it was obse~ed that TCA anion h~s a stronger binding to cytochrome c as compared to TFA anion. These studies suggest that TFA unfolding of cytoohrome c is induced by acid and is not due to any specific ionic effects, whereas the TCA induced unfolding o f cytochrome c is influenced by the binding oflrichloroacetate anion to the protein.

P89 Discriminating between .Correct and Incorrect Folds of Proteins

Akira R. Kinio and Ken Nishikawa Center for Information Biology, National Institute of Genetics, Japan

The applicability of fold recognition methods has been hindered by the inaccuracies and ambiguities in database-derived statistical potential functions. In order to eliminate such difficulties, realistic physieo--chemical potetial functions may be of use. First, the classical problem of discriminating the native structure from deliberately misfolded structures is revisited. Second, the problem is extended to address the following problem: how can we discriminate the correct (not necessarily the native) structures from the incorrect (misfolded) structures? The "correct" and incorrect structures were created by simulated annealing molecular dynamics simulations (in vaeuo) with the structural restraints derived from other proteins of the same or totally different fold, respectively.

For the first problem where all the backbone atoms were essentially fixed, we found that, in contrary to the previous results obtained by others, conformational energy alone can discriminate the native structure from the misfolded if the native structure was minimized to the same extent as the misfolded. For the second problem, the situation was more difficult: the energies of the misfolded structures were comparable to those of the correctly folded structures. The analyses of the optimized structures revealed an excess of the electrostatic energies and the negative correlation between the conformational energy and empirical solvation free energy. In order to reduce these artefacts of the simulations in vacuo, further calculations with implicit treatment of solvent effect were carried out. The intrinsic features of thus created correctly folded and misfolded proteins are discussed. The present study will also serve as the basis for "post-fold recognition modeling" in the era of structural genomics.

64


P 9 0 Residue Depth: A novel parameter for the analysis of protein structure and stability

$uvobrata Chakravartv and Raghavan Varadarajan Molecular Biophysics Unit. Indian Institute of Science. Bangalore-560012

Accessible surface area is a parameter that is widely used in analyses of protein structure and stability. However accessible surface area does not distinguish between atoms just belov) the protein surface and those in the core of the protein. In order to differentiatebetween such buried residues, we describe a computational procedure for calculating the depth of a residue from the protein surface. Residue depth correlates significantly better than accessibility with effects of mutations on protein stability and on protein-protein interactions. The deepest residues in the native state invariably undergo hydrogen exchange through global unfolding of the protein and are often significantly protected in the corresponding molten globule states. Measurement of protection factors of the deepest residues provides a convenient method of measuring the free energy of folding in the absence of denaturant. Depth is often a more useful descriptor of residue burial than accessibility. This is related to the fact that the protein interior and solvent differ significantly in polarib' and packing density.

P 9 1 Are there parallel folding pathways in human lysozyme, bovine c~-lactalbumin and LYLAI?

K. Noyelle, P. Haezebrouck, M. Joniau and H. Van Duel Interdisciplinary Research Centre, K.U.Leuven Campus Kortrijk, B-8500 Kortrijk, Belgium

LYLA1 is a chimeric protein obtained by the insertion of the Ca2+-binding loop and the helix C of bovine a- lactatbumin (BLA) into the homologous position in human lysozyme (HLY). The folding kinetics of HLY, BLA and LYLA1 are determined over a wide range of GuHCl-concentrations using stopped-flow fluorescence spectroscopy, leading to clear evidence for the population of an intermediate state along the refolding pathway. Interrupted refolding experiments on HLY, however, show that 13 % of the molecules folds through a fast direct pathway whereas for BLA and LYLA 1 no parallel pathways were detected. The insertion of a part of BLA into HLY disrupts the fast folding channel.

P 9 2 Thermal Stability Studies on Photosystem II-A Multicomponent Multimeric Protein Complex Sheffali Dash', Indra Brata Jha', Rajiv Bhat" and Prasanna Mohanty-. * Centre for Biotechnology, JNU, New Delhi - 110067. ** School of Life Sciences, JNU, New Delhi - 110067.

Photosystem (PS) II enriched membranes were prepared from beet spinach (Beta vulgaris palanga) leaves and its purity was checked by SDS-PAGE. Differential Scanning Calorimetry (DSC) of PS II complexes indicated five endothermic transitions. Each of these transitions correspond to the unfolding of various components viz., A], A2, B, C and D in the PS II complex. Transitions due to components C and D could not be resolved possibly because these transitions were closely spaced. Under stress conditions, plants produce various types of osmolytes in order to protect their vital enzyme systems. DSC was used to study the effect of naturally occurring osmolytes such as, proline, glycine, and trehalose on PS II complex stability. The highest transition peak for PS II complex, PS II + proline (1M), PS II + gIycine (IM) and PS II + trehalose (1M) was observed at 62.8~ 62.5~ 65.3~ and 66~ respectively. The results furthei indicated that these osmolytes have differential stabilization effect on the various components of the PS II complexes owing to different modes of interactions of the surface residues on the component proteins with the osmolytes mediated via solvent water.

65


P93 Ferredoxin from the extreme haiophile Halobacterium salinarum is stable in highly saline

brine: spectroscopic investigations

A.. K. Bandyopadhyay and H. M. Sonawat Department o f Chemical Science, T.I.F.R., Homi Bhabha Road, Mumbai-400005.

Halobacterium salinarum is an extreme halophilic archaea found in the habitat with saturated NaC1 concentration. As intracellular salt concentration is also very high, the proteins from this archaea are salt-adapted. We have purified ferredoxin from this archaebacterium and characterized the native state and salt dependent stability using absorbance, circular dichroism (CD) and fluorescence spectroscopy. The native protein exhibited an An2o:A277 ratio of 0.35, double mi~iraa at 210nm and 217rim in far-UV CD spectrum and a fluorescence emission maximum at 335 rim. These observations indicate an intact and functional [Fe2-S2] chromophore, predominantly p-sheeted secondary structure and the tryptophans in hydrophobic environment. The native structure is maintained in _> 1.5 M NaCI. Below this a decrease in A42o:A~7 ratio and ellipticity at 217 nm, and a red-shitted emission maximum is observed sis unfolding of the protein. Ferredoxin from this extremophile is, thus, stable and functional under conditions ofvery high salt, a reduction of which has drastic effects on its structure and function. These and other results will be discussed in detail.

P94 CHAPERONE-LIKE ACTIVITY OF APLHA-CRYSTALLIN AND ITS SUBUNITS

Ch. Mohan Rao and Siddhartha Datta Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

Alpha-crystallin is shown have chaperone-like property. We have shown earlier that structural perturbation of (~- crystallin enhances its chaperone-like activity several fold and proposed a hypothesis for its action ( JBC 269, 27264-27268,1994: FEBS Letters 365,133-136, 1995; JBC, 270. 19888-19892, 1995; JBC 271, 27595- 27600,1996; JBC 272, 23559-23565,1997; FEBS Letters,416,369-372,1997; In. J. Bio. Macromolecules 22, 271- 281,1998). We have investigated the chaperone like activity of (zA- and otB-crystallin homo-aggregates against thermal and non-thermal modes of aggregation. We find that the two proteins differ significantly in their ability to protect against a non-thermal mode of aggregation. Interestingly, such a difference in the protective ability of ctA- and (xB-crystallin homo-aggregates is totally absent against a thermal aggregation of 13L-crystallin. o~B-Crystallin shows significant protective ability at sub-physiological temperatures at which both (zA- or hetero multimeric cz- crystallin show very little chaperone-like activity. To investigate this differential behaviour, we have monitored the temperature dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Our study demonstrates that. despite a high degree of sequence homology, the two proteins have some distinct differences in their structural stability and chaperone-like properties. These differences appear to be relevant to temperature dependent enhancement of chaperone-like activity of ot-crystallin. These results also indicate different roles for the two proteins both in (~-crystallin and as separate proteins under stress conditions.

P95 STRUCTURAL FEATURES OF THE TARGET PROTEIN-ALPHA CRYSTALLIN INTERACTION

K. Rajaraman, B. Raman, T. Ramakrishna and Ch. Mohan Rao Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

To investigate the conformation of the target protein bound to the chaperone o~-crystallin we studied the interaction of carbonic anhydrase with c~-crystallin. Fluorescence and circular dichroism spectroscopy revealed that the chaperone-bound carbonic anhydrase exposes hydrophobic surfaces, has minimal tertiary structure and retains sufficient secondary structure similar to the molten globule formed in 1.5 M GdmCl (J. Biol. Chem. 1996, 271, 27595-27600). This result prompted us to look at the additional requirements for efficient target protein-cc-crystallin interaction. We studied the interaction of c~-crystallin with a) the compact, molten globule state of cc-Iactalbumin (apo- lactalbumin), b) the expanded, flexible molten globule state of ~-lactalbumin (reduced ~-!actalbumin) and c) the aggregation-prone molten globule formed at pH 6.0 in the presence of DTT. Our results indicate, that only the aggregation-prone molten globule state of ~-lactalbumin interacts efficiently with ~-crystallin. Extended or random coil states of the protein donot interact with ct-crystallin (BBRC 1998, 249, 917-921). These results help us understand the chaperone function in general and the role of cc-crystallin in maintaining the lens transparency.

66


P96 ~/CRYSTALLIN MUTATIONS AND OCULAR LENS OPACITY - A. Pande*, J. Pande #, S. Betts*, N. Asherie #, O. Ogun #, J. King* and G. Benedek #. Departments of Biology*, and Physics #, M.I.T., Cambridge, MA, U.S.A.

The lens ), crystallins are implicated in cataract formation. The ), crystallin family of proteins is structurally very similar, both in sequence and in conformation. Yet they exhibit significant differences in their thermodynamic behavior. On the basis of the critical temperature for phase separation (Tc), these proteins have been classified into high-Tc and Iow-Tc groups. An increase in the proportion of the high-Tc crystallins in the lens is believed to promote cataract formation. A comparison of the sequences of the low and high-Tc ), crystallins, has led to the identification of amino acid substitutions likely to raise Tc. Using site-directed mutagenesis, we have replaced selected residues in the Iow-Tc protein, ),B crystallin, and obtained single and double mutants of ),B (e.g. S130W, K163R, C15, 22H). These mutants indeed exhibit higher Tc values than that of the parent protein. These types of studies will provide the basis for understanding cataractogenic changes that result from genetic mutations in the human ), crystallins.

P97 Investigation of"without aldehyde" luminescence of recombinant Photobacterium leiognathi luciferase.

I.V. Sokolova, N. A. Tyulkova, G.S. Kalacheva. Institute of Biophysics Russian Academy of Science, SiberianBranch, Krasnoyarsk, 660036.

High-purificated luciferase from recombinant strain E.coli SL60, carrying plasmid pUC18, containing only lux A and B genes of Ph.leiognathi luciferase, in absence exogenous aldehyde has a luminescence, which makes 0.03 % from luminescence with exogenous substrate. The rate constant ofbioluminescent decay of reaction with photorecovered FMN makes 0.8 s ] and corresponds to those of "without aldehyde" luminescence of wild type luciferase. Extraction of lipid components from luciferase was carried out with use of the organic solvents. The removal of lipid components from lacfferase resulted in irreversible loss of enzyme activity. The qualitative and quantitative analysis of extracted lipid fraction was carried out by method gas-liquid chromatography. It was shown, that its structure includes fatty acids with chain length from 14 up to 18 carbon atoms and not identified aldehyde fraction, and also alcohols, ftolates and carbon-hydrates. It is supposed, that "without aldehyde" luminescence is caused by sticking of lipid components on laciferase, which

essentially stabilize its structure in vitro.

P98 Structural Stability of Bacteriorhodopsin at Alkaline pH

Masashi Sonoyama, Yasunori Yokoyama, Kunihiro Taim and Shigeki Mitaku Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nabaeho, Koganei,

Tokyo 184-8588, Japan

Bacteriordhedopsin CoR) is a light-driven proton pump that forms the 2D crystalline array on the cell membrane in Holobacterium salinarum. When the 2D crystalline state is not retained, however, it was reported that denaturation of bR is drastically accelerated by the irradiation of visible light. This strongly suggests that some of the photointermediate states are less stable than that in the ground state of blL In the present paper, the light- induced denaturation phenomenon and the 2D-crystalline structure are investigated for bR at alkaline pH where lifetime of some photointermediates is prolonged. Denaturation kinetics experiments indicated that even in the dark, bR is stalilized at pH 11, while the light-inducxxi denaturation occurs above the temperaatm ~40~ lower than at neutral pH. In addition, circular dichroism spectroscopy in the visible region revealed the grach,al melting of the 2D crystalline structure above 40~ It is suggested from these results that the protein-protein interaction in the 2D crystalline structure is crucial for the structural stabilization of hR.

67


P99 Effect of Detergents on Light-induced Denaturation of Soluhilized Bactriorhodopsin

Chicko Nakazawal, Takanori Sasakil, Yuri Mnkai 2, Naoki Kamo 2, Masashi Sonoyama ~ , Shigeki Mitaku ~ t Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan

2 Faculty of Pharmaceutical Sciences, Hokkaido University, Hokkaido 060-0812, Japan

The correlation between the light-induced denaturation phenomenon and the 2D-crystalline structure of bacteriorhodopsin was suggested from the denaturation experiments of samples solubilized by octylglucoside 1~ However, the solubilizing activity is different among various detergents. We have compared the characteristics of bacteriorhodopsin solubilized by octyiglucoside, dedecylmaltoside and Triton X'-I00, measuring the circular dichroism around 5.50 nm and the light-induced denaturation phenomenon. Any circ-lar dichroism band was not found at about 5.50nm for samples solubilized by octylglucoside and Triton 25(-100 and significant light-induced denaturation phenomenon. In contrast, a sample solubilized by the mixture of octylglucoside and dodecylmaltoside showed an exciton band, and the effect of light illumination on the denaturation of bacteriorhodopsin was suppressed. These results indicated that the protein-protein interaction contributes to the stability of bacteriorhodopsin particularly under the illumination of visible light. l) Mukai, Y., Kamo, N. and Mitaku, S. (1999), Protein. Eng. To be published

P I O 0 INTERCONVERTING a-HELICES AND [3-SHEETS.

Seema Dalai and Lynne Regan. Yale University, New Haven, CT-06511 .In response to the "Paracelsus Challenge" [G. D. Rose and T. P. Creamer, Proteins Structure

Function Genetics 19, 1 (1994)] we have successfully transformed a predominantly [3-sheet protein (the B 1 domain) into a stable ix-helical protein while retaining 50% sequence identity to the B 1 domain [S. Dalal, S. Balasubramanian, and L. Regan, Nature Structural Biology 4, 548 (1997)]. Using the designed protein, which we have named Janus, as a starting point, we have constructed a series of mutants to evaluate the balance of forces necessary to stabilize an or-helix vs. a 13-sheet fold. These mutants include a series in which we have assessed the contributions of the various design considerations to the overall stability and fold of Janus. One goal is to determine the maximum number of B1 domain residues that can be accommodated while maintaining a helical fold, a variant that would be a minimal version of Janus. As a part of this study, we have created, a protein that has 61% identity to the B 1 domain, but continues to adopt a helical fold. Another aim is to induce a conformational switch from a-helix to [3-sheet, perhaps by changing a few'residues in minimal Janus. We have discovered variants that form ordered fibrils and we are currently studying their properties to gain a better understanding of the molecular basis of fibril formation.

Our study emphasizes that a subset of a protein's amino acid sequence is required for determining its fold and has. important implications for structure prediction and design.

P101 Light-induced denaturation of bacteriorhodopsin solubilized by octyl-l~-glucoside

Yuri Mukal 1, Naoki Kamo I and Shigeki Mituku 2 i Faculty of Pharmaceutical Sciences, Hokkaido University, Japan

2 Department of Biotechnology, Tokyo University of Agriculture and Technology. Japan

The structural stability of bacteriorphodopsin (bR) solubiiized by octyl-~l-glucoside was studied by measuring tile denaturation kinetics under visible light irradiation as well as in the dark. The denaturation of bR solubilized by 50 mM octyl-13-glucoside was very slow at room temperature, when it was left in the dark. However. its spontaneous denaturation was accelerated when the solubilized bR was irradiated by visible light. The denaturation kinetics under visible light irradiation as well as in the dark could be well described by a single decay constant_ The activation energy for the denaturation of bR was estimated from the temperature dependence of decay time constants. The activation energy under visible light" irradiation was 12.5 kcal/moi which was much smaller than the corresponding value in the dark 26.2 kcal/mol. These results strongly suggest timt some of the photointermediate states are less stable than the ground state of bR. The critical temperature and the activation energy for denaturation of bR in the solubilized state were much lower than those in the 2D-crystalline state. Comparing the denaturation behavior in the 2D-crystaUine state and that in the octyl-13-glucoside-solubilized state. our findings suggest that protein-protein interaction contributes to the solubility of this protein.

68


P 1 0 2 A comparative study of conformational selectivity of dehydro phenyl alanine derivatives

by theoretical methods

MIHIR ROYCHOUDHURY AND DEVESH KUMAR Department of Physics, DDU Gorakhpur University

Gorakhpur (U. P.) India

Dehydro amino acids are well knoss~ for their role in modifying the actisity of peptides. specific secondary structures have been of interest of many experimental workers. In the present work, molecular orbital calculations using PCILO (Perturbative Configuration Interaction using Localized Orbitals) method have been carried out to investigate the conformational behaviour of some peptides with either dehydro-Phe or dehydro -Leu at the corner position i+l or i+2 of the 13oturn. It has been observed that the conformation around the dehydro is mostly rigid and exhibits very few minima. The conformations observed through crystallographic studies quite often correspond to the second or third minimum due to packing requirements. A comparative analysis of these results along with experimental results of other workers will be presented.

P103 EFFECT OF NUCLEOTIDES IN MUSCLE FIBRES BY DSC, TMDSC AND EPIL

lD~nes L6rinczy, 2Franciska KOnczOl, 3L~szl6 Farkas, 4joseph Belagyi and SChristoph Schick ~Inst. of Biophys., 2Inst. of Forensic Medicine, 3Dep.t of Urology and 4Central Res. Lab. of Univ.

Med. School ofP6cs, SInst. of Polymerphysics Univ. of Rostock. Electron paramagnetic resonance spectroscol~y (EPR and ST EPR) and differential scanning calorimetry (DSC) were

used in conventional and temperature modulated ~node to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Skeletal muscle fibres isolated from psoas muscle of rabbit were spin - labelled with isothiocyanate-based probe molecules at the reactive ~fhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMP.PNP) and ATP or ADP plus orthovanadate, the conventional EPR spectra showed large changes in the ordering of the probe molecules in fibres, and a new distribution appeared and AMP.PNP increased the orieutational disorder of myosin heads, and a random population of spin labels was superimposed on the ADP-like spectrum. DSC profiles were deconvoluted (Jandel Peakfit). The higher temperature transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin, which was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region. (National Research Foundation: OTKA TO 17099, CO-272; INCO COPERNICUS EUERBIC CT960821)

P 1 0 4 Hsp47 May Prevent Self-Association of Collagen in the ER

Christy A. Thomson and Vcttai S. Ananthanarayanan, McMaster University, Hamilton, ON, Canada Hsp47 is a collagen-binding protein with a suggested role as a molecular chaperone for collagen. As yet, the nature of hsp47's interaction with collagen and its role in collagen assembly are unresolved. It has been shown that hsp47 binds collagen in the ER and releases it in the acidic cis-Golgi. As such, hsp47 may help prevent the premature serf-association of collagen into fibrils. Using turbidity measurements, we have monitored the effect of hsp47 on the serf-association of soluble calfskin collagen. At pH 7 and 34~ we found a marked time-dependent increase in absorbance of collagen at 313 nm indicating fibril formation. Addition of hsp47 at pH 7 effectively abolished the absorbance increase. When the pH was reduced to 6 (where hsp47 is known not to bind collagen), fibril formation occurred after a short lag. This pH dependence may be correlated to the effect of pH on the conformation of hsp47. At pH 7, hsp47 was found to have alpha- helical content while on reduction to pH 6, it underwent a structural rearrangement with an increase in the I~-sheet form. Fluorescence emission spectrum of hsp47 at pH 7 showed a broad peak around 336 nm indicating exposure of some hydrophobic residues to the solvent. Treatment with ANS at pH 7 caused a significant increase in the emission intensity and a blue shift indicating that hsp47 does have exposed hydrophobic sites in the native state. An increase in emission intensity upon ANS addition at pH 6 was also observed. Together, our results show that hsp47 may bind collagen in the ER via exposed hydrophobic groups of both the proteins and help prevent premature collagen association. The release of collagen in the acidic Golgi may be due to the reduction in hydrophobic interaction caused by conformational change in hsp47.

69


P105 PROPERTIES OF FICIN, THE IMMOBILIZI~.D ON KM-DIASORB, FROM LEAVES OF FIG

Alirzayeva, E.G., Baba-Zade S.N. Institute of Botany, Azerbaijan Academy of Sciences, 2 Matbuat Avenue, Baku-370073, Fax: 99412-381570, E-

mail: alirzayevaeg@aznet org

Ficin, a partially purified from leaves of fig was immobilized on the ionchange was so much that it was failed to desorb enzyme even 1M NaCI. The concentration of ficin bounded with the resin was 0,1 g/g of the resin. It was determined that the activity of enzyme increased by increasing concentration of the casein from 0,125 to 2%, reaching maximum at concentration 0,5%. The ficin sorbed on KM-diasorb show the activity in wide interval of pH from 6,0 to 9,0. The effect of tempeF, aure on the activity of the sorbod ficin in interval fi'om 20 to 80~ was investigate(t It was shown that the activity of the enzyme increased with the rise of the temper-,aure and reached maximum on 70~ The effect of the concentration NaC1 on the activity of the ficin was stodiect The activity of the enzyme increased in the lxesence of the salt reaching the maximum at 0,5M, the~ gradually decreased. The results show that the immobilization of the ficin on the KM-diasorb raise its stability.

P106 Development of Protein Thermodynamic Database and Analysis on Protein Stability

M. Michael Gromiha. M. Oobatake, H. Kono, J. An, H. Uedalra and A. Sarai RIKEN Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan

Predicting protein stability upon mutation is one of the greatest challenges in molecular biology. We developed a database, "ProTherm: Thermodynamic database for proteins and mutants" [1]. It contains numerical ~AtA for several important thermodynamic parameters, structural data, experimental methods and conditions, functional and literature information. The database is available at http://www.rtc.riken.go.jp/protherm.html. By using the data we analyzed the correlation between stability changes caused by mutations and changes in 48 amino-acid properties [2]. In buried mutations, we found that properties reflecting hydrophobicity strongly correlated with stability, and there was a direct relation between the property values and the number of carbon atoms. Classification of mutations based on their location within helix, strand, turn and coil segments improved the correlation of partially buried mutations with stability. The stability changes by mutations at ordered structures were mainly governed by hydrophobicity whereas those at coil were mainly influenced by effects of entropy. Further classification of mutations within coil based on hydrogen-bond forming capability led to much stronger correlations. Information about local sequence and structural effects were more important for predicting stability changes caused by partially buried mutations than for buried mutations. In essence, hydrophobicity was the major factor determining the stability of buried mutations, whereas hydrogen bonds, other polar interactions, and hydrophobic interactions were all important determinants of the stability of partially buried mutations. References: [1] Gromiha et al. Nucl. Acids Res. 27, 286 (1999). [2] Gromiha et al. Protein Eng. in press (1999)

P107 Role of Amino Acid Residues at Turn in the Structure, Stability, and Folding of Human Lysozyme

Kazufumi Takano t, Yuriko Yamagata s, Katsuhide Yutani 1 ]Institute for Protein Research, and SGraduate School of Pharmaceutical Sciences, Osaka Univ.,

Yamadaoka, Suita 565-0871, JAPAN To investigate the role of turns in the stimeture, stability, and folding process of a protein, mutant human lysozymes (i) deleted or (ii) substituted at the turn structures were constructed. (i) For the deletion mutants, A47-48 and A101, the deletion residues are deleted in human a-lactoalbumin which is homologous in sequence and structure to human lysozyme. X-ray analysis revealed that each turn in both deletion mutant structures was shortened like human wlaetoalbumin, and DSC experiments showed that the stability of both proteins was slightly decreased. A47-48 increased the rate of unfolding by GuHCI monitored by fluorescence, while A101 decreased the rate of refolding. (ii) For the single mutants, RSOG, Q58G, H78G and G37Q, the substitution residues located at turns have positive ~) and V angles in the wild-type structure. Although the overall structures and the main-chain conformation of the substituted residues of the single mutants were similar to those of the wild-type protein, the changes in stability and unfolding-refolding kinetics upon mutation were significantly different f~om each other. These results suggest that the deletion or substitution at turns hardly affect the structure, but the protein stability and folding are affected, depending on the location.

70


P108 EFFECT OF VISCOSITY ON THE KINETICS OF e-HELIX AND [3-HAIRPIN FORMATION

Gouri S. Jas, Victor Mufioz, James Hofrichter, and William A. Eaton Laboratory of Chemical Physics, NIDDK, NIH, Bethesda

Knowing the effect of viscosity on protein folding kinetics is necessary for obtaining the magnitude of the energy barrier, because the temperature dependence of the rates must be corrected for changes in solvent viscosity. The major problem encountered in such studies, however, is that the agents used to change the viscosity also alter the folding-unfolding equilibrium, preventing the determination of the pure dynamical effect of viscosity on the kinetics. We have found that in simpler systems, or-helices and [~-hairpins, it is possible to observe the effect of viscosity on the folding kinetics under conditions where equilibria are unaffected. Both sucrose and glucose produce up to three-fold changes in the solution viscosity with almost negligible effect on the thermodynamic stability of a 2 l-residue alanine-based helical peptide (Ac-WAAAH*(AAARA)3A-NH2), as determined from far ultraviolet circular dichroism measurements. The kinetics as a function of viscosity were measured for this peptide using nanosecond laser temperature jump. The relaxation rate slows with increasing viscosity, and has been fitted to the function ~" with K < I. For sucrose (342 Dalton) K---- 0.60 + 0.02 while for glucose (180 Dalton) K = 0.65 • 0.02, suggesting that K may increase with decreasing Size of the viscogenic agent, but is still less than t.0 for pure water (18 Dalton). These viscogenic agents also produced little or no change in the folding-unfolding equilibrium of a 16-residue 13-hairpin peptide (GEWTYDDATKTFTVTE-OH) as observed from measurement Of tryptophan fluorescence. The relaxation rate slows with increasing viscosity, and is proportional to ~" with K = 1.0. For sucrose z = 0.95 • 0.05 while for glucose K = 0.92 + 0.02. Our results suggest that for the helix internal friction of the peptide, as well as solvent friction, contribute to the dynamics of the helix-coil transition. In contrast, internal friction of the hairpin peptide makes-only a small contribution to the dynamics.

P 1 0 9 Secondary structural studies of a schistosoma mansoni protein (Smp50) using Circular dichroism (CD) and Fourier Transform Infra-red (FTIR) spectroscopic techniques. Jonathan Penoyar, Thamarapu Srikrishnan and Philip T Lo Verdet, Department of Molecular & cellular Biophysics, Roswell Park Cancer Institute and Department o f Microbiologyt, School of Medicine & Biomedical Sciences, State University of New York Buffalo, Buffalo, New York 14263, USA Recently a 50 idlodalton FKBP protein(Stop50) from the trematode Schistosoma mansoni was produced in bacteria and shown to have biological activity. There is a 42% homology between Stop50 and the yeast FKBP1 and 44% homolgy between this protein and the human FKBPI 3 proteins. We have undertaken a structure-function study of Stop50 with a view to understand the aminophyllin-ligand interactions and to compare it with the human FKBP protein. We present here a detailed analysis of the secondary structure of this protein using Fourier Transform Infra-red (FTIR) recorded on a Perkin- Elmer Spectrum 2000 spectropolarimeter and circular dichroism spectroscopy (CD) recorded on a JASCO 500A spectropolarimeter. From an analysis of the FTIR spectrum, Smp50 has 28% c~- helix, 42% 13- structure and 30% randon coil structure. These results are in excellent agreement with the results from CD. At room temperature and in a phosphatebuffer (pH 7.1) Stop50 has 26% a-helix, 42% 13-structure and 31% random coil structure. When the protein is cooled to 10 ~ the amount ofct-helix increases to 32% with a 36% 13-structure and a 33% random coil. When the pH is lowered to 4.0 with an acetate buffer, the c~-helix decreases to 21%, whereas at high pH (borate buffer), the co-helix increases to 29%. Our results indicate that the protein has an highly extended 13- structure in solution and gives a high degree of stability to the protein, based on our temperature and pH variation studies.

P l 1 0 Structure stabilising function of propeptides in zymogen and mature pancreatic serine proteases

.l. Kardos, ,~. Btdi, I. Venekei, P. Zdvodszky and I.. Grdf Department o f Biochemistry, EOtv0s Lontnd University, Budapest, Hungary, E-maii:[email protected]

Institute o f Enzymology, Hung. Acad. Sci., Budapest, Hungary

Chymotrypsinogen and proelastase-2 are the only known zymogens o f pancreati~ proteases that have propeptides bound also by a disulphide bridge and therefore remain attached to, and can have function in the active enzyme. We have recently demonstrated that chymotrypsinogen propeptides neither have any role in the catalytic performance or !n substrate discrimination o f chymotrypsin, nor in the proper folding o f the protein [I. Venekei et ill. 1996, FEBS Leaers, 379, 139-142]. Here we investigate the possibility o f a structure stabihsing function o f this propeptide which is significant for the mature chymotrypsin, too, by comparing wild-type rat chymotrypsinogen, chymotrypsinogen containing a trypsinogen propeptide and the active forms o f these. Chymotrypsinogen and chymotrypsin that do not contain propeptide interactions showed an increased sensitivity for guanidite- hydrochloride finfold|fig, a lower heat stability and a faster loss o f activity at extremes o f p H . The f indlngthat the presence o f the propeptide makes chymotrypsinogen resistant against pepsinolysis at acidic pH suggests that this might be the biologmcal function from which the evolutionary conservation o f chymotrypsin-like propeptide-enzyn~e interactions arises. From the guanidine hydrochloride denaturation experiments we estimated the AAG free energy o f the extra stahilisation conferred by the propeptide to be 24 kJ/mole. Further observations suggest that the propeptide restricts the relative mobility between the two domains o f chymotrypsin, which is in accord~/nce with its position and interactions in the molecule. This function appears to be important even in the active form o f pancreatic serine proteinases and is accomplished through other interdomain contacts in those enzymes that do not contain disulphide linked propeptide.

71


P l l l ~ u n d dmmac~ ~ ~ n ~ o p ~ snd ~ p~ot~ s u i m ~

P, mule, of a mmpNhms/ve s~ ,e7 ~ Palr Z~voDszKY

Inu/mt, of~/og~, HMg. ~ &~., ~dap~u, Hungary. s ~aOog/m.;m

In 9 symmmtie study, we cmmrum~l a damb,~ that oo,~_m;,,, all themmphilie pmmi:,,, with imowa thzm-di:::mmouat stmmme~ along with all their mmophi]ir homologum with known swamm, m. Tim damba~ is non-redundant and~ mpremmtativ~ contsining 64 mesophilir and 29 thennophil~ structures in 25 protein families. 13 structm~

in $ catmgorim wm'e eaL,:,,l-*-,~_ for i l l mbmim in the ~ . They were: nnmb~, vol,,,,'- and ares ofl ~vit i~; munb~ of hydrog~m bonds and ~ ~ h ~ bored ckmom and a o : e p ~ , numb~ of mong and wealc iou pe/m; polariS, of exlm~l aud buried sur&ms; ~ of vsdom momdm7 mummal cdmmm. The dam w~o mmlymd by smtisu'cal methods. Tlm only ~ that showed a sigu/flaug dif&nmm becwem the mmoph/h'c m i thennophilic strummm w u the mnnber of iea ~ mpeeially the mamber of wink iea lmim. Much leu a i ~ S m m diff, mmm. w m o i ~ v ~ l in the ~ l ~ r ~ , although dmmmpi~c protein w m observed to havo a m~Im~y to conm/n lower caviars, morn ~ bonds, m~l to Imve a morn polar sur&m ~ a ~ mdemd meondary mucmm thau their mmophillc ~ ~ pmmia ~Mily seems to utilize a different me~animn for smbiliz~on. The order of imporUmm m t the intermlsfiomhip of the mdied propertim with regard to their role played in stabilization ~ wel l as the _l~3~___'ble evolutionary aspects of the remdts are discussed.

P l 1 2 Prediction, Analysis and Characterisaflon of tRmA-bimlkmg Coiled-con Motifs.

JUl , .hl imb J. Walshaw and D.N. Woolbon Cenlre for Biomolecular Design & Drug Devdopmmt, School of Biological Sciences,University of Sussex, Bml 9QG,UK Aminoacyl-tRNA synthetases (aa-tRNAs) are responsible fix charging tR~A with the correct amino acid. The recognition of tRNA by the approlria~ synthetase is therefore cru~aI for faithful Uanslalion of the gem~tic code. To examine tRNA recognition we aligned and compared multiple sequences of Set- and Phe-tRNAs, which share an unusual tRNA-recognition motif, r~mely an antipmallel coiled coiL Compute~-gemerated heptad assignmeats concmn~ with those made manually on the basis of the Set- and PIw-tRNAs crystal structures. These assignments and the aligned sequences were used W produce an amino acid profile for the mt/pmal~l co,ileal coil. The profile displayed clear similarities with the amino acid IX'ofile of known paralkl d/meric coiled coils, but ~ differences from a IXofile for pmallel trimms. These observalions could be rat/o,mliqed in structmal terms, and provided a route to the pred/ction c~ other an "tipmal~l coiled coils. For instamce, am analysis of all other aa-~MAs identified in compleUxl gencmm located an matipmallel coiled- coil motif at the C-teminus of Val-tRNAs. 1"he strucnne and the mechnni~ of ~JqA n~cognition of VaI-tRNAs is nnk'nqwn. Our observation sugges~ that recognition of IRNA by Val-tRNAs may be facilitate! through a C-tefmimm, a u m ~ o3iled-o3il ~ in a similar lammef to that effec2ed by the N-t~minai region of Set- and Phe-IRNAs. We have begun to test this hEpetlz~. DNA encoding the C-terminal ,mlipmallel coiled-coil region of the E. co//Val-tRNAs was mnplified aad the protein product ~ Ch'cular dichroimn measumnents showed that this did indeed fold co-operatively into air a - helical ~ with a stability ~mmilar tO the isolated, N-tennl.~l anl~parallel coiled coil from E. coli Ser-tRNAs.

P 1 1 3 Effects of Solvent-Exposed Hydrophobic Residues on Conformational Stability of Human Lysozyme

Jun Funahashi ', Kazufumi Takano t, Yuriko Yamagata 2, and Katsuhide Yutani ~ 'Institute for Protein Research, Osaka Univ., 2Graduate School of Pharmaceutical Sciences, Osaka Univ.

It has been generally accepted that surface amino acid substitutions in globular proteins have little effect on protein stability. But mutagenesis studies show that solvent-exposed residues also affect protein stability. In this study, hydrophobic mutants (Val to Giy, Ala, Ile, Leu, Met, and Pbe), substituted at Val 2, Val 74, and Val ! 10 of human lysozyme, were constructed. Positions 2, 74, and 110, which are located in ~-sbeet, loop, and c~- helix, respectively, are completely exposed. Their thermodynamic parameters of denaturation and crystal structures were studied by calorimetry and X-ray crystallography at 100K, respectively.

The denaturation Gibbs energy of mutant proteins with the same substitutions at each position showed different values. In the case of the mutants at position 74, the stability increased with increasing the volume of substituted residues and total ASA,o,.pot~ values also increased with. This might be caused by structural changes in the denatured state. On the other hand, almost mutant proteins at position 2 and 110 were destabilized and stabilized, respectively, depending on changes in the secondary-propensity by the substitutions that are unfavorable for [~-sbeet and favorable for c(-helix. In a poster, we will discuss the relationship between changes in stability and mutant structures.

72


P l 1 4 Conformational Stability o f Binuclear Copper Center in Subunit II of

Cytochrome c Oxidase from Paracoccus denitrificans Sayan Gupta and Shyamalava Mazumdar

Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005

The Subunit II in cytochrome c oxidase consists ofa dimeric copper complex, called CuA with six ligands, two cysteines bridging the copper ions and two histidines, a methionine and a glutamic acid as terminal ligands. The temperature and pH dependent optical and circular dichroism spectroscopic studies at visible region show that the conformation of this binuclear copper center changes reversibly with temperature and pH. At neutral pH and room temperature, two conformations of CuA co-exist. The 'low pH' conformation also predominates at room temperature. The 'high pH' conformation is similar to that at high temperature. AH~ and Tm of the binuclear copper site measured in our study suggested lower stability of the 'low pH' conformation. Unfolding of the Subunit II by urea at different pH has also been monitored by far- UV CD and tryptophan fluorescence, which shows higher conformational stability (AGH~ o) of the 'low pH' species.

73

A3 : Proteins : Design and Ligand Binding

P l 1 5 Docking of flexible ligands to flexible receptors in solution by Molecular Dynamics Simulations

A. Di Nola l, M. Mangoni ~ and D. Roccatano 2 Department of Chemistry, UniverSity of Rome "La Sapienza", P. le A. Moro 5, 00185. Rome, Italy

2 University of Groningen, The Netherland

In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of the algorithm previously reported by Di Nola et al. (Proteins 19: 174-182, 1994). The method allows a fast exploration of. the receptor surface using a high temperature of the center of mass Iranslational motion, while the ligand internal motions, the solvent and the receptor are simulated at room temperature. In addition the method allows a fast center of mass motion of the ligand even in solution. The dampening effect of the solvent can be overcome by applying different weights to the interactions between system subsets (solvent, receptor, fig,and). Specific ligand-receptor dislances have been used to compare the results of the simulations with the crystal structure. The method is applied, as a test sy/aem, to the docking of the phosphocholine to the immtmoglobulin McPC603. The results show that the structure of the complex in solution and in the crystal are similar.

P l 1 6 S T R U C T U R A L D E T E R M I N A N T S O F M A N N O S E R E C O G N I T I O N

Gosu Ramaehandraiah 1 and Nagasuma R C h a n d r a .2 1Molecular Biophysics Unit, 2Bioinformatics Centre, Indian Institute of Science, Bangalore 560 012, INDIA

Mannnose, an abundant cell surface monosaccharide binds to a diverse set of receptors, which are involved in a variety of important cellular mediated processes.A structural analysis has been carried out on all mannose binding proteins in the Protein Data Bank to identify common recognition principles. The superfamily of bulb lectins, highly specific to mannose are found to contain a consensus sequence motif QXDXNXVXY, which has been identified to be essential for mannose binding. Structural analysis of this motif in the crystal structure of garlic lectin and other related proteins has led to the understanding of the contribution of indvidual residues in mannose recognition. Comparison with other mannose binding proteins reveal common hydrogen binding patterns in all of them despite differences in sequence, overall fold and the substructures at the binding sites of individual proteins. A database analysis suggests that although the topology of the backbone, as at the binding site in bulb lectins can generate mannose binding capability in a few other proteins, tertiary interactions of not only the conserved residues in the motif, but also the non-consensus part play a crucial role in allowing that property to be retained.

P l 1 7 Structural basis of inhibition of Integrase activity by ligands:

A computer modeling study. V.Kothekar, Srinivasan.M._Shakti Sahi. Department of Biophysics,

All India Institute of Medical Sciences, New Delhi, India AIDS is a deadly disease caused by the retrovirus HIV. One of the principal steps in the replicative cycle

of HIV is the integration of the viral DNA into the host genome. This step is catalysed by the enzyme Integrase(IN). IN cleaves terminal CA nucleotides from the 3' ends of the double stranded viral DNA to leave it with a two-base overhang (3' processing). The protein DNA complex is then transported into the nucleus where the host DNA is cut leaving a 5' overhang of five bases and the 3' ends of the viral DNA are covalently linked to the 5' ends of host DNA(strand transfer). IN has no known analogues in the host which makes it an attractive target for therapy. HIV-1 IN is a 288 residue protein with 3 domains namely N-terminal domain (1-49), C- terminal domain (213-288) and the catalytic domain(50-212). Using computer modeling technique we have studied the inhibition of HIV replication by disruption of the protein-DNA interaction of IN as a result of binding of inhibitors derived from Arylamides, Arctigenin as well as pharmacophore studies. We have also studied interaction of non-inhibitors of IN haying closely related structure to IN inhibitors obtained in phannacophore searching. The drugs ~vere docked to IN'using DOCK 3.0 and in house package IMF. Molecular Dynamics studies on each complex was done for 500picoseconds using AMBER 5.0. Structural basis for such differential activity in seemingly similar drugs is discussed in the paper.

7 4


P l 1 8 Structural and Interactional Homology of Clinically potential Trypsin Inhibitors : Molecular Modelling of Cucarbitacea family peptides using the X-ray structure of MCTI-II. S. Chakraborty, S. Bhattacharya, A. K. Beta, S. Ghosh, A. K. Pal. U. Haldar, B. P. Mukhopadhyay and Asok Baner/ee Department of Biophysics, Bose Institute, Calcutta 700 054, India

Clinically potential protease inhibitors are drawing attention recently. Several trypsin inhibitor peptides (28 -32 amino acid residues ) of Cucurbitacea family ( LA-1, LA-2, MCTI-I, CMTI- I, CMTI-III )with mostly arginine at their active centre, have been modelled using the high resolution X-ray structure of homologous inhibitor MCTI-II isolated from bitter gourd. The inhibitors have been modelled in their native as well as in complexing state with the trypsin molecule as observed in MCTl-II-trypin complex. These results support the binding constants (behaviour) of the inhibitor - trypsin complexes in solution state. The difference accessible surface area (DA) study of the trypsin with and without inhibitors shows subsites and throws light into the structure-function .It appears that the role of nature's mutation in these peptides is to modulate inhibitory function as needed rather than changing the overall structural folding characteristics which is thought to be highly conserved. So, the minor changes of amino acids in the conserved and non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complexing mode. P119

De novo design of pepfldes that switch conformafional state

Barbara Ciani and Derek N. Woolfson

Centre for Biomolecular Design & Drug Development, School of Biological Sciences, University of Sussex,BNl 9QG,UK

Protein design offers a mecbani.~m for testing our understanding of protein structure and folding. To date, several successful protein designs have been achieved for common protein-folding motifs. Here we present results on a rationally designed peptide sequence that codes for two sUmctural motifs simultaneously, namely, a ~- hairpin together with an c~- helix. Rules that relate sequence to structure for ~-- haiipin motifs have been developed by us [Hutchinson et al, Pro. Sci., 7, 2257-2300, 1998] and we have combined these with rules for ct- helix formation in peptide SWITCH_B 1. The terminal residues of this peptide are cysteines the intention being that the formation of an inWamolecular disulphide bridge together with other designed cross-strand interactions would constrain the peptide initially into a ~- hairpin conformation. Reduction of the disulphide bond should release the termini and then allow the embedded c~- helical motif to fold. Consistent with the design, circular dichroism experiments show that both the oxidised and the reduced states of SWITCH_B 1 are partially folded in phosphate buffer and these su~ctures denature cooperatively upon heating. In addition, in aqueous-alcohol solutions SWITCH_B1 shows predominantly [$-- sheet structure in the oxidised state and r helical conformation in the reduced form.

P120 STRUCTURAL ANALYSIS OF FUNCTIONALLY EQUIVALENT MOLECULES:TOWARDS

RATIONAL DESIGN OF PEPTIDE MIMICS OF OLIGONUCLEOTIDES Usha B. Hair, Kanwal J. Kaur, Dinakar M. Salunke,

National Institute of Immunology, Aruna Asaf Aii Marg, Hew Delhi-I 10 067 Molecular recognition holds the key to function in biological systems with noncovalent interactions

playing a crucial role. Investigating the nature of these interactions in instances where functional equivalence among dissimilar entities exists, should provide valuable insights into structure-function relationships. We have evolved one such novel system, wherein mimicry between peptides and oligonucleotides can be exploited to derive an empirical model applicable to other similar systems. A nine residue long peptido derived from Ribonuclease Inhibitor was found to competitively inhibit the activity of Ribonuclease A. The residues and the minimum length critical for binding were determined leading to rationally designed peptide inhibitors with optimal activity. The circular dichroism spectra of these peptides revealed their propensity to adopt a polyproline II conformation. To dissect the nature of the interactions involved in binding and also assist further design and optimization, modelling studies were carried out, as well as crystallization of the enzyme, free, and in complex with the peptides was set up by vapour diffusion in banging drops. X-my intensity data were collected with single crystals of the free enzyme as well as those of the complexes. The refined structure of the native enzyme (PDB code:7RSA) was used as the initial model for molecular replacement. Peptide models have been built into the electron density for two different complexes and refinement is in progress.

7 5


P121 WATER UPTAKE ASSOCIATED WITH SPECIFIC BINDING OF SUGARS BY LECTINS

Chittoor P. Swaminathan*, and Avadhesha Surolia ** t Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560 012, and t Jawaharlal Nehru Centre for Advanced

Scientific Research, Bangalore-560 064, INDIA. Fax: 91-80-334 1683, E-mail: swami(-~mbu.iisc.ernet.in It has previously been demonstrated by us that the specific recognition of the branched trimannoside, the

individual dimannosidic arms, and the monomeric unit D-mannopyranoside by the lectin Con A is accompanied by differential uptake of water molecules t. We show here that a coupled osmotic-thermodynamic approach using isothermal titration microcalorimetry (ITC) provides a useful handle to dissect out the involvement of water molecules in the recognition of sugars by plant lectins belonging to Leguminosae and Moraceae families as well as by an animal galectin. Sugar binding to these lectins is accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and are described by well defined negative slopes characteristic for each sugar, consistent with a net uptake of water molecules. As these changes are independent of the chemical nature of the osmolyte used, the results reflect a true osmotic effect. This study also addresses the role of water-reorganization in effecting the phenomenon of enthalpy-entropy compensation in lectin-sugar interactions in general, and perhaps protein-ligand interactions. In closing, this study demonstrates that the specificity of iectin-sugar recognition is characterized by a differential uptake of water molecules. Ref: 1. Swaminathan, C.P., Surolia, N., and Surolia, A. J. Am. Chem. Soc. (1998) 120, 5153.

P122 SPECIFIC BINDING OF PROINSULIN C-PEPTIDE TO HUMAN CELL MEMBRANES

R. Rigler ~, A. Pramanik -~, P. Jonasson 2, G. Kratz a, O. T. Jansson 3, P.-A. Nygren 2, S. St,elhl 2 , K. Ekberg ~, B.-L. Johansson 3, S. Uhl~n 4, M. Uhl~n 3, H. JOrnvall l, J. Wahren 3

1Dept. of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; 2Dept. of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden; 3 Dept. of Surgical Sciences, Karolinska

Hospital, Stockholm, Sweden; 4Dept. of Physiology, Uppsala University, Uppsala, Sweden Since the discovery of insulin, it has generally been held that C-peptide, the connecting segment of proinsulin, does

not possess biological activity of its own. However, several studies during the last years have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state. To examine the background to these effects binding of C-peptide to cell membranes has been studied using fluorescence correlation spectroscopy. Measurements of ligand-membrane interactions at single molecule detection sensitivity in 0.2 fl~ confocal volume elements showed specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface was ~ obtained at nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high affinity binding processes with K m > 3.3"109 M ~. Addition of excess unlabeled C-peptide was accompanied by competitive displacement. Cross-reactions were not obseryed with insulin, IGF-I, IGF-II, or proinsulin. It is concluded that C-peptide binds to specific receptors on human cell membranes, thus providing a molecular basis for the peptide's biological effects.

P123 MICROCALORIMETRIC STUDIES ON ANTIBODY PEPTIDE INTERACTIONS

Karin Welfle l, RolPMisselwitz 1"2, Wolfgang HOhne 2 and Heinz Welfle ~ tMax-Delbr0ck-Centrum for Molekulare Medizin, and 2Humboldt-UniversiUtt, Medizinisehe Fakult/tt (Charit6),

Institut filr Bioehemie, Berlin, Germany

The murine monoclonal anti-p24 (HIV-1) antibody CB4-1 binds to a linear peptide epitope of the capsid protein but also to several unrelated peptidos. The X-ray crystal structures ofCIM-1 Fab fragment alone and in eomplex with epitope-homologons and non-homologous peptides were solved at 2.6 A resolution [1], and recently we described physicochemical properties of CB4-1 [2]. We are studying the interaction of CB4-1 with peptides by isothermal titration calorimetry. From the binding curves of e-, h-, u- and d-peptide with CB4-1 and its Fab fragment the number of binding sites, binding constants, and binding enthalpies were determined, and binding entropies were calculated. The binding constants are in the order of 107 M "t, and except for the h-peptide, in reasonable good agreement with data determined earlier by ELISA techniques. Enthalpy and entropy contributions to the free energy of binding differ significantly from peptide.to peptide. [1] T. Keitel, A. Kramer, H. Wessner, C. Seholz, J. Schneider-Mergener,. W. H6hne, Cell 9t: 811-820 (1997) [2] K. Welfle, R. Misselwitz, G. Hausdorf, W. H6hne, H. Welfle, Bioehim. Biophys. Aeta 1431:120-131 (1999)

76


P124 Effects of Ligands Binding on Muscle Glycogen Phosphorylase b Internal Dynamics

Mazhul V.M.'-, Zaitseva E.M.', Mitskevieh L.G. b, Fedurkina N.V. b, Kurganov B.I. b ' Institute of Photobiology Natl.Acad.Sci.Belarus,Akademicheskaya Str., 27, Minsk, BY-220072, Belarus.

b A.N. Bakh Institute of Biochemistry, Russian Academy of ScienceLeninski pr., 33, Moscow, 117071, Russia. Using room temperature tryptophan phosphorescence (RTTP) technique, the effect of binding of substrate (glucose- l- phosphate) and competitive inhibitor (glucose) to glycogen phosphorylase b (EC 2.4.1.1) has been studied with respect to its internal dynamics. Recently, RTTP lifetime have been shown to be a sensitive monitor of flexibility of phosphorescent Trp residues environment. Earlier (1998) we have demonstrated glycogen phosphorylase b to exhibit long lived RTTP. It has been established that binding of glucose- l-phosphate (0.25-4 mM) to the enzyme active site results in increase of RTTP lifetime values up to 30% with the rising of the substrate concentration. The data indicate an increase of rigidity of the protein matrix in the vicinity of the phosphorescent tryptophanyls. Binding of glucose (0.5-amM) to glycogen phosphorylase b induced similar effects on the RTTP decay parameters. Thus we have concluded that the competitive inhibitor binding as a binding of the substrate makes the protein matrix more rigid. Correlation between protein internal dynamics and realisation of their functional activity is discussed.

P125 Effect Of Ligand Binding And Sub-Unit Dissociation On Intrinsic Fluorescence Of Yeast

Hexokinase. Gotam K. Jarori and Haripada Maity, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba,

Mumbai-400 005 India. Yeast hexokinase exists in monomeric as well as dimeric forms depending on the pH and ionic strength of

the medium. The enzyme also undergoes ligand induced conformational changes which may be essential for catalysis and regulation of its activity. We have performed steady state and time resolved fluorescence measurements on the enzyme to study the effect of ligand induced conformational changes on the fluorescence properties of the tryptophan (trp) residues. The fluorescence decay of trp residues in the enzyme is bi-exponential with life time components xt-2.2 ns and x2-3.9 ns. Dissociation of dimer to monomer does not have any significant effect on the measured life times. Binding of glucose to monomeric as well as dimeric forms resulted in significant quenching of fluorescence and a decrease in life-time component Xl, without any effect on x2. Interaction of nucleotide ligands (AMPPCP and ADP) to the enzyme in presence and the absence of glucose had no effect on life-time distribution and their magnitudes, suggesting that there is no significant conformational change during the formation of a ternary complex (E.Glc.Nucleotide) from the binary (E.Glc) cdmplex. Trehalose 6-phosphate, a strong inhibitor of hexokinase, was found to compete with glucose for binding and appears to stabilize 'open' conformation of the enzyme. Fluorescence quenching studies by acrylamide and KI indicate that glucose induced conformational changes alter the accessability profile of various trp residues. The mechanism of quenching will also be described.

P126 LUMINESCENCE STUDIES OF PROTEIN-FLAVONOID INTERACTION

I, GUHARAY, B. SENGUPTA and P. K. SENGUPTA Biophysics Division, Saha Institute of Nuclear Physics, Calcutta 700 037, India.

Recent studies have revealed that various therapeutically active flavonoids possess novel luminescence

properties which .can potentially serve as highly sensitive monitors of their micro-environments in biologically

relevant systems. We report a study on the interactions of Bovine Serum Albumin (BSA) with representative

flavonoids, using their excited-state proton-transfer (ESPT) luminescence parameters as probes. Upon addition

of BSA to the flavonoid solutions we observe remarkable changes in the ESPT emission and excitation profiles

as well as anisotropy values. The spectral data also suggest that in addition to ESPT, the protein environment

induces proton abstraction from the flavonoids leading to formation of anionic species in the ground state. All

these findings indicate significant protein-flavonoid interactions, implications of which are examined.

77


P127 S t r u c t u r e o f nue leo t ides b o u n d to Tubulin

K. Sridevi & S. R. KastuH Department of Chemical Sciences, Tata Institute of Fundamental Research,

Homi Bhabha Road, Mumbai, 400 005, INDIA

Tubulin, an a15 dimer protein of molecular mass 110 kD, self assembles into microtubules and is known to bind the nucleotides GTP and ATP. In vitro, tubulin has been shown to bind ATP and polymerize into ring like structures as opposed to the long tubule~ formed in the presence of GTP. For polymerization, Mg +2 is also required even though the nucleotides can bind to tubulin in the absence o f M g +2. It has been reported that Mg+2 induces a conformational change in the protein. To tmderstand the sU'uctural differences, if any, between the bound ATP and GTP and the effect of Mg +2, 2D-tranferred nOe experiments were carried out to determine the conformation of the bound nucleotide. NOE measurements on the nucleotide protons in tubulin-nucleotide complexes were made at 600 MHz, 15~ for different mixing times. The nOe build-up curves from such measurements were analyzed by a complete relaxation-exchange matrix calculation and various inter-proton distances were obtained. These distances were used in a restrained energy minimization to derive the conformation of the bound nucleotide. The differences in the bound nucleotide conf~ri-.afions and the effect of Mg+2 on the slruc~es will be discussed.

P128 QSAR Studies on HIV-I Protease Inbibitors: Modes of Enzyme-Ligand Binding

. S.P.Gupta

Depar tment o f Chemistry, B irla Institute o f Tec lmology and Science, Pilani - 333031, India

The quantitative structure-activity relationship (QSAR) studies have been made on several different series of human-immunodeficiency-virus type 1 (HIV-1) protease inhibitors, the prospective anti-AIDS drugs. Based on the results, excellent models for the binding of the drugs with the enzyme have been proposed, indicating the modifications in the drug struct0res leading to the increase in binding and thus to the increase in the efficacy of the drugs.

P 1 2 9 Melting Mechanisms Of Single HbS Fibers As Viewed By DIC Microscopy

Suzanne Kwong and Robin W. Briehl Physiology and Biophysics, Albert Einstein College o f Medicine, 1300 Morris Park Ave. Bronx, NY ! 0461

Sickle cell hemoglobin (HbS) polymerizes into long fibers in the deoxygenated state. These fibers distort and rigidify red-blood cells and induce pathogenesis in sickle-cell disease. The mechanism of polymerization consists of formation of new fibers via homogenous and heterogenous nucleation followed by fiber elongation ~'2 However, there have only been limited observations On polymer melting and the mechanisms are not well-defined; melting is critical in undersianding pathogenesis and treatment of sickle-cell anemia. We isolated and observed single fibers of HbS in real time by differential interference contrast (DIC) microscopy using photolysis of CO- figated HbS. Melting of HbS fibers due to religation of CO after removal of photolysis was observed at partial pressures of CO (pCO), ranging from 0.1 to 1.0 atm. Two mechanisms of melting are evident: (1) fibers melt from the ends and (2) fibers melt by a 'fading' mechanism in which the entire fiber diminishes simultaneously. Melting of fiber from ends is dominant at low pCO (< 0.2 atm). As pCO is increased, the melting rate increases, gradually leading to a transition to the faster mechanis~ of fading. An overlap of both the mechanisms is observed at intermediate pCO, while at higher pCO (>0.4 atm), only mechanism (2) is seen. 1. F. Ferrone, J. Hoffichter and W. A. Eaton, J. Mol. Biol. (1985), 183, 611-631 2. R.E. Samuel, E. D. Salmon and R. W. Briehl, Nature (1990), 345, 833-835

7 8


P 1 3 0 FLAVINMONONUCLEOTIDE ACTIVATED ON THE PHOSPHATE GROUP BY N-METYLIMIDASOL IN

REACTION OF BACTERIAL BIOLUMINESCENCE. O.I.Ismai!ova~N.A. Tyulkova. Institute of Biophysics Sibirian Branch of Rns.Acad.of Science, Russia. Krasnoyarsk

660036. The kinetic characteristics of bacterial biohiminescence reaction with flavinmononucleotide activated on the phosphat group by N-metylimidasol were investigated. Aktivated flavin is differed from native FMN on the physical and chemical properties: absorbfion spectra of recovered form, resistence to oxidation, relationship to luciferase and others. Luminescence is absent with use of photorecovered activated flavin as substrate. But luciferase gives long light emission with chemicaly recovered activated FMN . The use of mixing activated and not activated FMNH2 eccure set to competision for binding with luciferase. However in absence of exogenous aldehyde in the reaction with activated substrate the accumulation oxygen peroxide is not observed, which the reaction has with native flavin and it is unproductive decay of intermediate II. It is supported, that the active center of luciferase has the functional groups are strong nucleofils, which able to form covalent bond with activated flavin. This bond does not disturb the formationof excited emitter.

P 1 3 1 Time Resolved Fluorescence Study of the Molecular Interaction between Basic Fibroblast Growth Factor

and Novel Anti-Angiogeneic Drugs C. Harilmmn, D. Pines, E. Pines, M. Zamai, R. Cohen- Luria, A, Yayon and A.H. Parola

Department of Chemistry, Ben-Gurion University of Negev, Beer-Sheva, Israel

Vae molecular characterization of the interaction between angiogeneic growth factors (eg. BFGF and its m, tants) and small molecule anti-anglogeneic compounds (eg. Suradista) is fundamental for unraveling the basic mechanisms involved and their control, bFGF is a 146 amino acid protein with a single tryptophan and seven tyrosine residues. Suradista (PNUI45156E) is a naphthalene sulfonic distamycin A derivative which interacts with heparin binding growth factors in a specific and reversible ~vay and inhibits tumor angiogenesis with no caothropic protein aggregation [1]. A temperature profile of the picosecond time resolved fluorescence emission and anisotropy study invovling the tJ~tophan fluorescence of the protein is used to characterise the binding interactions between the protein and the drug as well as the docking surfaceon bFGF, Preliminmy experiments of this kind performed with the Trp and Tyr fluorescence of the bFGF yielded binding constant of 143,5 nM and 155,6 nM respectively [1]. The effective binding of the mutated bFGFs is also compared with that of the wild type. 1. Zamai, M., V.R. Caiolfa, D. Pines, E. Pines and KH. Parola (1998) Nature of interaction between basic

fibroblast growth factor and the antiangiogeneic drug PNU 145156E. Biophys. J. 75, 672-682.

P 1 3 2 S a f . A ~ m b , y of a ~gn,d Pret~.ImU~

G A Spooaer inK! D N W ~

Centre for B iomobcul~ Design & Drug Developmenl, School of Biological Sciences, University of Sussex, Fslmer, East Sussex, BN19QG, UK

Most natural coiled-coil pmteim have two Og thn~ a - helical s U m ~ that wind mound each ~ in-register to form a left-handed supcd~elix. Many such proteins extend over hundreds of residues and assemble further to form thicker, multi-stranded filaments. In an attempt to mimic extended-fibre fcmmalion, we designed two 28-r'~tidue peplides to form out-of-register betem-dimefic coiled-coil suructmes. ~ resulting complememmy ovedmnging ('sticky') ends were intended to promote fibre, assembly. Consistent with the design, circular dichroism spectroscopy demmmumed concentration-dependmt fotmation of 0t-helical stlxlctt~ fog peptide mixtule~ but only limited folding fog the isolated pel~dea Fmtbema~ the generation of long coiled-coil fibres was visualized directly by electron ~ . Compmison with Impomyos~ however, revealed that the fibge$ weld thlcke~ than a typical dinu~c coiled oo~ Assembly of these pclHides into hi~hcf-Otlka" was not expected and this result suggests a general m~haniem f0r ftbriliogenests: we propose that periodicities, inbenmt in rej~.atin 8 S ~ of thC type we designed, dh'c~ fullhef ot~smiT~tinn into thick r~IRiilCilIS.

7 9


P133 Synthesis and Physicochemical Studies of Glycoconjugates of Enkephalin Analogues

Panchan Bakshi, Deepak Sharma, Sunita Sharma, D.K. Bharadwaj and Santosh Pasha Peptide Unit, Centre for Biochemical Technology, D.U. Campus, Mall Road, Delhi-110 007, India.

Classes of neuropeptides known as brain's own opium, mimic some of the key actions of morphine and are clinically useful for management of pain. However their rapid degradation, inability to cross blood brain bamer (BBB) and rapid clearance through liver'limits their use as therapeutic drugs. To overcome these lacunae enkephalin analogues having glucose moiety were synthesized, as it is known that glucose act as Wansport vector and is brain's main nutrient. The glycosylated peptides were synthesized by solid phase methodology using the glycosylated amino acid employing penta chloro active ester method. It has been found that this method of coupling gave exceptionally good yield as compared to all other methods known. The peptides were purified using ion exchange chromatography and RP HPLC. Peptides were characterised using sequencing and biochemical methods. Their conformation at different pH, temperature, TFE concentration and in different solvents were studied using fluorescence spectroscopy, circular dichroism and FTIR. From the above structural studies it is established that the attachment of glucose moiety does not disturb the native conformation of enkephalin and above all, the gaVcoconjugate exhibits and enhanced helical order.

P134 A Comparatiue Stud~l of Solution Structures of Daunomycin, Adrlamycin and

4-- Epiadriamgcin by Proton NMR Spectroscopg. U. Sharma, N. Srivastava*, R. Barthwal* and N. R. Jagannathan.

Department of NMR, All India Institute of Medical .Sciences, New Delhi - 110 02% India. * Department of Biosciences and Biotechnology, University of Roorkee, Roorkee - 247 667, India.

The variation in biological activity of anticancer drugs is an important determinant to investigate their cozffonnational characteristics. We present here the results of our study oa the comparison of solution co~fformations of damlomycin, adriamycin and 4 t - epiadriamycin by proton NMR spectroscopy. Spectral assigranents were carried out using ID and 2D NMR techifiques. Coupling constants were measured by analysing the spin systems observed in 2D phase-sensitive COSY, while the interproton distances by NOESY spectra recorded at different mixing times. A significant difference in the chemical shift positions of sugar protons of 4 t- epiadriamycin were observed in comparison to daunomycin and adriamycin. Interproton distances calculated using NOE connectivities of sugar protons with ring A, clearly demonstrate a significant variation in the conformational

9 9 t ' . . . . . . . - 9

characteristics of4~ epsadnamycm m companson with the other two drugs, which may be a possible explanation for the improved activity of 4'- epiadriamycin compared to the other two analogues (1). Refereqces: 1. Arcamone, F. and Penco, S. (1988), in Lown, J.W. (ed.), Anthracycline and anthracenedione based anticancer agents, Elsevier, New York, pp. 1 - 53.

P135 EVOLUTIONAL PROCESS OF THE STRUCTURE OF ATP-BINDING DOMAINS

~ s ) , Takaaki Nishioka s), and Nobuhiro Go 2) Graduate School of Agricultural Science D and Graduate School of Science z~ , Kyoto University, 6068502, Japan

W e previously found that the topology and spatial arrangement of secondary slructural elements of the ATP-binding domain of glutathione synthetase is similar to those of D-AIa;D-AIa figase, succinyl-CoA synthetase, and acetyl-CoA synthetase biotincarboxylase subunit (Matsnda, et al.,1996). These four euzymes, which are functionally similar to each other, are supposed to be derived from a common ancestor. Their Uructoral features are quite different from those of the other ATP-binding domains. There arc at least 17 topologically different structures in.the 36 ATP-binding domains with the known 3D stru~ac. Did so diverse structures derive from a single common ancestor ? To analyze the cvolutional ~ of ATP-binding domains, we made a lineage map that shows topological relations among the 3D structures ofprotcin domains and analyzed the disu'ibution of the 3D slructures of all the ATP-binding domains on the map. Each 3D structure is relr~a,ented by ~ topology of the major [~-sheet and linked to otliers bythe topological relations. On the map, ATP-binding domains distribute on several independent structural lineage that are not mutually linked by the permutations of [~-strands. Our analysis suggests that ATP-binding domains' evolve independently and several times from different ancestors. Matsuda, K., et al., Plot. Eng,, 9, 1083-1092 (1996)

8 0


P 1 3 6 HOW DOES AMINO-ACID LANDSCAPE ~ PROTEIN LANSDSCAPE ?

AITA T*., URATA S. and HUSIMI y. Ftme. Mat. Sei., Saitama Univ (JPN)," Takarazuka Re~ Inst. Novartis Phanna K.K. (JPI'0

* present affiliatioll Evolution of a protein is regarded as an adaptive walk on a protein property landscape in a base sequence space (=DNA space) where the height corresponds to a measure of the protein function, such as a catalytic activity or affinity to a receptor. The architecture of this landscape can be translated primarily by an amino- acid property landscape in the codon space and.secondarily by a protein property landscape in an amino-acid sequence space (=protein space). We focused on the former and analyzed 11 amino-acid property (e.g. hydropathy index) landscapes in the 4-valued 3-dimensional codon space, based on a model of the Mt.Fuji- type landscape. We found that hydropathy landscapes and polarity landscape for the standard genetic code (SGC) are very dose to the Mt~Fuji-type. This implies that the additivity of the contribution from each letter holds for these properties. In order to clarify the meaning of the so-called mutational robus~ess of SGC, we next examined correlation between the ~mino-acid property and actual function of a protein, where we used, as a test case, a set of binding preference scores of amino-acid in MHC class I molecule binding peptides. We found that the conservativeness of an amlno-add property in SGC is correlated with the importance of the property in protein function. Adaptive walk simulation on .~firdty landscapes on the. base sequence space for these model peptides confirmed the better evolvability due to the introduction of SGC.

P 1 3 7 Biophysical studies of lectins from Saraca indica (Ashok)

Mainak Majumder and Bishnu P. Chatterjee Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Calcutta 700 032

Three sialic acid specific lectins were purified from different parts ofSaraca indica (Ashok), namely from

filament, gynaecium and seed integument. The molecular weights were found to be 63, 33 and 12.5 kD respectively

for filament, gynaecium and seed integument lectins. From fluorescence quenching studies it was found that all the

three lectins had two binding sites for Neu5Ac and the association constants were found to be 2.33, 12.6 and 1:44

~tM "1 respectively for the filament, gynaeciam and seed integument lectins. From circular dichroism study it was

found that all of them contained high amount of random coil structure though the seed integument lectin had a good

amount of 13-sheet. On binding with the inhibitor, Neu5Ac, the percentage of a-helix increased in all three cases,

suggesting that the lectins attained stable conformations.

8 1

B1 : Nucleic Acids : DNA Structure and Dynamics

P 1 3 8 Comparison between low and high resolution structures of the oligonucleotide d(CGTATATACG) as a site specific

complex with nickel ions Nioola G. A. Abrescia, Lucy Malinina and Juan A. Subirana Departament d'Enginyeria Quimica, Universitat Polit~cnica de Catalunya, Diagonal 647, Barcelona E-08028, Spain

We have determined the structure of the alternating (YR) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction in two different space groups. A high resolution structure (1.58 A) was obtained by complexing with the netropsin drug. Both structures were solved by molecular replacement and ref'med with either the X-Plor or the Shelx programmes. The general conformation is B-like with an end-to-end interaction which involves terminal guanines as described by Spink et al. (PNAS 92, 10767, 1995). One crystal belongs to space group P412t2 with a=b=53.46, c=I01.49 A. This is a new way of packing for decamers. A novel C-A.T triplet structure has also been tentatively identified. In the high resolution structure the overall conformation is B-like but the end-to-end interaction previously seen is different. In fact one of the terminal guanines stacks on a pyrrole ring of netropsin in the minor groove of the helix. The crystal belongs to space group P21212m with a=25.18, b=39.1 l, c=53.47 A. Both~tructures are characterized by the specific association with nickel ions, involving the N7 atom of every guanine. One of the Ni § ions in the P412~2 structure is shared between two guanines of symmetry related molecules. Until now no oligonucleotide has been crystallized in the presence of Hi § Thus it appears that such ions may be used as specific probes for guanine in nucleic acids.

P 1 3 9 Crystal stucture of a benzimidazole minor-groove binding drug eomplexed with

a non-self-complementary DNA dodecamer. Juan Aymami '~, Ram6n Eritxa 2 & Miquel Coil 3. I Departament d'Enginyeria Quimica, Universitat Polit~cnica de Catalunya, Diagonal 647, 08028 Barcelona, Spair~ 2EMBL, Meyerhofstrafle 85, 22603 Hamburg, Germany. 3Institut de Biologia Molecular de Barcelona, C.S.l.C., Jordi Girona 18, 08034 Barcelona, Spain.

The crystal structure of the non-self-complementary DNA dodecamer d(CG-CGAAAACG-CG) + d(CG(~C)GTTTTCGCG) has been solved at 1.8 Angstroms resolution, complexed with the minor- groove binding drug Hbis. This drug is a Hoechst 33258 analog in which the piperazine ring has been replaced by an hydrocarbon chain. The crystals have been diffracted in the Elettra synchrotron light source (Trieste, Italy) allowing high resolution data. The inclusion of a bromine atom in one strand of the structure allows the correct determination of each DNA strand and the possibility of disorder to be discounted. The structure is isomorphous with the Dickerson dodecamer and dearly shows the drug along the minor groove of DNA. The drug interacts in the central part of the oligonucleotide which contains a four .AT base pair tract.

P 1 4 0 Structure and Stereochemistry of Nucleic Acid Duplexes and Triplexes formed with

2',5' and 3',5' ofigonueleotides Premraj, B. J. and Yathindra, lq., Department of Crystallography and Biophysics, University of

Madras, Guindy Campus, Chennai 600 025, India.

Recently 2',5' nucleic acids have been receiving greater attention because of their new found importance in antisense applications. Several interesting experimental observations have been reported regarding the discriminating behaviour of 2'5' oligonucleotides with regard to their complex formation with 3',5'oligoribo and oligodegxyribonucleotides. We report here molecular modeling investigations of a number of mixed duplexes and triplexes comprising 2',5'and 3',5' linked oligonucleotides in order to provide a stereoehemical rationale for the ability or lack of it, for the formation of specific duplex and triplex complexes. While modeling these complexes the stereochemical information on the influence of type of sugar-phosphatd linkage not only on the shape and dimension of nucleotide repeats, but also on the preference of deoxy and ribose sugar conformation is kept in mind. Results of these studies provide structural clues for better design of 2',5' oligonucleotides that offer greater specificity of interaction against the target nucleic acids especially in relation to antisense applications.

8 2


P 1 4 1 Stereochemistry of DNA Triplexes formed by (AG). and (GT). oligonucleotides: Effect of residual

Hoogsteen / reverse Hoogsteen twist Thenmalarchelvi, R. and Yathindra, N., Department of Crystallography

and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.

It is known that d(AG), and d(GT), oligonucleotides form DNA triplexes despite the triplets T.A*A,C.G*G and T.A*T being non-isomorphous due to differences in their geometries and residual Hoogsteen/reverse Hoogsteen twist. To deduce stereochemical details of such triplexes, a variety of them formed by using the triplex forming oligonucleotides (GT). and (AG). are investigated using computer modeling procedures. It is found that residual Hoogsteen/reverse Hoogsteen twist varies from 16 ~ to 21 ~ depending on the base triplet. This leads to alternating low and high twist .angle at successive steps causing steric hindrance for formation of triplexes. However considerable rearrangements in triplet twists take place upon energy minimisation leading to stereochemically feasible triplexes. Hoogsteen/reverse Hoogsteen twists now assume values close to W&C twist but still retain some differences in the range 5-10 ~ depending on base steps. This causes non-uniform helical twist along the triplex. While d(GT)n supports both parallel and antiparallel triplexes, d(GA), supports only antiparailel triplex due to the constraints of T.A*A triplet. It is found that r(AG), and r(GT), for the third strand also support triplex. Detailed stereochemical features as deduced from molecular mechanics and some MD simulations of different triplexes are discussed.

P 1 4 2 Unusual Ring - Shaped OHgonucleotide Crystals

P.Satheesh Kumar and N. Gautham Department of Crystallography and Biophysics, University of Madras, Guindy Campus,

Chennai 600 025. E-mail : [email protected]

As part of our studies on the effect of a single AT base pair on the regular d(CG) repeat, we have grown crystals of d(CGCGCA).d(TGCGCG). This sequence yielded hexagonal shaped crystals of size 0.2 x 0.2 x 0.1 mm in the presence of MgCIz. The crystal diffracted up to 2.0 ,~,. While trying to improve the quality of the crystal with Co(NHe)3+ we have obtained hexagonal ring shaped crystals of size 0.05 x 0.05 x 0.005mM under specific and reproducible conditions. The hanging drop - vapour diffusion method was used for crystallization; the drop contained 2mM hexamer duplex, 100 mM sodium cacodylate buffer at pH 7.0, lmM spermine and 20 mM Co(NI-I6)CI3 and was equilibrated for four days against the reservoir solution of 50 % MPD. A gel electrophoresis experiment confirmed that these ring shaped Crystals are those of the DNA hexamer. Each ring shaped crystal could be the residue of a flat hexagonal plate, the, center of which has somehow evenly dissolved back into the solution.

P 1 4 3 The kinetic study of the Effect of polyamine on the Formafionof A-T base pair-rich DNA Duplexes by

Surface Plasmon Resonance.

Lou -Sing Kan 1"2, Ming-Hou 2, and Shwu-Bin Lin 3. qnstitme of Chemistry, Academic Smiea, 2Institute of Biuchemis~ry, national Chung-Hsing University, 3G _r-~h~:ae Institute of Medical Technology, National Taiwan University.

Polyamines are present in millimolar concei~aiion and can be as high as 5mM in the eukmyotes nucleus. Due to their chemical structure, it was suggested that the polyamine may stabilize the nucleic acid secondary structures, such as duplex and triplex. This report presents a novel strategy for using Surface Plasmon Resonance to detect the kinetic constant of the effect of polyamine, such as spermine and spermidine, on the formation of DNA ch~lexes with A l~tse-rich sequences. The synthetic oligowacleotide duplexes contain either a budged base 031), or a mismatch base pair(MI) or without lesions (1'I). Results showed that the association ml~ consents are in the range of l06]~'ls'l in al] duplexes and in various condition. This indicates that the formaticm of duplex is instantaneous. On the other hand, the dissociation rate constants have the following order. MI~BI>P1 w/th cations presende. However, the difference of the dissociation constant of M1, B1 and Pl are ~tm~hed. This indicated that polyamine can compensate the energy discrepancy caused by lesions (e.g bulged loop or mismatched base pairs) in the ~ e x formation. The same conclusimt was indicated from UV meiting tempenm~ data. (This work is sup~rted by NSC.).

8 3


P 1 4 4 GGATCC Repeats In Supercoiled Flasmid Form An Unusual Structure Of Biological Relevance

Tapas Saha and Knnal K Roy Centre for Biotechnology, Jawahaflal Nehru University, New Delhi 110067

d-GGATCC repeats are found in telomeric associated sequences and also, d-GGA repeats belong to the m/crosattelite group of mammalian genome known for genetic instability. A 36 mer syntbetic deoxyoligonuclcotide, 5' d- AATrC(GGATCC)~3 3' was inserted at EcoRI site of pUClg The insert region would thus contain seven palindromic repeats and could form a putative tehaplcx structm~ under supercoiling as was suggested by A. R. Morgan (1979). The structure formed in this ttgion under supercoiling was probed both in-vitro an in-vivo with chloroacetaldehyde (CAA). The CAA modification done at pH 5.2 or 7.0, with or withou'c MgCI2 followed by KMnO4 sequmcin8 reaction or primer extension gave bend patterns consistent with fold back intramolccular triplex s~actm~. Probing with S1 nuclease revealed restricted access to the unpaired region of the triplex. A pseudo tetraplex slructure with ~plex core is proposed, where the fotath strand is neither fully paired not fully unpaired. The slng-tme is similst to that reported by Kohwi and kohwi-Shigematsu (1988) for PolydG.PolydC sequence in ~ i l e d plasmid with the impommt diff~icr that the triplex c.or~ in ore" sU'uctt~ is a peuntllel triplex with aRenmte smmd pairing. Biological significance of such a structure in generalised recombination or triplet expansion is discussed 1. A.R. Morgan, (1979); Do Multisa'anded polynudleotides have a biological role?; Trends Biochem. N244-N247. 2. Kohwi, Y. and Kohwi-Shigematsu, T. 0988); Magnesium ion dependent triple helix stmctue formed by homopurine-

homopyrimidine sequmces in ~iled plasmid DNA, Proc. Natl. Acad. Sci., U.S.A., 850 l), 3781-3785.

P 1 4 5 FROM ATOMIC TO MESOSCOPIC DESCRIPTIONS OF THE INTERNAL DYNAMICS OF DNA

N. Bruant § , D. Hatters ~, R. Lavery # and D. Genest § : Centre de Biophysique Mol6culaire, CNRS UPR 4301. affiliated to the University of Ofldans. rue Charles Sadron, 45071 Orl6ans, France # : Laboratoire de Biochimie Th6oriqur CN-RS UPR 9080, IBPC, 13 me Pierre et Marie Curie, 75005 Paris. France.

An analysis of four one nanosecond molecular dynamics trajectories corresponding to two different 15-base pair oligonucleotides is presented for determining which groups of atoms could be considered as rigid bodies within a bead representation of DNA. The selected rigid bodies must not depend on the base sequence or on the conformations of the DNA. Five models with moderate intra-group deformations are proposed in which the groups are formed of atoms belonging to a single nuclcotide or to a complementary nuclcotidc pair, The influence of the deformation of the groups on the dynamics is checked for two of these models, using canonical correlation analysis. It is shown that the internal dynamics of DNA is dominated by the rigid motion of the defined groups of atoms. Finally, using one of the jmodcls within a bead representation of the DNA duplex, it is possible to extract the stretching, torsional and bending rigidities, and a reasonable agreement with experimental values is obtained.

P 1 4 6 Theoretical studies of methylene blue binding to DNA with u GC alternating base sequence

Remo Rohs', Heinz Sklenar I and Ri.'chard/.,avery z I Theoretical Biophysics Laboratory, Max Delbriick Center for Molecular Medicine, Berlin

2 Laboratoire Biochimie Thdorique, CNRS UPR 9080, [nstitut de Biologie Phy$ico-Chimique, Paris Theoretical and experimental investigations of complexes consisting of photoactive dyes and DNA-oligonuclcotides arc of special interest because of possible applications in gene therapy. We have searched for a photophysical mute to break oligonur backbones via photosensitizcd reactions with singlet oxygen generated by photoactive dyes. Methylene blue has been chosen for this task. It is capable of causing photooxidative damage in biological systems by the generation of singlet oxygen through energy transfer from the egcited triplet state of the dye to the triplet ground state of O z. Absorption and luminescence studies of this dye have shown that the singlct oxygen quantum yield depends on the conformation of the dye-oligonuclcotide complexes, In order to understand this mechanism, we have used the JUMNA internal and helicoidal coordinate molecular modelling program (Lavery et al., Comp. Phys. Comm. I995, 91, 135-158) in conjunction with a finite difference Poisson-Boltzmann treatment of the solvent to compare dye interealation with minor and major groove binding sites. The calculated binding energies ofmethyleae blue to a double stranded dccamer with a GC alternating sequence indicate a prcfercnco for intercalation versus groove binding. The results, which correlate with circular diehroism d~t~ obtained by E. Tuite and B. Nord~n (J . / /~ Chem. Sot:. 1994, 116, 7548-7556), should be helpful in the design of new active complexes for specific sequences.

8 4


P147 Sequence Directed Flexibility of DNA with Environmental Effects

Sudip Kundu 1, Dhananjay Bhattacharyya ~, Debashree Bandyopadhyay 2, Ashoke Ranjan Thakur 1 and Rabi Majumdar 2

1Department of Biophysics, Molecular Biology and Genetics, University of Calcutta, 92 Acharya Prafulla Chandra Road, Calcutta 700009, INDIA z Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Calcutta 700037, INDIA

The sequence directed structural flexibility of DNA is known to modulate its biological function through ligand induced structural alterations. The macroscopic parameters like the persistence length (P) and the torsiona! rigidity (C) are measures of such flexibility. These flexibility indices for B-DNA have been calculated by analysing crystal structure database. In order to evalute the effects of environment on DNA flexibility, the values of the parameters P and C for different structural forms like A-DNA and Z- DNA have also been calculated byusing the corresponding databases. The flexibility of B-DNA is found to be highly sequence specific and its values are intermediate between those of the more flexible A-DNA and the rigid Z-DNA. The protein bound DNA structures are also analyzed and the effects of non-crystalline solution environment discussed.

P148 ON THE HETEROGENEOUS BINDING OF BERENIL TO DNA F. Barcel6 (Department of Biology.Universitat de les hies Balears. Palma ofMallorca-0707l. Spain), J. Portugal (Instituto Biologia Molecular, CSIC. Barcelona. Spain)

Nucleic acid binding ligands are classified by the mode by which they interact with the nucleic acid target. Berenil is a minor-groove binding ligand, which recognizes AT-rich tracts in DNA (1,2). However, recent studies (3,4) have shown that some ligands, including berenil, might show a mixed binding mode that directly depends on the nucleotide composition of the host DNA. We have used ITC and DSC calorimetric techniques to sight new information into the characteristics ofberenil binding to either synthetic DNA polymers, or natural sequence-averaged DNAs. ITC preliminary results indicated a rather complex interaction involving more than one kind of non-identical binding sites, which show some peculiarities in the sequence-dependent recognition of DNA by berenil. (1) J. Portugal and M.J. Waring, Eur. J. Biochem. 167(1987)281 (2) F. Barcel6 and J. Portugal, Biophys. Chem. 47(1993)251 (3) D. S. Pilch, M.A. Kirolos, X. Liu, E. Plum and K.J. Breslauer, Biochemistry 34(1995)9962 (4) Ch. Bailly, J-P H6nichart, P. Colson and C. Houssier, J. Molecular Recog. 5(1992)155

P149 A Novel approach for uni form 13C and XSN label ing of DNA

Sunita Ram~l~than , K.V.R. Chary and B.J. Rao Tara Institute o f Fundamental Research, Mumbai 400 005, India

A novel method has been proposed for a large-scale biosynthesis of ]3C and ]~N labeled DNA for NMR studies. In this methodology, endonuclease 6ensitive repeat amplification (ESRA), a modified PCR strategy was used to amplify tandem repeats of a given target DNA soquence. The template used for ESRA was designed such that the restriction enzyme (RE) sites separated repeats of the target sequence. The ESRA product was then cloned into a suitable vector. The E. coli cells harboring the plasmid were grown in a medium that contained ~3C-lab61ed glucose and ~N-labeled ammonium chloride as the sole sources of carbon and nitrogen, respectively. Cells were harvested and the doubly labeled plasmid, thus obtained, was subjected to RE digestiorL The target sequences were purified from 12 % polyacrylamide 6M urea gel.

8 5


P 1 5 0 Structural Basis for Uracil DNA Glycosylase interaction (UDG) with DNA: NMR

Characterization Mahua Ghosh t, N. Vinay Kumar 1, Umesh Varshney z and IL V. R. Chary t"

'Department of Chemical Sciences, Tam Institute of Fundamental Research, Mumbai 400 005, INDIA 2Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, INDIA

2D NMR. and molecular dynamics simulations have been used to provide a structural basis for understanding the interaction between UDG and DNA. In this direction, we have determined the 3D structures of three hairpin DNA (22 mers) which differ in their tetra-loops, whose nucleotide sequences are UI2-TI3-TI4-TIs, Tl2-Ul3-Tl4- Tl5 and TI2-TI3-TI4-UIs. In all the structures, the nueleotides at position 12 and 15 stacks on the top of the respective stems of the hairpin DNA. Further, stacking of individual bases at positions 15; 14 and 13 continues on the 5' end of the stem. Such structural characterization explains the discrepancies seen in UDG activity for various tetraloops with U at different positions in the loop of a hairpin DNA.

P151 STRUCTURAL STUDIES ON ENHANCED MYOCLONUS EPILEPSY REPEAT SEQUENCE

Shashank S.Pataskar 2 and Samir K. Brahmachari t'2 t Functional Genomics Unit, Centre for Biochemical Technology, Mall Road, Delhi I I0 007, INDIA. 2 Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, INDIA.

Progressive Myoclonus Epilepsy [EPMI] is an autosomal recessive disorder caused by mutations in cystatin B gene [CSTB]. Majority of EPMI alleles contain expansion of dodecamer repeat, d[CCCCGCCCCGCG]n/d[GGGGCGGGGCGC]n present 70nts upstream oftho +I site ofCSTB gene where n=l to 3 & nffil6 to 75 for normal and mutant alleles respectively.

Oligonucleotides containing I, 2 & 3 copies of G-rich as well as C-rich strand of EPMI repeat sequence were synthesized. The structural characterization was done by CD spectroscopy, UV melting, electrophoretic mobility in native pol|,acrylamide gels (PAGE) and chemical probing etc. CD & gel electrophoretic studies suggested that depending on the ionic strength of the solution and repeat length the G-rich strand oligonuleotides exhibit conformatinnal equilibrium between folded hairpin and tetraplex structure. With increase in the length of the repeat G- strand oligonucleotides tend to fold back and predominantly form intramolecular structures involving Hoogstein type of G-G base pairing. C-rich oligonucleotides exhibited characteristic CD spectrum for "i-motif' at pH 5.0 while with increase in pH (7.2) blue shift was observed.. PAGE experiments, P1 nuclease and hydroxylamine probing indicated that the C-rich oligonucleotides adopt unique, compact, intramolecular structures. Thus we show that both the strands of EPM I repeat sequence adopt different conformations in a length dependent manner which could provide nucleation for the expansion of these repeats.

P 1 5 2 Structure and dynamics of the double - stranded DNA from Rice Leaf

Saro/ini. R ~, Selvasekarapandian. S z, Kolandaivel. P,2 and Sukumar. S 3 IDepartment of Physics, Kongu Nadu Arts & Scie~ace College, Coimbatore 641 029 2Department of Physics, Bharathiar University, Coimbatore -46, 3Centre for Plant Molecular Biology, Tarnil Nadu Agricultural University, Coimbatore - 641 003. Tamil Nadu, India

The solution secondary structure and nucleotide conformations in the double-strandard DNA isolated from rice leafs by a modified standard method of Sambrook et al. have been determined by FT-Raman spectroscopy. For both 1-120 and D20 solution, the experiments are performed at 25~ containing effective dsDNA concentrations of 50 mg/ml. The spectra are excited with the 1064 nm line of Nd : YAG laser using 200 mW of radiant power and collected on FRA 106 RAMAN MODULE equipped with an intensified Ge diode array detector. The method is exploited for monitoring hydrogen-isotope exchange dynamics of DNA to confirm definitive vibrational assignments. The following results are obtained : 1) The 8CH protons of adenine and guanine are exchanged as evidenced by the pronounced shifts (722 ~ 710, 684 --~ 670 cm ~) of respective Raman band frequencies. 2). The spectra also exhibited shifts of 784 --, 775, 1341 ~ 1346 and 1363 --, 1350 cm -~ frequencies upon H ---, D exchange of the amino protions of cytosine, adenine and guanine respectively. 3) Raman frequency shifts of 675 ~ 662, 758 ~ 749, and 1182 ---> 1155 cm -t bands indicate thymine N3H --, N3D exchange. 4) But the present results lack any marker of dG imino (NIH NID) exchange. The results thus confirm that the B-form secondary structure of dsDNA of rice leaf is essentially fully conserved in aqueous solution.

86


P153 Effect of Transition metal Ions Binding on the FT-Raman Spectrum of D N A

Selvasekarapandian, St., Saro/ini, R. 2, Kolandaivel, pl and Sulatmar, S ~. tDepartment of Physics, Bharathiar University, Coimbatore ~16, 2Department of Physics, Kongu Nadu Arts & Science College, Coimbatore 641 029, 3 Centre for Plant Molecular Biology, Tamil Nadu Agrieullaral University, Coirnbatore - 3, Tamil Nadu, India.

FT-Raman spectroscopy has been used to investigate interaction of DNA in the presence of the chloride salts of Zn (II) and Cd (II). Metal-DNA interactions have been observed in aqueous solutions at 25~ containing 5% by weight of high molecular weight DNA iso|ated from rice leaf by a modified standed method of Sambrook et al. The spectra are excited with the 1064 nm line of Nd :YAG laser using 200 mW of radiant power and collected on FRA 106 RAMAM MODULE equipped with an intensified Ge diode army detector. These spectra reveal that both the ions interact with the phosphate groups indirectly as evidenced by the intensity increase and lower frequency of the phosphodi0xy marker band near 1093 cm -~. The DNA undergoes significant structural changes, upon binding to both the ions; most notable of these changes is the destacking of the bases reflected by increase in intensity in the 1185 (dT), 1338 (dA, dG), 1297 (dC), and 1488 (dG, dA) cm ~ bands. Zn 0I) ions interact strongly with the N7 atom of guanine thus induces anti --) syn reorientations of the guanine nncleoside as evidenced by the presence of both 686 (C2' endo/anti) and 675 (C2' endo/syn) cm -1 marker bands. Whereas Cd (II) ions interact with the N (1) atom of guanine thus alters the hydrogen bonding states of the carbonyl groups of guanine.

P154 FT-Raman spectroscopy of the Rice Leaf DNA and complexes with AI (HI) and Pb 0I)

Saro/ini, Rl., Selvasekarapandian, S 2 Kolandaivel, 1 ~ and Sukumar, t ~ of Physim, Keagu Nada Arts & Seienoe College, Ceimlmore 641 029, 2 ~ of Physi~ BIm'alhi~ ~ , ~ --46, 3 Centre f~r Plant Molecmlai" Biology, Tamil Nadu Agrieultu~ University, Coimbatore - 641 003, Tamil ." Nada, India.

The interaction of rice leaf DNA with Al(m) and Pb(II) has been ~ in H20 soh~m ltdng FF-Ranmn ~ . The high molecula~ weight DNA from rice plant leafs are ~ bY a medified standard meeaenl of Stun" brook etal. The spectra are excited with the 1064 nm line ofNd :YAG laser tt~ing 200 mW ofradi~ Fower and collected on FRA 106 RAMAM MODULE ~ with an i~ten.~fied Ge diode anay delect~. The results _c~nfinn that in AI-DNA complex, the melal ligafien at N7 sites, of paine slrongly IXnteb the dect~o~e sm~aJre within the six- membered xing, tiros shifting the pufine markers (1336 --~ 1341 cm "t, 1489 ~ 1476 ~an "l) in the O - x t i f ~ Th~ complexatio~ with the hnidazole ~ reflects tl~ following results: ( 1 ) the C2'-endo / anti mnfonn~en is deslabaqized. (2) The intensity of the phosphtxiiest~ mad~ band (826 c~n 4) decreases and is _~n/buted to ~m in.ease in t~mfirn~n~. flexibfl~ of the DNA backbcme. (3) Howev~, the plmsplmdioxy mafl~ band near 1093 t in "l ~ no spectral ~ suggesting highly localizedvibraticm OfPO2 gro~..... Ramanbands q the _b~__ at_ 1306(.dA), 1238(d~ and 1248 can" (de) exldbit higtgg freq-ency ~ reflecting racial b l m ~ atNl o f ~ anti N3 c[eytosme ana Uxymme in the destabilized DNA- PIKI0 imm, en the ~ are found to irdemct ~ ly weakly wiih the nudeic bases. But the iule~tyinczeaseand lower frequency shill of the 1093 can "~ band indicates interaclien ofFo (fi) ions withthe charged phosphate groulm of DNA through water molecule~

P155 A FT-Raman study of the effects of Alkaline-Earth Metal Ions on Double-Stranded Rice Leaf D N A

Selvasekarapandian. S l, Saro/ini. 1~, Kolandaivel. 1 ~ and Sukumar: S 1Department of Physics, Bharathiar University, Coimbatore ~6, 2Depa~rnent of Physics, Kongu Nadu Arts & Science College, Coimbatore 641 029, 3Centre for Plant Molecular.Biology, Tamil Nadu Agricultural University, Coimbatore - 641 003, Tamil Nadu, India.

Vibrational spectra of DNA in aqueous and alkaline earth metal [Mg (II), Ca (II), Sr (II), and Ba (II)] chloride solutions are compared by FT-Raman spectroscopy. The high-molecular weight DNA are extracted from rice leaf by. a modified standard method of Sambrook et al. and excited with the 1064 nm line of Nd:YAG laser using 200 mW of radiant power. The spectral changes observed for the phosphodioxy band (1093 cm "1) reveal that all of these ions bind to the phosphate groups of DNA~ Marginal intensity changes within the envelope of the phosphodiester band (825-845 cm "]) are consistent with a net narrowing of the minor groove of B-form DNA in metal-DNA complex. The purine marker bands at 1338 and 1488 cm -~ exhibit intensity change and frequency shift in all metal-DNA complexes indicating that alkaline metal ions interact moderately with acceptor sites on the purine N7 rings. This makes the torsional rotations about the glycosyl bond more flexible, as evidenced by the occurrence of both C2'-endo/anli (686 cm t) and C3'-endo/syn (623 cm "]) dG conformers. A notable exception is an extensive perturbation by Ca (II) ions on pyrimidine (N3) rings, causing the greatest effect.

8 7


P 1 5 6 C o n v e r s i o n o f S a n g u i n a r i n e A l k a n o l a m i n e F o r m to I m i n i u m F o r m b y D N A

Anjana Sen and Suman Das Biophysical Chemistry Laboratory

Indian Institute of Chemical Biology, Calcutta 700 032, India

Sanguinarine exhibits pH dependent structural equilibrium between the iminium form (charged) and alkanolamine form (neutral) with a pK value of 7.5. The alkanolamine form of sanguinarine does not bind to DNA. In presence of high concentration of various natural and synthetic B-form duplex DNAs of high P /D value (i.e., nudeotide phosphate/drug molar ratio), this form is transformed to the imim'um form and then it binds, as evidenced from the appearance of characteristics band for the bound imint'um form in the wavelength region where the alkanolamine form has no spectrum from the measurement of spectrophotometry, spectrofluorimetry and circular dichroic study. This conversion is inhibited in presence of sodium salt concentration of 0.5 M or higher. These results suggest that the conversion of alkanolamine form to iminium form is due to the formation of an ion-pair with the N5 of alkanolamine and the polyanion phosphate backbone of DNA structure.

P 1 5 7 Repression ofe-Ki-ras promoter activity by triplex-forming oligonueleotides endogenously generated in

human 293 cells Elisa Del Terra, Cliiara Suraci, Silvia Diviacco, Franco Quadrifoglio and Luigi Xodo

Department of Medical Sciences and Technologies, School of Medicine, Universify of (/dine, 33100-1taly

Triplex-forming oligonucleotides (TFOs) may haveChe potential to manipulate in vivo the expression of individual genes. In previous studies We showed that synthetic GA and GT motif TFOs directed against a critical polypurine- polypyrimidine [poly(RY)] located in the c-Ki-ras promoter were able to inhibit in HIH 3T3 cells the expression of CAT placed under the control of the c-Ki-ras promoter: Here we addressed the question whether the TFOs can be generated directly in cells by using specific expression vectors~ Two vectors were constructed in order to generate in human 293 cells CU and GU rTFOs which are potentially capable to bind to the c-Ki-ras target, via triple-helix formation. The affinity of the rTFOs for the c-Ki-ras poly (RY) target was investigated by DNase I footprinting experiments, while their capacity to inhibit in 293 cells the expression of CAT was investigated by transient transfection experiments. Results form several independent transfections showed that the expression vactors generating the GU and CU motif rTFOs significantly inhibit the expression of CAT driven by the c-Ki-ra., promoter.

P 1 5 8 Effect of Flanking Residues on Base-pair Stacking OrientaUon: A Molecular Dynamics Study.

Debashree Bandyopadhyay and Dhananiav Bhattachatyya Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road,

Calcutta 700037, INDIA

It is generally believed that base-pair stacking interaction in DNA double helices is one of the strongest interactions that governs sequence directed structural variability. However, X-ray crystal structures of afew base-paired doublets have been seen to adopt different structures when flanked by d{fferent base-pairs. DNA crystal database however, is still too small to make good statistical inference. Influence of neighboring residue on the local helical geometry of a base-pair step in B-DNA has been investigated here using molecular dynamics simulation. We have generated ensembles of structures for'd(CA).d(TG) and d(AA).d(TT) base-pair steps located at the centers of d(CGC~GCG).d(CGCTrT_C~GCG) and d(CGCGA~AACGCG).d(CGCG~CGCG) sequences and their analogs which are varying with" the bases either in 5'- or 3'- position to the central doublet. Comparison of base paired doublet parameters for the ensemble of structures show that stacking geome~y of d(CA).d(TG) doublet depend on some of the flanking base-pairs, d(AA).d(Tr) doublet remains nearly unperturbed when the flanking residues are changed.

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P159 Effect of flexibility on denaturation of supercoiled DNA: a Monte Carlo study

Sudip Kundu and Ashoke Ranjan Thakur Department of Biophysics, Mole clflar Biology and Genetics. Umversil 3 College of Science, Universily of Calcutta, 92 APC Road. Calculla 700 009. INDIA

A Metropolis Monte Carlo algorithm has been developed to examine the thermal and alkaline detmturation profile of supercoiled DNA: The denaturation probabilities of each base pair at various degrees of supercoiling have also been calculated. Altlmugh one can qualitatively reproduce the alkaline denaturation profile, the agreement with experiment is not as good as in the case of therlnal denaturation. This disagreement lms been shown to be due to DNA fl, exibility. Results show that with change of flexibilitY parmneters, the denaturation profile changes. The cooperativity also changes. The flexibility' undergoes drastic change with the change in effective average distance between base pairs,

P 1 6 0 Theriimdynamics of the Interaction of Sanguinarine with Triple Helical DNA

Suman Das, Arghya Ray & Motflal Maiti Biophysical Chemistry Laboratory

Indian Institute of Chemical Biology, Calcutta 700 032, India

Using spectrophotometric technique we report, for the first time, the thermodynamic characterisation of sanguinarine binding to DNA triplex poly(dT).poly(dA)xpoly(dT) over a wide range of temperature. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature dependent circular dichroic measurements. Sanguinarine forms very stable complex with the DNA triplex. The binding isotherms fit reasonably well to the neighbour exclusion model. Temperature dependence of the binding constants is used to estimate the thermodynamic parameters. Negative enthalpy and positive entropy changes characterise the intermolecular interaction of sanguinarine at the interaction site of the triplex, which indicates that the process of binding is enthalpy driven. Possible molecular contributions towards sign and ma~,,nitude of the thermodynamic oarameters are discussed.

P161 Fold-back structures at distal end influence DNA slippage at proximal end during mononucleotide repeat expansions

Karthikeyan G, Kandala.V.R.Chary#& Basuthkar J Rao. Department of Biological Sciences, Department of Chemical Sciences#, Tara Institute of Fundamental Research, Homi Bhabha Road, Bombay 400005, INDIA Abstract: Polymerase slippages duringDNA synthesis by Klenow across A, C, G and T repeats (30 bases) have been studied. Duplexes that contain only repeats (30 base pairs) expand dramatically to several hundred base pairs longwithin minutes. Rate comparisons in a repeat duplex, when one strand against that when both strands are expanding, suggest a model of migrating hairpin loops which in the latter case coalesce into a duplex: Moreover slippages (at 3' or proximal end) are subject to positive and negative effects from the 5'-ends,(distal) of the same strand. Growing T and G-strands generate T.A:T and G.G:C motif foldback structures at distal ends that hamper slippages at the proximal ends. On the other hand, growing tails at the distal end upon annealing with complementary templates accentuate proximal slippages se-,eral fold.

89


P 1 6 2 HIGH RESOLUTION NMR AND COMPUTATIONAL INVESTIGATIONS ON CONFORMATIONAL PLASTICITY IN

AN ANTISENSE DNA~ HYBRID DUPLEX

Anwer Muleeb and Thomas L. James Department of Pharmaceutical Chemistry, Univ6a'sity-of California at San Francisco, San Fraaciaco, CA 94143-0446, USA

Antisense DNA technology employs oligodeoxyribonucleotides with modified phosphate backbone as potential therapeutic agents to target specific mRNA sequences for inhibiting protein synthesis and, thus, regulating gone expression. Methylphosphonate oligodeoxyribonucleotides, with methyl group modification on phosphate, are of particular interest due to their stability, enhanced rcsistanc= towards nuclease activity, and efficient permeability through cell membrane. To improve the efficacy and functionality of antisense approach, it is important to understand the structure and dynamics of antisense DNA-target RNA complexes. We have been investigating the NMR structure of a hybrid duplex containing [Rp]-methylphosphonate moieties on alternating phosphate centers on DNA strand. Quantitative analysis of high resolution NMR data revealed variations in interpmton distances and sixth-root R factors that were indicative of enhanced confonnatiunal plasticity in anfisense DNA strand [11. This observation prompted us to apply the computational methods of structtm: refmement and conformational sampling which allowed us to construct a dynamics picture of this hybrid at high resolution. Conformational sampling was achieved by applying time-averaged restrained molecular dynamics (MDtar) with particle mesh Ewald (PME) method in presence of explicit water. The generated pool of conformers was subjected to PARSE (Probability Assessment via Relaxation Rates of a Structural Ensemble) analysis that determines the probabilities of conformers which best account for NMR data. An ensemble of calculated structures with non-zero probabilities was thus ganm-ated. This ensemble embodies our NMR observations and presents an envelop of dynamic events. A detailed analysis of structural features of the ensemble will be presented. [ 1 ] Anwer Mujeeb, Made A. Reynolds, and Thomas L. lames, Biochem~try, 1997, 36, 2371-23792

P 1 6 3 DNA CONFORMATION IN COMPLEXES W I T H THE COORDINATION COMPOUNDS

N. Kasyanenko, E.E.F. Haya, A. Bogdanov, A. Zanlna Laboratory of Molecular Biophysics, Department of Physics, St.-Petersburg State UniversiO, Russia

DNA interaction with the coordination compounds of Pt(II), Pt(IV) and Co(III) in solution has been studied. It was shown that the composition of the first coordination sphere of compounds under study defines the mode of interaction. At the first step of interaction the electrostatic attraction of complex ions and DNA molecule is primarily responsible for their binding. The nature and relative positions of the first coordination sphere ligands determine the kind of binding. The coordination linkages between DNA and complex ions can be produced after the formation of aqua-ions in solution. The sterical correspondence between the elements of DNA double helix and the positions of replaced ligands in the coordination sphere gives the stability of DNA structure during the interaction. The capacity of the coordination compound for the variation in its first coordination sphere dominates in binding in contrast to the initial charge and composition of a complex ion. For example, the compound [Co(NO2)6]Na~ dissociates in solution with the formation of negative ion. However, it interacts with polyanionic DNA and produces rather strong binding. The binding of trivalent complex ions to D N A is accompanied by the packing of macromolecule. The trivalent ions tie the separate DNA segments. This conformational change initiates the appearance of mutually oriented parts of double helix. Comparison studies of DNA binding with Pt(II) and Pt(IV) compounds have been done. The compounds with two Platinum ions were used

P 1 6 4 TORSION-CONFORMATIONAL STUDY ON DNA BY PHOTOACOUSTIC SPECTROSCOPY

BUGS, M.R. and ~ (UNESP - PHYSICS DEPARTMENT - S. J. DO RIO PRETO - BRAZIL) Non radiative decayment is the primary source of the photoacoustie effect, here used to investigate the torsion- conformational changes induced on DNA, by ethidium bromide (Et-Br) intercalation and ionic strength. Monitoring singlet and triplet non radiative decayments of Et-Br free and bound to DNA, and quenching the triplet contribution using 02, we found a photoacoustic phase lag response due to unquenched and quenched samples. That is related to the triplet lifetime. Which value for Et-Br free was (1.28 ms), and when bound to DNA at different concentrations (0.2, 0.54, 0.81 mM of DNA)a t 10raM of NaBr were (2.22, 3.13, 6.99 ms) respectively. Increasing the ionic strength to 50mM we obtained to Et-Br free (3.1 lms) and bound(0.13, 0.34, 0.51 mM of DNA) were (1.66; 1.73, 2.21ms), and at 100mM of NaBr the value for Et-Br free was (5.21ms) and bound (0.15, 0.4, 0.6 mM of DNA) (5.24, 5.39, 6.29 ms). The increment on the triplet lifetime is understood as the biding of the drug and its loss of mobility, besides that the influence of the ionic strength is detected with its increasing, inducing DNA migrates from high flexibility (10mM NaBr) to lowflexibility (100re_M). At 50mM the results seem to be non linear as expected what configured a region where DNA is semi-flexible and the torsion-conformational change was detected. These results obtained through photoacoustic spectroscopy (PAS) were also confLrmed with anisotropy and variable frequenoy cross-correlation phase fluorescence, both indicated DNA unwinding under these conditions. The application of PAS investigating drug-DNA interaction is promissory even when the drug does not has emission.

9 0


P165 Tandem lesions in DNA oligomers and duplexes: Detailed analysis of their structure, conformation and energetic studies. Thamaranu Srikrishnan, Department of Molecular & Cellular Biophysics, Rosweil Park Cancer Institute, Elm & Carlton streets, Buffalo, New York 14263, USA. We undertook a systematic investigation of the structure, conformation and energetics of tandem lesions of DNA induced by reactive oxygen species (ROS). Tandem lesions are lesions in which adjacent nucleotides are both damaged. and is the consequence of a single free radical initiating event. ROS includes mainly hydroxyl radicals, hydrogen peroxide, superoxide and singlet oxygens and are produced by many environmental factors such as ionizing radiation, sunlight and smoking. There is a host of diseases, in addition to cancer, where ROS has been implicated. The most common free radicals induced DNA damage products invloving a single nucleotide are 8-hydroxypurines and 5- formamidopyrimidines. A large number of the tandem lesions have been isolated, identified and quantitated in our laboratory, both under oxic andanoxic conditions by Dr. Box and his coworkers. Some of the important lesions studied by us are two G.T lesions found in the model tetramer CGTA and one C.G lesions found in cyclic CATG. These studi.es yield very valuable information that these lesions link the C8 of the purine to the C5 of the pyfimidines. Detailed study of a whole host of these lesions are underway and attempts to crystallize some of these lesions are in progress. The major target of ROS is DNA and they produce a plethora of nucleic acid base modifications as well as DNA strand breaks. Some of the newly found lesions support the concept of electron tran.sfer between non-adjacent bases. Our results suggest that the formation of tandem lesions is structurally and energetically feasible in duplex DNA and that these lesions are likely to have significant mutational consequences in biological systems.

P166 Conformational Dynamics of Nucleic Acids in Wetted Samples

M.Ye. Toistorukov Chair of Molecular and Applied Biophysics, Kharkov State University,

Svobody Sq.4, 310077, Kharkov, Ukraine

A mathematical model of the conformational transitions of a nucleic acid (NA) molecule induced with changing its water content is presented. The model takes into account mutual dependence of water sorption and conformational transitions of a NA molecule in an explicit form. The model is suitable for describing NA with both homo- and heterogeneous primary structures. The model allows one to describe sorption and conformational hysteresis phenomena observed experimentally as a consequence of cooperative conformational transitions of NA. The numerical results are in good agreement with experimental ones. In the frame of the model the evolution of the conformational perturbation can be described. It can collapse for finite time or, growing, occupy the whole NA molecule (conformational transition). The choice of the scenario is determined by the water content of the sample. Propagation of a new conformation along flae NA molecule is a process of the travelling front type that is in agreement with Zipper model of conformational transitions. The existence of a domain wall separating regions of a nucleic acid molecule with different conformations is possible for biopolymers with heterogeneous primary structures.

P167 A theoretical study of d r u g / d y e - DNA base pair interactions

N i t i s h K. S a n y a l " and S .N . T i w a r i Department of Physics, DDU Gorakhpur University, Gorakhpur- 273 009

"Vice-Chancellor, U.P. Rajarshi Tandon Open University, Allahabad - 221 001

Most of the members of acridine and ellipticine family elicit their biological properties by intercalating in between a dinucleotide unit of DNA double helix. In this paper, stacking interactions between a large number of acridine derivatives such as proflavine, acridine orange etc., and the DNA base pairs have been extensively studied using modified Rayleigh-Schr0dinger second order perturbation theory valid at intermediate range distances. In addition to acridine, certain members of ellipticine family have also been studied. On the basis of the results obtaine~ relative stability of various stacked complexes, preferred binding sites of drugs/dyes etc., have been examined and elucidated. It has been observed that merely intercalation can not be alone held solely responsible for the biological activity of either acridines or ellipticines.

91


P 1 6 8 Stacking interactions in nucleic acid bases and base pai rs - a theoretical study

S.N. T i w a r i and Ni t i sh K. Sanyal" Department of Physics, DDU Gorakhpur University, Gorakhpur- 273 009

"Vice-Chancellor, U.P. Rajarshi Tandon Open University, Allahabad - 221 001

Stacking interactions influence the secondary structure of nucleic acids and confer stability to these structures. In view of this, a detailed and systematic analysis of stacking interactions in various nucleic acid bases and hydrogen-bonded base pairs have been carried out nsing modified second order pe~dzation theol . Net atomic charge and dipoles located at various atomic centres of bases and base-pairs have been computed .~ng CNDO/2 metho(l All possible combinations of staeki.~ have been taken into account. Results have been discussed in the fight of experimental as well as other theoretical observations. The possibility of relative lzeference of the left-handed conformation for alterrmri,~ sequences has been ~mlitatively explore(t

P 1 6 9 Theoretical Considerations of DNA Triplex Formadoa

Mihir Roy Choudhury, Devesh Kumar and Nitish K. Sanyal Del~tment of Physics, DDU Gorakhpur University, G o ~ CLI.P.) India.

An attempt has been made to estimate the probabifity of formation of triple stranded DNA for homonucleotide sequence, alternating sequence and mixed sequence. Second order penmbalion method with multicentred multipole expansion has been employed to compare the association e~ergies under different conditions of interactions. The stabilizing and destabilizing forces have been analyzed and the results have been discussed with a view to understand the condition of tri~le helix formation and their possible biological significance.

P 1 7 0 NMR Observation of C-Tetrad in a DNA Quadruplex

P. K. Patel, Ned S. Bhavesh and R. V. Hosur Department of Chemical Sciences, Tata Instititute of Fundamental Research

Homi Bhabha Road, Mumbal 400 005, India

Our understanding of DNA structural polymorphism, especially with regard to multistranded structures, has undergone rapid enrichment in recent years with the discoveries of many new st ructural motifs, such as, G-tetrad, G:C:G:C tetrad, i-motif, T:A:A triad, U- tetrad, A-Tetrad, T-Tetrad, etc., to name a few. We report here the observation of a new motif, namely, the C-tetrad, which we discovered in the sequence d-TGGCGGGT, during the course of our structural investigations by NMR on many telomerie DNA sequences. The structure of the molecule is a parallel stranded quadruplex having five G-tetrads and one C- tetrad. The G-tetrads stack well over each other. The C-tetrad causes a bulging of the quadruplex in the middle

92


P171 THE ESTIMATION OF NUCLEIC ACID'S MODEL COMPOUNDS' VANToHOFF'S

THERMODYNAMIC TRANSFER PARAMETERS.

Anna GABRIELIAN

Institute of Molecular Biology, NAS, Armenia.

To clear up the influence details of the solvent and its ingredients (Polyethylenglycol (PEG), high concentrations oi

neutral salts, various alcohols and protein-ligand) on DNA conformation and stability, the temperature dependence

of relative solubility data, for various bases, nucleosides, nucleotides and di-, three-nucleotidas have been obtained.

Corresponding thermodynamic tr~msfer functions were calculated, using Vant-Hoff's curves. The consideration of

AFtr, AH tr, AStr signs and values allowed to elucidate strong dehydrating PEG-influence on the phosphate groups,

the nature of salt anions effect, the specificity of DNA-binding membrane protein action etc. The peculiarities ot

nucleic acid components' hydration, the affect mechanism of different factors, as well as solvent-water structure

shades have been discussed.

P172 Non ergodic behaviour in conformational

transitions of single DNA molecules

Stefan Wennn~lm Lars Edman, and Rudolf Rifler. Department of Medical Biophysics, Kamlinska Institutet, Stockholm, Sweden.

Conformational fluctuations in single nucleic acid molecules were observed as fluor~cence intensity fluctuations. The sample molecule, a 217-bp DNA oligonucleofide with tetramethylrhodamine linked via a carbon-atom-linker to one end and biotin attached to the other end, was immobilised on a streptavidinised glass surface. The-sample molecule fluctuates between two conformations; TMR attached or not attached to the specific quencher guanosine. 37 molecules showing fluorescence intensity fluctuations were detected. Within the time window of observation the reaction rates differ between the molecules, but stay constant within a single molecule. In addition, the distribution of relaxation rates between the molecules correspond to the distribution seen in a bulk measurement on a similar system. We therefore conclude that within the observation time window the single DNA molecules behave in a non ergedic way.

P173 Chromatin irradiation by fast neutrons: modifications and radioprotection

B. Constantinescu. L. Radu*. I. Radulcscu* and D. G~.daru Institute of Atomic Physics. PO BOX MG-6, Bucharest. Romania

*Victor BabEs Institute. Bucharest 76201, Romania

Fast neutrons action on normal and tumoral chromatin was studied using intense fast neutron beam produced at Bucharest Cyclotron via d(l 3 MeV) + Be thick target reaction (irradiation doses between 10 and 100 Gy). Chromatin structure modifications were tested ~ using determination of absorption and emission characteristics of chromatin complexes with a specific DNA ligand, analysis of chromatin intrinsic fluorescence and measurement of Forster energy transfer efficiency between two fluorescent iigands coupled to chromatin. Complex structure modifications as DNA strand breaks with the decrease of the double helix proportion, acidic and basic proteins destruction and changes of global chromatin structure were observed. Tumoral chromatin has a greater fast neutron r than nbrmal chromatin, due to its greater euchroamtin proportion. For radioprotcction purposes, the effects of OH radicals scavengers - captopril and glutathione - and of Cs § and AI 3+ metallic ions were analyzed. They protect chromatin DNA from strand breakage by scavenging the hydroxyl radicals produced in water radiolysis. The results suggest possible interference between treatments with drugs bearing free sulfhydryl groups and radiotherapy by fast neutrons.

9 3


P 1 7 4 Imino proton exchange in DNA base-pairs catalyzed by ammonia and tr imethylamine.

Evidence for a secondary long-lived open state.

Sebastian Warml~d~. & Mikael Leijon*

Department of Biophysics, Arrhenius Laboratory, Stockholm University, S-106 91 Stockholm, Sweden.

By comparison of dsDNA imino proton exchange catalysed by ammonia and trimethylamine we find that the two catalysts sense the same open state(s) of the base-pair. At intermediate catalyst concentrations we observe a nonlinearity in the exchange times that we explain by the existence of a second opening mode with an opening rate circa ten times lower than that of the commonly observed opening. For both the original and the novel mode the catalytic efficiency of TMA seems to be reduced by roughly a factor of three compared to ammonia, taking into account the difference in the molar transfer rates from the mononucleosides for the two catalysts. We interpret this as TMA having a constant lower accessibility to the open states of the base-pairs, Comparing the dissociation constants of the two opening modes and the two catalysts we then find that the open state lifetime of the novel mode is 10-100 times longer than for the commonly observed mode. This new type of long-lived open state may be of relevance in DNA-protein recognition and interactions with ligands.

P 1 7 5 Circadian Clock-Controlled Genes in the Cyanobacter ium SynechoFystis PCC 6803.

Setsuyuki Aoki 1, Takao Kondo 2, and Masahiro Ishiura ~ ]Graduate School of Human Informatics, Nagoya University

2Graduate School of Science, Nagoya University

An increasing number of genes have been revealed to be controlled by a self-sustained biological oscillator, circadian clock. Circadian clock regulates various physiological rhythms through the expression of such clock-controlled genes (ccgs), and thereby allows organisms to adapt to daily changes of environment. We have developed an efficient method for isolating the promoters of ccgs in the cyanobacterium Synechocystis PCC 6803. In this method, we used the Vibrio harveyi luxAB gene set, which encodes bacterial luciferase proteins, as a bioluminescent reporter. We transformed the wild-type Synechocystis cells with a iuciferase expression library carrying the Synechocystis genomic DNA fragments upstream of the lu, rAB. Out of approximately 10,000 transformed clones, at least 55 exhibited circadian rhythms of bioluminescence with various waveforms in continuous light. By analyzing three clones (No. 49, 52 and 58) selected among these rhythm-expressing clones, we identified the clpP gene, the ORF slr1634 and the rpb gene as ccgs which showed different phase relationships, and localized their promoters within short DNA fragments.

P 1 7 6 Peculiarities of the hydration of pyrimidine bases of DNA upon field ionisation mass spectrometry

conditions V.A. Pashinskaya, M.V. Kosevich, V.S. Shelkovsky, Yu. P. Blagoy B. Verkin Institute for Low Temperature Physics and Engineering

of the Nalional Academy of Sciences of Ukraine, 47, Lenin avenue, Kharkov 310164, Ukraine

Interaction of nucleic acids with water solvent is one of key factors which determine their spatial structure and properties. The study of molecular processes upon partial dehydration is also very important for understanding of the role of water in DNA biosynthesis, nucleic acids-protein interactions, etc. The condilions of mass spectrometry allow to model partial dehydration conditions and to investigate complexes of DNA components with a.few water molecules. In this work we examined the effect of water on tautomeriTation of nucleic acid bases during processes, in which only occasional water molecules can approach the incorporation site. Fidd ionlsation n ~ s spectrometry experiment revealed that upon interaction between thymine (or l-mcthylthymine), cytosine (or l-methylcytosinr and heavy water in vacuum, a deuteron is transferred along the bonds, inducing tautomeriTation. The interaction energy was calculated by atom:atom potential functions method for different relative positions of the base (thymine) and water molecules and a model for the resulting complex was proposed. The base and water molecules were found to be coplanar, Formation of this complex during nucleic acid biosynthesis may induce point mutations, the probability of the latter being dependent on the structure of the DNA-enzyme-nucleotide complex.

9 4


P177 The comple te symmetry o f t h e I s e n t r o p i c E q u a t i o n s o f DNA sequences J i - h u a W a n g Depar tment o f phys i c s , Dezhou t e a c h e r ' s c o l l e g e , De Zhou 253023, Shan dong, China

The I s e n t r o p i c E q u a t i o n s have been deduced and u sed t o s t u d y m o l e c u l a r evo lu t ions (Wang &Zhang, J. t heo r . Biol . 181.1996) . The DNA group ( t h e o r d e r i s 12)has been i n t r o d u c e d t o s t u d y symmetry ofDNA sequences by Zhang(Zhang, J. t heo r . Bio l . 187.1997) . We expand t h e DNA group o f t h e o r d e r be ing 12 t o a c o m p l e t e s y m m e t r i c a l g roup o f DNA of t h e o r d e r be ing 24 ba~ed on t h e comple t e s y m m e t r i c a l g roup o f a r e g u l a r t e t r a h e d r o n . The 24 e l e m e n t s o f t h e comple t e s y m m e t r i c a l group o f DNA a r e I , R , , R T ...... Nx, Ny, Nffi, N~,N~,N~,Pffi .. . . . . . r e s p e c t i v e l y . I t i s p roved t h a t t h e I s e n t r o p i c E q u a t i o n s keep i n v a r i a n t u nd e r t h e o p e r a t i o n s o f t h e comple te s y m m e t r i c a l g roup o f DNA.

9 5

B2 : Nucleic Acids : Ribozymes and RNA Structure

P178 Self Splicing in Group-i intron of Tctrahymena rRNA and its possible inhibition when reacted with

platinum complexes

R. Malathi and K. Chandrasekhar Department of Genetics. Dr. AML PG Institute of Basic Medical Sciences. University of Madras. Taramani

Campus. Chennai-600 113, India

Mechanism of self-splicing in group-I mtron of Tetrahymena thermophilo, a ciliated protozoan was studied by setting up an in vitro splicing assay. Initially the plasmid PT-7 [gift from Prof. Thomas Cech] containing the genomic fragment of 26s ribosomal RNA was transcribed using T7 RNA polyn~rasr yielding precursor rRNA of length 566 base pairs including cxon 1, intron and cxon2. After purification the splicing reaction was carried out and the products were analyzed. In addition to catalytic activity, group-i intron ribozyme is also known to act as a target for a number of therapeutic agents. In view of this and recent findings that cis-DDP a potential anticancer agent bind to rDNA genes disrupting the rRNA synthesis, we have studied the binding of cis-DDP and its analogues with pre-rRNA. The nature of splicedproducts under varying concentration of drug and time is discussed.

P179 NMR studies on 2',5' Nucleic Acid Fragments: 3D structure of 2',5' r(GCCGCGGC) and

2',5' r(CCGGCGCCGG) Premraj, B.J.', Patel, P.K. r Kandimaila, E.R. $, Agrawal, S. s, Hosur, R.V. # and Yathindra, N." *Department of

Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025. *Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005, SHybridon Inc., Milford, MA 01757.

Nucleic acids with 2',5' links have assumed importance due to their implication in antisense application besides the intriguing question as to why Nature did not use them for biological information storage and transfer. The important steaeochemical finding that nucleotide dimensions depend on the type of sugar-phosphate linkage facilitated modeling studies of 2',5' nucleic acids on par with those with 3',5' links. It has been shown that they can form stereochemicaily feasible A, B, and Z duplexes, and also triplexes. To delineate their detailed structural features we have investigated by NMR two self-complementary oligonucleotide fragments, 2',5' r(GCCGCGGC) and 2',5' r(CCGGCGCCGG). 1D H20 exchangeable spectra reveal imino proton resonances implying Watson & Crick base pair formation. The uninterrupted sequential cross peak connectivity of H8/H6 with HI' protons in the 2D-NOESY spectra suggests a right handed helical structure and their intensities indicate that all the nucleotide bases are in the anti conformation except for the surprising syn conformation of the 5'end residue in both the oetamer and the decamer. Structure solution and refinement have been carried out and detailed stereochem'~cal features are discussed.

P180 Synthesis and characterisaflon of complex of Co01) with 6-azauracii

M Alcolea Palafox* and Chatar Singh** Department of Physics, L R College, Sahibabad-201 005, India

* Departmcnto-de Qulmica-Flaica I, Universidad Compluteuse, Madrid-28040, Spain ** Department of Physics, D A V (PG) College, Muzaffarnagar, India

The biological activity of aza analogues of uracil has been investigated intensively. 6-azauracil causes severe ii,h~ition of RNA formation and gives stable complexes with first row transition metals. The present work re1~orts the Synthesis and characterisation of complex of Co(H) with 6-azauracil.

The complex was isolated by the method described eisewberc I. IR and Raman spectra of the complex and ligand in the solid phase were recorded on Beckman !R-12 spectrophotometer in KBr pellet in the region 200-4000 cm -i and on a Spex spectrophotometer in the region 50-3500 cm -I respectively.

On the basis of various physicochemical studies it was found that the complex possesses the molecular formula [CO(6-AU).3H20] and a tetrahedral structure around the metal ion. 1 Rasto~ etal, in Spectroscopy of Biological Molecules, Kluwer Academic Publishers, Netherlands, 1995.

9 6


P181 A P S E U D O K N O T - C O M P A T I B L E UNIVERSAL SITE L O C A T E D IN THE R I B O S O M A L

PEPTIDYLTRANSFERASE CENTER

A.D. Beniaminov, S.A. Bondarenko, E.M. Zdobnov, E.E. Minyat , N.B. Ulyanov and V.I. Ivanov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov st., 117984 Moscow, Russia

Using a computer p rogram for searching the genomic ,sequences potential ly adopt ing pseudoknot structure, 419 pr imary structures o f 23S and 23S-like rRNAs from different k ingdoms (prokaryota, eukaryota. archaebacteria) as well as plast ids and mitochondria were analyzed. A universal site was found in the peptidyltransferase center (PTC) capable o f folding to a pseudoknot of 48 nucleotides in length. Phylogenetic conservation of its hel ices (concurrent replacements with no violation o f base pair ing,- covarial ion) together with other f indings conf i rmed folding o f this ear l ier unknown pseudoknot. We suggest the reversible folding- unfolding of the pseudoknot for certain stages of the ribosome functioning.

P182 Vibrational Spectra of Bin-molecules: 5-Bromo-Uracil

J.S. Singh* Depamnent of Physics, BHU, Varanasi 221 005, India

* Correspondence address - Dept. of Physics, GLA Institute of Technology and Management, Mathura 281 001, India

Infrared (IR) ~ of 5-Bmmo - Uracil was recorded at room temperture in the region 400-4000 cm-' on a Fourier Transform Infra Red (FrIR) ~trophotometer Model - 5300 in nujol. The accuracy of the measuremenl was within ~:3 cm and resolution was better than 2 cm "t. The IR speclrum shows complex structures in regions 600-900, 1300-1900 and 2500-3400 cm "~. The nucleic acid base derivative, 5-bmmo-uracil has 30 normal or fundamental modes of vibration, assuming the pyrimidine ring as a planar structure, are distributed as 21a'+9a". Hence, it belongs to the Cs point symmetry group. The complex IR spectrum of 5-Bromo-Uracil, due to the C=O and N-H/C-H stretching groups are presented in regions 1600-1800 and 3000-3300 cm "~. The two C=O stretching are observed in the region 1600-1800 cm -~ at 1665 and 1700 cm m. The two N-H stretching and one C-H stretching frequencies are distinctly separated and observed at 3170, 3140 and 3050 cm l. And also, the one C-Br stretching mode due to Bromo group is observ~ at 1330 cm ~ due to heavy substituent at H atom. The nucleic acid base derivative has a pyrimidine, is similar to the phehyl ring. However, the some difficulties have to assign the ring breathing and Kekule stretching modes due to complexity owing to the mixing of the ring stretching (C-N, C=N, C-C and C=-C bands of the ring) with the other modes. The breathing and Kekule modes appear at 735 and 1225 cm" in the present case which are lower in magnitudes compared to their counterparts of the phenyi rings.

P183 Conformation of anitcodon adjacent hypermodified nucleoside N6-hydroxy isopentenyl adenosine, io6A

Kailas D. Sonawane ~, Henri Grosjean 2 and Ravindra Tewari j ' Physical Chemistry Division, National Chemical Laboratory, Ptme 411 008, India

2 CNRS Laboratoire d'Enzymologie et Biochemie Structurales, F91198 Gff-sur-Yvette. France

The preferred conformation of the hydmxyisopentenyl substiment, in the hypermodified nucleoside io6A which occurs 3'-adjacent to anficodon in specific tRNAs of aerobic bacteria, and other accessible conformations have been studied using quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. The hydroxyisopentenyl substituent in model anticodon segment Me-p-io6A-p-Me orients away from the five membered ring imidazole moiety of adenine "distal orientation', irrespective of the pmtonation status or the site of protonation in the adenine ring. Thus essentially the preferred conformation of the substituent in the free modified base is retained in the anticodon segment as well. The preferred conformation about the glycosyl bond is antL The preferred orientation of the hydroxyl group in the substituent depends upon the cis or trans isomeric form of the hydroxyisopentenyl modifiecJ adenine. For the trans isomer the hydroxyl oxygen is placed folding towards the lsopentenyl double bond, whereas m case of the cts isomer ~t Is placed extending away instead. A weak intramolecular hydrogen bond is indicated between the N(l) of adenine and the hydroxyl group for trans isomer, Interesting similarities and subtle differences in the preferred orientation of the N6 substituent are noticed when 2-methylth/olation is further introduced as in model anticodon Me-p-mS:io6A-p-Me segment.

9 7


P184 Confoma/ion of Hypemodifk.d Nuclenside Threonylcarbamoyl Adenosine t ( A in Model Anticodon Loop

~gments Uddhavesh B. Sonavane ~, Annie Morin 2, Henri Grosjean 2 al~ Ravindra Tewari ~ Physical Chemislry Division, National Chemical Laboratory, Pone 411 008, India

2 CNRS Laboratoire d'Enzymologie et Biochimie Structurales, F91198, Gif-sur-Yvette, France

The preferred ori~tation of the threonylcarbamoyl substituent, in the hypermodified nucleoside t 6 A which occurs Y-adjacent to anticodon in specific tRNAs from all life forms, and other alternative accessible stable conform afious have been studied using quantum chemical.Perturbative Configuration Interaction with Localised O1bitals (I)CILO) method. The threonylcarbamoyl substituent in model anticodon segment Me-p-t6A-p=Me preferen~lly spreads away from the five membered ring imidazole moiety of adenine "distal orientation', except on N(7) protonation when 'proximal' orientation (towards imidazole moiety) is preferred. The preferred orientation about the glycosyl bond is z=ll2 ~ for onprotonated nucleoside, but is Z--322 ~ for N(3) protonated nucleoside, yet Z=292 ~ is preferred for N(7) protonated t6A. The preferred orientation of the threonylcarbamoyl substituent in the unprotonated model segment is similar to the crystal structure conformation of tSA nucleoside. While the N(3) protonation does not have a major influence on the threonylcatbamoyl orientation, although it alters the glycosyl orientation substantially, but the N(7) pmtonation drastically changes the threonylcarbamoyl orientation.

P185 ASSEMBLY OF AN EXCEPTIONALLY STABLE KNA TERTIARY INTERFACE

Elizabeth A. DoherW and Jennifer A. Doudna, Yale University, New Haven, CT, 06520, USA

Like proteins, RNA molecules readily fold into specific structures adapted for ligand binding and catalysis. Large catalytic RNAs such as group I and group II introns and RNase P have compact interiors that involve close association of several segments of secondary structure. However, at present only four classes of complex RNA folds are known in atomic detail and little is currently understood about how tertiary contacts, charge screening and other factors stabilize the close packing of RNA helices. The X-ray crystal structure of the P4-P6 domain from the Tetrahymena thermophila group I intron [J.H. Cate et al. Science 273 p. 1625 (1996)] provided the first detailed view of an extended helical interface in RNA. The U-shaped'domain is characterized by parallel association of two sets of coaxiaUy-stacked helical segments. We have examined the formation and stability of this tertiary interface by providing one of the helical segments, called P5abc, in trans to a Tetrahymena ribozyme construct that contains the other, E ~162 Equilibrium gel shift experiments show that the P5abc and E ~ 5 ~ RNAs assemble into an exceptionally stable complex, with a / ~ o f - 100 pM at l0 mM MgCl2 (at 37"C). Using this two-component system we are now quantitatively examining the role of individual tertiary contacts that stabilize the folded intron.

9 8

B3 : Nucleic Acids : Nucleic Acid - Protein Interactions

P186 Solution Structure of the ~.Cro-Operator Complex and the V55C Mutant: Origin of the Operator

Recognition Specificity H. Tochio 1, S. Sato, H. Matsuo, M. Shirakawa 2 and X,_K.v_og.oJ~

tInstitute for Protein Research, Osaka University, Suita, Osaka 565-0871, 2Nara Institute of Science and Technology, Ikoma, Nara 630-0101, 3Fukui Institute of Technology, Fukui 910-8505, Japan

The solution structure of the ~.Cro-operator DNA complex was solved by using heteronuclear multidimensional NMR and the simulated annealing calculation. As the operator DNA, a symmetrical 17mer DNA with the consensus sequences of the right operators (OR) and G at the center was employed. From the intermolecular NOE between Cro and the operator DNA, the key bases for interaction were identified. By the comparison of the base sequences of OR s, OR 2 and O R 3 with the position of the key bases at each operator, the order of affinity of Cro to each operator was well explained.

When valine at the position 55 was replaced by cysteine, Cro spontaneously formed a dimer with a inter- subunit S-S bond. The structure of the dimer was solved and compared with that of the wild Cro. The molecular dynamics was also analysed. But there is no difference between the wild and mutant Cro. However the mutant Cro lost the affinity to operator. It is explained by the stiffness of the molecule which inhibits induced fitting to the operator.

P187 A Consensus View of the Energetics of Protein-DNA Recognition

B. Jayaram, Surjit B. Dixit, Piyush Shukla, Parul Kalra and Achintya Das Deparm~ent of Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi-1 ! 0016, India

and Kevin McConnell and David L. Beveridge,

Department of Chemistry, Wesleyan University, Middletown, CT-06459, USA. Protein-nucleic acid interactions are a key feature in numerous biological processes associated with the regulation of gene

expression. Despite the accumulating structural information, a molecular level understanding of the thermodynamics of specificity in these systems is sparse and sketchy. Employing some state of the art energy expressions for atomic level interactions combined with a novel technique for incorporating solvent and salt effects, we have investigated the binding energetics of about 40 different protein-DNA complexes representing a variety of structural motifs and functions. We present the first comprehensive semi-quantitative view of the flee energy components of protein-DNA complexation, identifying the major contributors and highlighting the consensus'elements. Packing and hydrophobic effects are found to be favorable to complexation whereas the electrostatics, when initial and final states are considered, show a net unfavorable contribution to binding. Our results dramatically illustrate the necessity to consider the diverse competing effects in constructing a structure- based interpretation of binding free energies in protein-DNA complexes.

P188 Dis tsm~ in a TyrR Repressor-DNA Complex

W.H. SaWyer, R.Y.S. Chart, LF. Eccelston*, YulingYan**, and B.E. Davidson. Dept. of Biochemistry & Molecular Biology, University of Melbourne, Parkville, Australia 3052, *Dept. of Physical Biochemistry, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK, and the

**Dept. of Cell Biology, Max Planck Institute for BiochemiStry, 82152 Martinsried, Germany.

TyrR i(a regulatory protein from Escherichia coli that controls the expression of 8 unlinked genes that code for proteins responsible for the synthesis and transport Of aromatic amino acids. We report the use of fluorescence resonance energy transfer (FRET) to measure the distance between the ATP site on the central domain of TyrR and four separately labelled sites on the DNA. The donor consists of a 30-mer oligonucleotide labelled with fluorescein at specific amino uridine residues in the sequence. The acceptor is rhodamine-ATP bound to the specific ATP site on TyrR. Assuming that monomers assemble in a head-to-bead fashion and that the monomers are roughly spherical in shape, transfer efficiencies were calculated for four separate donor points on the DNA to all points on the TyrR monomer surface. Three-coordinate geometry was used to define the points on the TyrR surface and the distances between donor and acceptor. A'comparison of the predicted efficiencies in the resulting database with the experimental transfer efficiencies for each of the four donor-acceptor pairs allowed the position of the ATP site on the TyrR protein to be precisely defined. We conclude that the central domain of TyrR is located distally from the DNA binding site and that the nearest distance of the ATP site to the DNA is approximately 48-50 A.

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P 1 8 9 Peptide Nucleic Acid 0PNA) influences the ReeA filament formation on double-stranded DNA template: Arehva Ra~ t, Elmer Tuite 1, Bengt. Norden ], Peter Nielsen 2, Masayuki Takahashi 3

IDept. of Physical Chemistry, Chalmets University of Tedmoiogy, S-412 96, G6teborg, 2Center for Biomoleculat RecogMtion, Dept. of Medical Biochemistry & Genetics, Bio(:hemitry Lab B, The Penum Instit, ne, Blegdamsvej 3c, DK-2200, Copenhagen N, 3Group d'Etude Mutag me se, UMR216, CNRS and lnstitut Curie, F-91405, Orsay, France

RecA protein can bind to double-stranded DNA or double-stranded DNA with a single-stranded gap to form helical nufleoprotein filaments. Any molecule that can selectively enhance the binding of RecA to DNA is of immense biologic;il importance. Peptide nucleic acid (PNA) is a DNA mimic in which the entire sugar phosphate backbone of DNA is substituted by a chain of peptide like N-(2-aminocthyl)glycine units. We have investigated the possibility of ntilising PNA to enhance the binding of RecA to DNA from circular dichroism, linear dichroism and gel electrophoresis ~audies. The cationic pseudoisocytosine-containing linked PNA (bis-PNA) used for this study binds to the fully matched., target in both circular and linear double-stranded DNA resulting in a stable bisPNA-DNA invasion complex and a looped out short stretch of single-strand. The kinetics of RecA filament formation is strongly influenced by the presence of bis-PNA. Also bis-PNA renders a better protection against enzymatic cleavage. The type of complex formed by bis-PNA, its use as an enhancer for RecA mediated biological functions and corresponding development for 'future biotechnological tools' will be discussed.

P 1 9 0 DNA local parameter variation in free and bound oligomer crystal structures and correlation with groove dimensions. Anirban Ghosh & Manju Bansal, Molecular Biophysics Unit, hldian Institute of Science, Bangalore-560012, India.

Analysis of 35 free oligomer and 20 protein bound complex crystal structures show that due to relative unwinding in GC, TA and GA dinucleotide steps the mean twist changes from 35.7 ~ to 34.1 ~ in going from free to bound state. Also the mean roll is 2.8 ~ as against 1.5 ~ in free oligomer dataset, while the mean slide of 0.33A in free oligomer dataset shifts to -o.12A in bound dataset. The minor and major groove widths are l l .4Aand 17.2A for B-DNA oligomer dataset and the corresponding dimensions in bound state are 12.3A and 17.1)L The minor and major groove depths are 8.2A and 8.8A in free oligomer while the coffesponding depth values are 7.7A and 10.2A in complex dataset. The minor groove width and depth has changed marginally while the major groove depth has deepened by 1.4A without any noticeable change in its width. The change in minor groove width has been affected through roll and the major groove depth has been deepened through twist and slide movement in the bound Oligomer dataset. Thus slide, roll and twist in the DNA sructure undergo a transition from the naked B-DNA fragments, thus adjusting the major groove dimensions to enable the o~-helix to bind tightly onto the DNA surface. Our recent analysis shows that optimal interaction between exocyclic groups across groove edges in each dinucleotide step, are responsible for the observed sequence dependent variability in the B-DNA crystal structures.

P191 Specif ic Interactions of the Homing Endonuclease PI-Scel with its DNA Substrate

Frauke Christ, Hubert Thole, Wolfgang Wende, Alfred Pingoud and Vera Pingoud Justus-Liebig-Universitaet Giessen~ lnstitut f. Biochemie, Heinrich-Buff-Ring 58, 33392 Giessen~ Germany

The PI-Scel nuclease from Saccharomyces cerevisiae is a protein-intron encoded highly specific endonuclease (1 cleavage/107 bp) which requires Mg 2§ ions as a cofactor for DNA cleavage and yields a staggered break with 3"overhangs of four bases. It belongs to the group of homing endonucleases which can mediate transfer of their own coding sequence into alleles that lacks it. The PI-Scel crystal structure which was recently solved indicates that this enzyme has a bipartite structure. In order to obtain structural information regarding the specific PI-SceI-DNA complex and to identify amino acids responsible for binding and cleavage, sequence-specific photoaffinity labelled DNA substrates were synthesized containing either iododeoxyuridines (base contacts) or single sulfur substitutions in the phosphate backbone with chemically coupled azidophenacylbromide (phosphate backbone contacts). The photoreactive DNA molecules form specific complexes with PI-Scei and,.following irradiation, specific crosslinks to amino acid residues of the PI-Scel are produced. Isolation of the covalent complexes, followed by proteolytic digestion and Edman- degradation of the HPLC purified crosslinked peptides, amino acids which are involved in DNA-binding and cleavage have been detected. Based on our crosslinking data as well as mutational and biochemical studies we propose a new docking model for the interaction between PI-Scel and its recognition site.

100


P192 Studies on the Interaction of Carbazole derivative with D N A

Pratibha Mehta Luthra and Ramesh Chandra Dr. B. 1L Ambedkar Center for Biomedical Research University of Delhi Delhi - 110 007.

DNA topoisomerases are nuclear enzymes that catalyze the concerted breaking and rejoining of DNA strand.q, thereby controlling the topological states of DNA. Topoisomerase I catalyzes the passage of a DNA strand through a transient single-strand break, while topoisomerase II catalyzes the passage of DNA double strands through a transient double strand break. Enkaryotic topoisomerases is the target for the antiturnor plant alkaloid camptothecin and its synthetic derivatives. Thus, the identification of new drugs which induce cleavable complex formation with toposiomerase I seems to be a promising approach to find clinically effective antitumor agents. It is known that synthetic derivatives of indolocarbazole antibiotic K 252a are potent inducers of the DNA cleavage complex with topoisomerases. We report the synthesis o f DNA affinity binding carbazole.derivative that do not resemble the classical paradigm for minor-groove interactions at AT sequences in DNA. Its interaction with bacterial and mammalian D N A has been studied.

P193 Interaction of an anlitenninator RNA with elongating RNA polymerase

Ranjan Sen, Rodney King and Robert Weisberg Laboratory of Molecular Genetics, NICHD/NIH, Ma~land, USA

The transcription antitermination in iamdoid phage HK202 requires two transcribed sites, putL and putR, one located in each of the early operons. These sites are predicted to form two stem-loop structures, which has been confirmed by RNA footprinting studies. Genetic studies indicated that this stem-loop structures interacts with elongating RNAP, consequence of which enables the enzyme to by pass the downstream terminators. In order to understand the mechanism of antitermination by put RNA, here we intend to demonstrate in vitro the direct interaction of put RNA with the elongating RNAP in cis and also the mode of this interaction. We have probed this putative interaction by antisense DNA oligos complementary to different regions of putL RNA. Oligos complementary to the ascending and descending part of the stem I and stem II, respectively, inhibited the antitermination, when they were added before the put sequence was transcribed. In contrary to this, once the put RNA is made and the interaction with RNAP had been established, oligos complementary to stem II only inhibits the antitermination. We also demonstrated that the stem II of put RNA in a road- blocked ternary complex downstream of the put sequence was inaccessible to RNAseH cleavage. Furthermore, we found that once put RNA is made, put mediated antitermination could be still obtained, even if the put RNA beating 5'-half of the nascent RNA chain is detached from its 3'-end. These results suggest: a) put sequence is functional as RNA, b) interaction is persistent, and c) final form ofput-RNAP interaction may involve only stem II of the put RNA, whereas the role of stem I is to either fdld the put RNA into its matured form or to alter the conformation of RNAP to interact specifically with stem II.

P194 Picosecond fluorescence dynamics of the HIV-1 Rev-RRE complex

Olaf F.A. Larsen -t, Jos Berends 2, Hans A. Heus 2, Cornetis W.- Hilbers 2, Ivo H.M. van Stokkum I , Bas Gobets 1, Rienk van Grondelle 1, Herbert van Amerongen 1

1: Department of Biophysics, Division of Physics and Astronomy, Free University Amsterdam, The Netherlands 2: NSR Center, Department of Biophysical Chemistry, Universtity of Nijmegen, The Netherlands

in this study the dynamics of protein-RNA interactions are investigated. The attention is focussed on the HIV-I Rev (protein) - RRE (RNA) complex, which is involved in the posttranscriptionai regulation of protein synthesis. Using a streak-camera system, polarized time-resolved fluorescence spectra were measured with a resolution of about 3 picoseconds. In our experiments, a small model system comprising the high affinity binding site of the RRE and the Rev protein was investigated. The tryptophan residue of the peptide was used a spectroscopic probe. The measurements were also performed on preparations where specific nucleotides of the RRE were replaced by the fluorescent nucleotide-analogue 2-aminopurine. In this way, the dynamics of different parts of the complex could be revealed.

101


P 1 9 5 Col)per and Zin c Porphyrins and DNA Binding HE. Sngrvan. Yu. S. Babayan. N.V. Khudaverdian

Cimir of Molecular Physics..verevan State Uifiversi .ty. A. Manoogian St. 1. Yerevan 375049. Armenia

We investigated tile interaction of calf thynms DNA with a group of copper an Zinc containing melallop,'Lrphyrins derived from the freebase porphyrin meso- tetra (4-N-oxyethylpyridyl) porphine tetrachloride, by means of absorptton spcclroscopy and c~rcular dichroism. II is showtL thal even at low concentrations of porphyrins. abscrved the inlcrcalalive external, or groovc, binding modes.

P 1 9 6 Target Recognition by Transcription Factors: Developments of Prediction Methods and Database

H. Kono, M. Gromiha, F. Pichierri, M. Aida', P. Prabakaran, K. Sayano 2, J. An, H. Uedairaand A. Sarai (The institute of Physical and Chemical Researeh(RIKEN), IHiroshima Univ., zAIST)

Genome project has disclosed complete genome sequences of several organisms. This has opened a possibility to study biological functions at genome level. Regulation ofgene expression is one of the most important biological functions, and to understand its mechanism we need to identify tens of thousands of transcription factors, their target genes and their interactions. The researchat this level will require the integration of huge amount of data and new tools foranalyzing the process by computer as well as experimental analysis. We have been developing methods for predicting targets of transcription factor, and database system to integrate various data on transcription factors. Here, we describe two methods for the prediction: structure-based method[1 ]; and ab-initio method[2]. The first method analyzes structural data ofprotein-DNA complexes and derive empirical contact potentials between bases and amino acid and use those potentials for predicting target sequences by combinatorial 'threading procedure. The second' method uses conformation sampling of amino acids around base pairs to derive base-amino acid interaction potentials. We also describe the "Protein-Nucleic Acid Recognition Database", which is a collection of structural data, information ofspecificinteractions and thermodynamic data on protein-nucleic acid interactions. References: [l] Kono and Sarai, Proteins 35, 114 (1999). [2] Pichierri et al. E. Am. Chem. Soc. in press (1999).

P 1 9 7 Energetic and kinetic aspects of DNA base-flipping by methyltransferases.

Saulius Serva I, Egle Merkiene I, Giedrius Vilkaitis I, Elmar Weinhold 2 & Saulius Klimagauskas 1 Institute ofBiotechnology, Viinius, Lithuania ~

Max-Planck-institut fdr molekulare Physiologie, Dortmund, Germany 2

Rotation of a nucleotide out of the DNA helix (base flipping) has been" first observed for the Hhal methyltransferase followed by numerous other DNA'modification and repair enzymes. To gain further insight into the mechanism of base flipping by site-specific enzymes, interactions of M.HhaI with a series of DNA substrates, in which 2-aminopurine replaces certain bases in the recognition site G_C.GC, were studied using stopped-flow, rapid-quench,and equilibrium fluorescence titration techniques. We find that the methylase-induced flipping of the target nucleotide can be as fast as 1000 s ~. Structural variations in the M.HhaI-DNA complex show, that the equilibrium befween the extra/intra-helical states of the target base is dependant on the strength of the target base- pair and the specific interaction of the partner base vcith the enzyme, whereas the position of the flipped-out base in the catalytic center is fine-tuned by the conserved Thr-250 in the protein. Similar experiments employing fluorescence of a uniqure tryptophan residue permitted to monitor the enzyme-cofactor interactions under non- catalytic and single-turnover conditions. In aggregate, kinetic parameters for the individual steps of DNA binding, co factor binding, target base flipping, catalytic methyl transfer and the release of methylated DNA have been elucidated which suggest a detailed mechanism for base flipping and catalysis by the tihaI methyltransferase.

102


P 1 9 8 A Nuclear Protein That Recognises the Single Stranded Telomeric Dna Motif D(CCCTAA).

Eleonora Marsich. Anlonella Bandiera. Luigl "Xodo and .Giorglo Manzini

Department of Biochemistry'. Biophysics and Macrolnolecular Chemistry. Uluversitv of Trieste. Via L. Giorgieri 1.1-34127 Trieste. Italy

Through Electr6phorclic Mobilitx Shift Assays and UV-crosslinkmg experilnents nuclear proteins capable of specifically binding the smgle-stral~ded telomenc DNA motif d(CCCTAA),, have been found in nuclear extracts from several vertebrate sources, from tissue as rat and pig liver, and chicken er}.1hro~1es, as well as from cultured cells as HeLa. FIL6(). and CHO. In particular the attention has been focused on a component with high speclficil3.' toward tins repeated molif, being not displaced by excesses of up to 1000 times of ss DNA of tmrelated sequences Its molecular v~eighl has been eslmmted through GPC to be about 40 kDa. The protein. ~vhose amount hag been eslmmted to be ~veli belmv one thousandth of the non-histonic nuclear protein componcl~I, has been recently isolated through affinity chromatography, using an avidinated resin fimctionalised ~vith the biolmvlalcd oligomer d(CCCTAA),, and the sequence of the N-terminal peptide has bcen obtained Work is m progress It filrther characterise this protein.

P199 The determination of speed constants of integration and disintegration of etidium bromide with DNA

from analysis of correlation functions

G. Potikyan. V. Arakeqyan. Yu. Babayan Molecular Physics Dept.. Yerevan State University, Yerevan 375049, Armenia-

Theoreticalh calculated that correlation functions and spectral density, of the number of molecules of etidium bromide joined ,~'ith DNA with small completions.

It is shown that in calculating of dependence of correlation function from concentration of etidium bromide in solution, it is possible to derive the additional information related to joining of etidiurn bromide with DNA. particularly kinetic constants of speed integration and disintegration of complex of etidium bromide with DNA. The analysis of spectral density allows distinguishing "'fast" and "slow" adsorption of ligands of macromolecule.

P200 THE BIOLOGICAL IMPLICATIONS OF DAMAGE TO DNA INCORPORATING AN 8-OXODEOXYGUANINE Alex Ninaber and Julia M. Goodfellow, Department of Crystallography, Birkbeck College, London

8-oxo-dGuanine is one of the major lesions due to the effects of oxygen radicals, whether from radiation or chemical damage, and has been implicated in cancer, aging and mutagenesis. The recent elucidation of the MutY repair protein structure has marked a major step forwards in our knowledge about this lesion, and we have now come to a point where theoretical approaches like Molecular Dynamics (MD) can make a valuable contribution. We have developed the force-field parameters of 8-oxo-dG for inclusion into standard MD package and performed simulations of the modified base incorporated into duplex DNA. Of primary interest is the base pair readout mechanism by repair proteins. For this, a precise description of the local structure and dynamics is needed. So far, we have found that 8-oxo-dG can be stabilized within a normal sequence and that the conformation of this base relative to the sugar residue depends on many local interactions not accessible to the isolated nucleoside. For the investigation of the dynamics, we have used the essential dynamics technique and have shown that there are only subtle changes on inclusion of 8-oxo dG. A possible factor in repair protein recognition is the structure of the first solvent layer on the DNA surface. We have developed an analysis tool using a modified form of the graph-set theory that enables us to compare hydrogen bond patterns on the surface of biological structures in a statistical manor, and have found significant differences.

103


P201 Evolutionary molecular engineering for E.cofi promoter

Yoichiro Ito, Shigeru Ohta and Yuzuru, Husimi Department of functional materials science, Saitama University, Urawa, 338-8750, Japan

We took lac promoter as a starting sequence for optimization of promoter activity and for exploring its fitness landscape on the base sequence space. We used GFP as a reporter and a fluorescent-activated cell sorter as a cloning/screening machine. GFP used was a novel mutant we discovered, which had the brightest fluorescence excited at 488nm among mutants reported so far. Selection was done for a random cassette mutant pool with diversity of'about 106. Plasmids with 100, 15, and 5 copies were used in order to avoid cell-killing by over expression of GFP. As a results of two rounds of adaptive walk performed by the mutation-selection cycle, we got mutants having promoter activity about 20-fold stronger than the lac promoter. We also preliminary indicated that the effect of advantageous mutations is roughly additive.

P202 Electron microscopic imaging of E. coil D N A transcription

J. Usukur{l !, H. Tagami 2, H. Aiba 2 : School of Medicine I and Science 2, Nagoya University, Nagoya, Japan 466-8550

Closed (non-specific; control), open and elongation complexes of RNAP and DNA in E. coli transcription system are analysed morphologically by stereoscopic imaging using improved low angle rotary shadowing. The rotary shadowed RNAP consisted clearly of two large blobs (presumed to be ~,13' subunits) stacked on two small blobs (presumed to be 20c subunits), forming a channel between them, 3 nm in width. Total appearance was similar to the RNAP model proposed by Polyakov et al.. DNA strand was passed through this large channel in both nonspecific and specific binding with the promoter region. RNAPs bound nonspecificcally faced each other at different angle. This suggest that transcription is carried out with rotation. In spite of dissociation of nonspecific bound RNAPs when applying heparin, specific binding (open complex) was remained there while kinking DNA strongly in the 3' flank. We succeeded in the direct visualization of mRNA elongating from template DNA. Newly synthesizing mRNA seemed to be coming out through the same large channel between 13, J]'and 20~ subunits.

P203 XS9 (=PRPI 9) gene of Saeeharomyces cerevisiae is involved in general stress response Mougli Suarez and Ella Nones Facultad de Medicina. Dpt. Biofisica. Univ. Republica. Montevideo. URUGUAY. ([email protected]) Stress response mechanisms aim to protect cells against potentially lethal effects of physical or chemical environmental perturbations, to repair molecular damage and to acquire tolerance against new exposures. Previously, it was shown that XS9 gone product is involved in mutagonesis, recombination and spomlation. Furthermore XS9 gene is allelic to PRPIg, involved in intron processing. Present study aims to further characterize XS9 gone function. Wild type yeast strain SC6 and its corresponding isogonic mutant strain xsg/x~9 were exposed to different stress treatments. The xsg/xs9 strain exhibited increased sensitivity to the following stress conditions: heat shock (38*(2, 0-20 hs), UV irradiation (0-120J/mZ); starvation (incubation in distilled water or in sporulation medium, 0-4 days); osmotic stress (0.7-2M sorbitol, 0-4 hs) and oxidative stress (l-4mM HzO2, 0-4 hs). Transcript analysis (Northern blot) showed induction of the XS9 gene during sporulation conditions, xs9 mutant strain, as is the case for ubi and ubc mutants, exhibits stress sensitivity and DNA repair deficiencies. These re.sults indicate that the product of the XS9 gone could be involved in a stress-activated signalling pathway (probably HOG and/or Ras- cAMP). It may act in a common step previously to the interaction of transcriptional activators with the STRE, HSE and ARE promoter sequences. We also propose that the 9~9 gone product could be involved in DNA postreplication repair, specifically interacting with ubiquitin and ubiquitin-conjugating enzymes. In both cases, the suggested functions may be accomplished directly or indirectly, i.e. through intron processing. We ate indebted to PEDECIBA.

104


P204 DIFFERENTIAL DNA REPAIR INDUCED BY HEAT SHOCK AND BLEOMYCIN IN YEAST CELL POPULATIONS Deborah Keszenman, E. Carmen Can&eva and Elia Nunes Dpt. Biofisica. Faeultad de Medieina. Montevideo Uruguay. [email protected]

To study some mechanisms underlying the stress responses we investigated the effect of heat shock (I-IS) on the induction of DNA double strand breaks (dsb) as well as on potentially lethal and mutagenie events induced by the radiomimetie antibiotic bleomycin in Saccharorayces cerevisiae. Haploid wild-type yeast cells in the logarithmic phase of growth were exposed to different concentrations of bleomyr (0 - 30 Ixg/ml, 1.5h) without and with a previous HS (38~ lh). Immediately after treatments, survival and mutation frequency determinations as well as quantitative analysis of chromosomal DNA by laser densitometry were performed. Our results indicate that HS induces resistance to potentially lethal and mutagentc effects of bleomycin. Quantitative analysis of chromosomal DNA performed im mediately after trealnaents showed the same DNA fragmentation, either upon bleomycin or bleomycin proceeded by HS. Incubation in nutrient medium during 4 hours alter treatments resulted in differential efficiency of dsb repair. The number of dsb is significantly lower upon HS pretreatment. These results indicate that the observed heat shock-induced resistance to bleomycin depends on a regulatory network acting after DNA-induced damage, which includes genes involved in DNA repair, HS response and DNA metabolism. We are indebted to PEDECIBA for support.

P205 Fluorescence correlation spectroscopy with global analysis of

multiple components with different quantum yields of fluorescence. Per Thvberg. Zeno FOldes-Papp, Rudolf Rigler

Karolinska Institutet, Medical Biophysics, MBB, 171 77 Stockholm, SWEDEN

Fluorescence Correlation Spectroscopy (FCS) can be used to study the fast degradation of DNA by an exonuclease enzyme. Three fluorescent components are involved: Full length DNA, free nucleotides and free dye molecules. With Cy5 as a 5'-fluorescent marker all three components had approximately the same fluoreseen~ quantum yield (photo counts per molecule and second). This system is compared with the study focused on how the data should be analysed when the components have different relative quantum yields of fluorescence. Traditional analysis of separate measurements is impossible because the quantum yield and the fraction of each component are too correlated to be determined at the same time without ambiguities. We showed, however, that both variables can be determined simultaneously if common parameter analysis of several measurements together, so called global analysis, is applied.

P206 How does the Eo coli MutS modulate its binding to mismatched DNA?

Amita Joshi and Basuthkar J Rao Dept. of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road. Colaba, Bombay-5

The E. coil mismatch repair protein MutS is likely to be a universal prototype of any mismatch specific protein from eukaryotes. Current models of the postreplication mismatch repair pathway suggest that MutS binding undergoes a cyclic transformation from a specific lodging onto a mismatched duplex to dislodging from the same, in favour of a homologous duplex. ATP is required in this pathway. We have addressed the role of ATP/ADP binding and ATP hydrolysis in modulating this reversal of roles that MutS undergoes. We find that both binding of ATP and its hydrolysis causes a dislodging of MutS from heteroduplexes. In contrast. ADP brings about a stimulation of binding to a mismatch. MutS shows increased ATP hydrolysis in presence of mismatch which has a relevance in its m vivo function. Quantitative comparison of the relative binding efficiencies of MutS to twelve different mismatches, an analogues of base-pairs suggest that the binding is mediated by a combination of DNA distortion and base-mismatch chemistry.

105


P 2 0 7 High Mobility group chromosomal proteins : Expression in skin carcinoma in Rats

Dinesh Singh and M.1LRajeswari Department Of Biochemistry, All India Institute Of Medical Sciences, New Delhi 110029 (INDIA)

High mobility group of proteins (HMG) are a class of low molecular weight non-histone, chromosomal proteins that bind to DNA and function as transcription cofactors. The HMG I group is known to have an important role in tumorogenesis. We have undertaken the present study to see the level of expression of HMGs and HMG I in particular in skin carcinoma in rats. Skin cancer was induced by carcinogens in swiss albino mice. The isolation of HMGs were also done in melanoma cell line. The results are discussed in comparison with the dose dependency etc..

106

Cl : Membranes : Structure, Dynamics and Functions

P 2 0 8 Membrane Dynamics and Heterogeneity Probed by Time-Resolved Fluorescence

Ira and G. Krishnamoorthy Department of Chemical Sciences, Tata Institute of Fundamental Research,

Homi Bhabha Road, Mumbai 400 005, INDIA Membranes exert their control on various functions of a riving cell through (i) their fluidity, and

permeability characteristics and (ii) spatial and temporal heterogeneity, which includes bilayer asymmetry and microdomain formation. In our laboratory, we have been using a large number of fluorescence probes such as Nile red, Fluorescein-PE, Rhodamme-DHPE, RH421, etc., for probing membranes. We use the technique of Time-Resolved Fluorescence Microscopy for achieving both temporal and spatial resolution of information. Fluorescence decay kinetics were analyzed by Maximum Entropy. Method (MEM) resulting in distribution of fluorescence lifetime of the probe in membranes. We have characterized a large number of fluorescence probes for reporting various specific aspects, of membrane characteristics. Our systems cover a wide range such as supported planar bilayers, vesicle membranes and single living cells in culture. Comparison of the dynamics and heterogeneity of supported membranes with those of vesicle membranes has brought out the more heterogeneous and less dynamic nature of supported membranes. In vesicle membranes, proton permeability was correlated with their polarity and water content. In a single living cell we could map out the cholesterol content and fluidity of various cellular membranes during various stages of cell cycle.

P 2 0 9 TIME-RESOLVED X-RAY DIFFRACTION DISPLAYS THE KINETICS OF THE NEUTRAL CORE

LIPID TRANSITION OF LOW DENSITY LIPOPROTEIN M.Pregetter, R.Prassl, R. Schwarzenbacher, H.Amenitsch, J.Chapman*, P.Laggner

Inst. Biophysics and X-Ray Structure Research, Austrian Academy of Sciences, Graz, Austria, *INSERM U.321, Paris, France

Low density lipoproteins (LDL) show a very special physical feature, which make them unique among all structural elements of blood: They undergo a major structural transition of the apolar core, from an isotropic towards a liquid- crystalline state, closely below physiological body temperature. The dynamics of several metabolic processes and susceptibility to oxidation have been related to the state of the LDL core. In the outer extremities (finger tips, toes) the blood temperature allows for a transition of the LDL core towards its more rigid and radially ordered state. Time-resolved measurements provide evidence whether the kinetics of the structural transition permit the transition during the time LDL spend in cooler parts of the human body. High precision small angle x-ray scattering (SAXS) curves in the range of 1.5.10"- < h < 0.2 A "t were measured at the SAXS-Beamline at the synchrotron source of ELETTRA (Trieste, Italy) on well defined subspecies of native LDL. Temperature jumps and flash cooling elucidated the kinetics of the core-transition. It can be stated that the transition from the rigid towards the fluid core of LDL takes place in less than the 10 ms time-resolution. The cooling process combined with the rearrangement of the lipid core proceeded within the time resolution of I s.

P210 Membrane alterations in gamma.irradiated mouse thymocytes : A fluore~ence study

B.N. Pandey and K.P. MiCra Radiation Biology Division, Bhabaha Atomic Research Centre, Mumhai 400 085, India

Changes in plasma membrane permeabifity and DNA fragmentation in immature mouse thymocytes caused by y-irradiation have been investigated by fluorescence technique employing fluorescein in diacetate (FDA) and gel electrophoresis. Thymocytes suspended in culture medium at 4~ were irradiated for various doses of radiation up to 10 Gy and these cells were analyzed'after incubations wither at 4~ or at 37~ Normal or irradiated cells were labeled with FDA and associated fluores~nce was measured spectmfluorimetricaliy. FDA uptake by thymocytes at ice temperature showed a rapid increase initially but saturation occurred after 30 mivL FDA labeled control and irradiated thymocytes displayed similar fluores~nce intensity on incubations up to 24 h at 4~ On the other hand, incubation of cells at 37~ showed a marked difference in the degree of fluorescence; controls were stable up to 7 h but the irradiated cells showed time dependtmt leakage of FDA after 2 h. Further incubations of these samples up to 24 h showed loss of fluorescence by 35% and 85% from control and irradiated cells respectively. It needs to be noted that viability determined by Trypen blue exclusion revealed an insignificant change in control while it changed within 10% in cells irradiated by 10 Gy. Preliminary experiments have shown a correlation between induced permeability and DNA fragmentation in cells. These results suggest a possibility of sensitively detecting injury to membrane of cells by FDA at low radiation doses and these changes seem to be an early event in radiation induced cellular apoptosis.

107


P211 Lectin induced alterations of membrane dipole potential: Comparison of fluorescence and electrical measurements. E. E. Pohl, d. Sun, I. 1. Agapov, A. G. Tonevitsky, P. Pohl Martin-Luther-University, Institute for Medical Physics and Biophysics, 06097 Halle. Germany.

The lectins ricin and viscumin are the most potent toxic compounds known to inactivate ribosomes. After linking them to antibodies these proteins can selectively kill specific cell types, such as cancer cells. The mechanism of their translocation across the membrane of an intracellular compartment is not understood yet. Because recently the toxins were shown to dehydrate membranes (Pohl et al, Biophys. J. (1998) 75, 2868) and to cause lipid dependent fusion (Pohl et al, BBA (1998) 1371, I1) they were expected to provoke dramatic rearrangements in membrane structure that should be accompanied by a drop of membrane dipole potential (~d). ~b,t was monitored by capacitive current measurements and by fluorescence measurements with 8-di-ANEPPS. Although the dipole potential is known to be partly generated by membrane bound water, it is changed by no more than 20 mV as revealed by electrical mesuarments. From fluorescence measurements undertaken with lipid vesicles quantitative different results were obtained. The origin of this discrepancy is discussed in terms of protein induced change.s in membrane hydrophobicity. the mechanical properties of the bilayer and the location of the fluorescence dye in the vesicle membrane. The impact of~d measurements as an indicator tbr lipid-protein interactions is evaluated. (Financial support of the DFG (PO533/1- 2) and of the Kultusmi,listeritml Sachsen-Anhalt. Germany is grateful acknowledged)

P21 2 Studies on a novel fluorescence probe in unilamellar liposomal membranes

S. M. Dennison, J. Guharay and P. K. Sengupta

Biophysics Division, Saha Institute of Nuclear Physics, Calcutta 700 037, India.

We have examined the fluorescence emission properties of a synthetic flavonol namely 4'-N,N-

dimethylamino-3-hydroxyflavone incorporated in small unilamellar liposomal membranes formed by

phosphatidylcholines. The simultaneous occurrence of excited-state charge transfer (CT) as well as proton

transfer (PT) fluorescence emissions from this fluorophore has enabled us to utilize several discriminating

emission parameters to explore its microenvironments in the membranes. Moreover, the temperature

dependence of the anisotropy (r) of the PT fluorescence has been utilized to obtain reliable measurements

of them~otropic gel to liquid crystalline phase transition temperatures.

P213 FLUORESCENCE QUENCHING STUDY OF THE MODEL PROTEIN - LIPID SYSTEMS

Gorbenko G.P., Dyubko T.S." Kharkov State University, Svoboda gq.4, Kharkov, 310077, Ukraine; "Institute for Problems of Cryobiology and Cryomedicine of the Ukrainian Academy of Sciences, Pereyaslavskaya Str., Kharkov, Ukraine

Model protein - lipid systems including cationic proteins and negatively charged liposomes are presently widely used for gaining insight into the nature of the interactions between peripheral proteins and biomembranes. One important aspect of such studies concerns the peculiarities of the lipid-induced structural changes of the proteins. Some factors governing these changes can be elucidated through the quenching of intrinsic protein's fluorescence. In the present study the quenching of tryptophan fluorescence of the cationic proteins ribonuclease and lysozyme in complexes with liposomes composed of phosphatidylcholine (PC) and cardiolipin (CL) has been examined to ascertain the mechanisms underlying the lipid's influence on conformational state of the proteins. Modified Stern -Volmer analysis of the data derived from the double quenching of the protein's fluorescence by ionic (iodide) and nonionic (acrylamide) quenchers revealed increasing accessibility of tryptophanyl residues to iodide and increase of the dynamic quenching constant for acrylamide in the presence of liposomes. The magnitude of these effects has been found to be dependent on CL content ofliposomal membranes, being varied in the range 10-70 mol %. The results obtained provide evidence for the 'loosening' or partial unfolding of the protein's molecules upon the formation of protein-lipid complexes.

108

C1 : Membranes : Structure, Dynamics and Functions

P 2 1 4 CARBAZOLE AS A FLUORESCENT PROBE FOR LIPID BILAYERS IN ALKALINE MEDIUM

N.PaDDavee & A.K.Mishra, Department of Chemistry Indian Institute of Technology, Madras, Chennai - 600 036.

In our effort to look for novel ESPT fluorescent probes in the alkaline pH ranges we have examined carbazole as a possible candidate because of its high quantum yield, extinction coefficient and the larger ApKa (pKa - pKa*) value. The photodissociation of carbazole was studied in liposomes by steady state and time resolved fluorescence measurements. The neutral form and the anionic form emit at 360nm and 417nm resp. This large shift in emission makes it convenient to monitor the physical properties of liposomes. Time resolved measurement showed evident for two localisation sites in the liposomes. Temperature effects showed a decreased emission intensity of the neutral form above and below the chain melting temperature (Tc) but a dramatic increase at Tc. This .variation of intensity can be explained due to the redistribution of probe between the surface and the core of the liposome. The cholestrol induced phase changes on liposomes were also explainable by the ESPT ofcarbazole.

P 2 1 5 Kinetics of lipid phase separation. Application of fluorescence techniques.

Prieto, Manuel;; Alineida, Rodrigol; Loma, Luis ]'2 lc~tl~ de Q,imica-Fisica Molecuiar, IST, Av. Roviseo Pais, 1049,4)01, Lisbon, Pon'ugal; 2 ~ o de ~dmica,

Universidad_e de Evora, 7000 Evora, Portugal e-mail: prieto~al fa jSt.utl.pt

Structural studies on membranes in order to obtain topological information (e.g., microdomain formation), can be carried out it~ing photophysical techniques.

In this work, the kinetics of phase separation Cdemixing') of a mixture of two lipids with a significant tail mismatch (DiC]2PC-DiC1sPC) was studied using photophysical techniques. For this pmlx~, a random distribution is induced at a higher temperature where the system is in the fluid state, followed by a sudden thermal quench into the fluid and gel phase coexistence region. Two methodologies were used: 1) Fluorescence intensity and aniselropy of a probe that partiu'onates into the fluid phase (N-BD-C]2 lipid derivative), 2) Time-resolved energy franker from a probe that prefers the gel phase (t- parinaric acid), to an acceptor that preferentially incorporates into the fluid phase (NBD-C]2 lipid derivative).

The phase separation is described by first order kinetics with a lifetime of the order of hours, and its rate shows an inverse dependence on temperature. These observations will be rationalised within a recent theoretical framework using Monte-Carlo simulations (Jorgensen, K. et al, Biophys. J., 95, 942, 1995; J. Phys. Chem., 100, 2766, 1996). This work was carried out under Projects: JNICT, PRAXIS/PCNA/P/BIO/56/96; JNICT/PRAXIS/SAU/14025/1998; CRUP-E-41/99

P 2 1 6 MEMBRANE TRANSPORT OF SPIN LABELED NONELECTROLYTES

L. Ya. Gendel Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia

A new approach is proposed to study the transport of nonelectrolytes (antioxidants, pharmacological compounds, etc.) into a biomembrane and the distribution of these substances within the intramemranc space. It is based on the application of ESR method and paramagnetic models of nonelectrolytes - homologous series of spin probes with different hydrophobic properties.

Using the erythrocyte membrane we have shown that nonelectrolytes, different in their hydrophobic properties are selectively distributed within various regions of the membrane structure.

The principle of the physicochemical conformity of a substance (drug, antioxidant and the like) to the biomembrane region where its biological activity, is realized, has been proposed. It explains the dependency between the structure of a substance, its distribution in a biomembrane and realization of the biological activity, which is important for the drug design.

109


P217 The Antagonistic Action of Tin and Lead Organic Compounds and Quaternary Ammonium Salts on

Membranes Przestalsld S., Kuczera J., Kleszczyliska H., Kral T. Department of Physics and Biophysics, Agricultural University, Norwida 25, 50 -375 Wroclaw, Poland

The destructive effect of both n-alkyltin and n-alkyUead compounds on liposome and etythrocyte membranes, and a protective effect of amphiphilic ammonium salts against that action, were studied. In particular, the effect of changing membrane polarity induced by quaternary ammonium salts (e.g., trimethyldodecylammonium bromide) was investigated. An increase in positive charge of membranes caused a marked decrease in the access of organic cations of tin to the membranes. The access of organolead compounds was also inhibited but to a lesser degree. Hence it may be concluded that the interaction of organotin compounds with membranes are of predominantly electrostatic character, whereas.in the case of organolead compounds not only electrostatic interactions are important.

The work sponsored by the Polish Research Committee, grant No 6 PO4G 04313.

P218 Vesicular-Competitive Hemolysis

Cbernitsky E.A., Senkovich O.A., Rosin V.V. Institute of Photobiology, Natl. Acad. Set. Belarus, Akademicheskaya Str., 27,

Minsk, 220072, Belarus. [email protected].

It is shown that slow hemolysis of human erythrocytes by sodium dodecyl sulfate follows the colloid-osmotic mechanism owing to formation membrane pore with diameter about 4 nm, whereas rapid hemolysis taking place at the greater detergent concentration is caused by the opening of large membrane pores, sufficient for the release of hemoglobin molecules from erythrocytes. It is found that the detergent-induced erythrocyte vesiculation is coupled with the formation of modest-sized pores, defends the cells from hemolysis and represents the process competitive with rapid hemolysis. The influence of osmotic protectors on the vesiculation rate makes this method unsuitable for the estimation of pore sizes responsible for rapid detergent-induced hemolysis. The properties of the new, vesicular- competitive hemolysis are described. Its graded nature (when only part of the cells can be lysed) is explained by the competition between hemolysis and vesiculation without resorting to view of eryth'rocytes heterogeneity. On the basis of analysis of the literature dat~ it is shown that the vesicular-competitive hemolysis takes place not only under the action of detergent, but under the different other influences, if the rate of vesiculation induced by some influence becomes comparable with hemolysis rate. P219

Glycinebctainc stabiliz~es photosy~em ! and photosystem I1 electron transport in spinach thylakoid membranes against heat inactivation

Y.M.Allakhverdieya,* G.C.Papageorgiou**& R.A.Gasanov* *Institute qf'Botany, Azerb. Acad. o.f Sci., Balm, Azerbaijan Republic, * *NR( ; Demokritos, Institute o.['l~iology, Athens, (.;reecr

The stabilization by glycinebetaine of the photo-induced electron transport in spinach thylakoid membranes and in Pholosystem 1 and Photo.system II particles, against thernud inactivation, was examined systenmtically. GlyCinebelainc, a compatible osmolyte of halololerant plants and bacteria protected parli~fl Photosystem I and Pholosystem II electron transport reactions against thermal inactivation but with different effieiencies. In its presence, the temperature for half-maximal inactivation was generally shifted downward by 3-12~ Glycinebetainc stabilized photo-induced oxygen evolving reactions of Photosystem II by protecting the tetranuclear Mn cluster and the extrinsic proteins of this complex as reported by Papagcorgiou, G. C. and Murala, N. (Photosynth. Rcs. 44 11995.] 243-252). A weaker, although noticeable, stabilizing effect was observed in photo-induced Photosyslem 11 electron transport reaclions that did not originale in OEC. This weaker protection by glycinebclaine is probably exerted on the Photosystem I1 reaction center. Glycinebetaine protected 'also photo-induced electron transport across Pholosystem I agait~sl thermal inaclivation. The protective effect was exerted on plastocyanin, the mobile protein in the lumen lha| carries electrons from the integral Cylochrome be/f complex to the Photo.system I complex.

110


P 2 2 0 FLURESCENCE QUENCHING AS A TOOL MEMBRANE FLUIDITY MEASUREMENT

Calin Apetrei, Tudor Savopol, Marius Balea "Carol Davila" Medical University, P.O.B. 15-205, Bucharest , ROMANIA

A new technique for measuring rod outer segment membrane fluidity is proposed, based on the fluorescence quenching phenomenon.

Our experimental results shows that rod outer segment membrane is labelled with 1-(4-trimetylammoniumphenyl)-6-phenyl-l,3,5,-hexatriene p-toluensulphonate, (TMA-DPH) presents a decrease of the fluorescence intensity, depending on the degree of rhodopsin bleaching. The dependence follows a typical Stern- Volmer plot. We conclude that this phenomenon is due to bleached molecules of rhodopsin which act as quenchers for the TMA-DPH molecule. This allows calculating the viscosity coefficients of the membrane. The obtained

results are in good agreement with similar estimations based on other methods. Some considerations about the generality and limits of this new method are made.

P 2 2 1 Electrical Potential Oscillations Studied in Two Model Membranes: Comparative Biophysical and

Molecular Dynamics Simulation Study Cucu 19, Mihailescu D., University of Bucharest, Faculty of Biology, Spl. Independentei 91-95, 76201 Bucharest, Romania. 9 Electrical potential oscillati0n~were studied in two different systems: a) A lipid impregnated model membrane interposed between two solutions with the same alanine concentration, which performs temporal changes of electrical potential. The lipid used is L-~ Phosphatidilcholine and the system is sensible in amplitude and frequency to different physical and biochemical parameters. b) A triphasic liquid system composed of nitrobenzene/picric acid placed between two aqueous phases in the presence of alanine, but also with other taste (salty, bitter) substances. 9 Oscillatory behaviour was studied using Fourier analysis. For the liquid membrane Fourier spectnun is

a "fingerprint" of the substances that could be correlated with taste index. 9 Preliminary data of molecular dynamic simulations consisting of an explicit representation of all atoms

forming the system of L-ct Phosphatidilcholine, water and alanine were obtained. Dynamical parameters of a trajectory of 1 Ns length are correlated with the time dependence of the electrical potential characterising the experimental system.

P 2 2 2 A novel method for the estimation of local order through cross-polarisation from dipolar reservoir

~K.V. Ramanathan, Indian Institute of Science

Bangalore 560 012

The determination of the local order of the hydrocarbon chains provides information on the mobility of these chains in Iipids and model membrane systems. The method widely used for this purpose is the estimation of the order parameters along various C-D bond axes in specifically or perdeuterated molecules (1), though it is possible to use other methods such as Hartmann-Hahn cross-polarisation (2). We here propose a method in which initially, an adiabatic transfer of magnetisation in the rotating frame from the proton Zeeman reservoir to the dipolar reservoir is effected. A subsequent transfer of magnetisation to carbons through a cross-polarisation process under various matching conditions provides information on the proton dipolar reservoir, from which an estimate of the local order can be obtained. The method is demonstrated in the case of a liquid crystal oriented in its nematic phase by a magnetic field. I. I.C.P. Smith, in Nucl. Magn. Reson. Liq. Cryst., Ed. J.W. Emsley, D. Reidel Publ. Co. 1985, 533. 2. R. Pratima and K.V. Ramanathan, J. Magn. Reson. AllS, 7, (1996); C.S.Nagaraja and K.V.Ramanathan,

Liouid Crvst. 26, 17, (1999).

111


P 2 2 3 SHAPE OF HYDROPHOBIC BARRIER OF PllOPllOLIPID BILAYERS:

A COMBINED NMR AND EPR STUDY Goron B ~ct~

Faculty of Physical Chemistry., University of Belgrade, Yugoslavia

The purpose of this study was to study the depth to which water penetrates phospholipid bilayer using paramagnetic stable free radicals (nitroxides) and combining EPR sectruscopy with measurements of proton TI HMR dispersion. Large unilamcllar liposomes were made by the reverse phase evaporation process using a 4:1 mixture of DPPC/DPPG or DMPC/DMPG. Different fauy-acid nitroxides (lipid-to- nitroxide molar ratio of 25:1) were used to label various positions (1-16) along the lipid chain. EPR spectra were obtained at 9.5 GHz The HMRD profiles, i.e. plots of T1 relaxation rate vs magnetic field strength, were obtained using a field-cycling relaxometer over the range of proton Larmor f~luences of 0.02-50MHz. These measurements showed ten-fold higher relaxivity of nitroxides located at carbon atoms 1-5 than those located deep in the hydrocarbon portion of the membrane. Although the relaxivity of nitroxides decrases with the increase of their mobility, EPR measurements showed that the difference in relaxivity is too high to be explained on the basis of the difference in their mobility. Consequently, the observed differences in velaxivity dominated by the different accessibility of nitroxide group to water. In conclusion, it seems that water molecules penetrate into the pete phospholipid bilayers to a considerable depth beyond carbonyl oxygens and that the shape of hydruphobic barrier of phopholipid bilayers can be assessed from T1 NMR measurements.

P 2 2 4 MODELING neu/ErbB-2 TRANSMEMBRANE DOMAIN DIMER

Nicolas Sajot#, Norber t Garnier ~, Serge Crouzy s and Monique Genest # ~Centre de Biophysique Mol6culaire, CNRS, rue Charles Sadron, 45071 Orl6ans C&lex 2, France SD6partement de Biologie Mol6culaire et Structurale, CENG, rue des Martyrs, 38054 Grenoble C6dex 9, France

Dimerization of neu/ErbB-2 receptor tyrosine kinase is necessary but not sufficient for signaling. To understand the mechanism of receptor activation it is essential to identify the molecular interactions responsible for receptor-receptor contacts, particularly those involving the membrane-spanning domain recognized as a mediator in dimerization process. For this aim, molecular dynamics simulations are used to predict the dimeric structure of the transmembrane domain in both wild and oncogenic forms. In vacuo simulations on nanoseconds time scale show that the two helices preferentially wrap in left handed interactions With a packing angle at about 20 ~ Interfacial residues conform to the knobs-into-holes packing mode of transmembrane helices and we show that the transforming Glu residue side chain interacts directly with its cognate or with carbonyl groups of the facing backbone. Similar stabilizing hydrogen bond interactions are Observed in a membrane-like environment. The predicted dimer interface shows strong similarities with the inter receptor contact face proposed from Cys mapping approach.

P 2 2 5 ADAPTATION OF THE PHOTOSYNTHETIC MEMBRANE OF CYLINDROSPERMOPSIS

RACIBORSKII ACT 9502 CELLS TO EXTREM ENVIROMENTAL CONDITIONS V/wk0nyi, Zs? , Z~ros, 0.1 , Fad(as, T. 2 and Combos, Z?

I Biological research Center, Hung. Acad Sci. Inst. o f Plantbiology, 2Biological research Center, Hung. Acad Sci. Inst. o f Biochemistry, Szeged, Hungary We measured the temperature and light intensity dependence of photosynthesis of cyanobakterium strain, C. raciborslai isolated from Lake Balaton, Hungary. The temperature sensitivity of photosynthesis was monitored by oxygen evolution of the cells with Clarke type electrode. On low temperature, when the cells were grown at low light inten~ty the cells survived, but the growth rate was very low. We analysed the lipid composition by Hewlet Packard gas cromatogaph, and found that the unsaturated fatty acid component of glicerolipids of the thylakoid membrane increased. When the temperature and the light intenzity were increased - the degree of unsaturation of the fatty acids of the photosynthetic membrane decreased. Paralelly with this expe~en ts we analysed the pigment composition of the C raciborskii cells by HPLC and FTIR spectroscopy. Our ~ t~ suggest that this cyanobacterium has a remarkable susceptibility to adapt to the extrem enviroraental conditions and that was the reason of selection of this strain to be the dominent species during the eutrophication period of this lake.

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P226 FUSION AND HEMIFUSION : AN APPROACH WITH GIANT UNILAMELLAR VESICLES

Sophie Cribier - Institut de Biologie Physico-Chimique - 13, rue Pierre et Marie Curie - 75005 Paris - France

Interactions between biological membranes are of major interest in biology as they are involved in adhesion and fusion processes. Even though ~ huge amount of proteins involved in these processes have been identified, the mechanisms are far from being understood. We have used Giant Unilamellar Vesicles (GUVs, 10 to 150 microns in diameter) as model systems for biological membranes in order to better understand these phenomena at a molecular level. GUVs are produced by an AC electric field controlled swelling of a lipid film.

In a first study, GUVs have been used in experiments investigating the possible role of tetrameric Annexine II during fusion. We have shown that GUVs and LUVs (Large Unilamellar Vesicles, 100rim in diameter) containing both PS (15%) are able to fuse when Annexine II is added in presence of calcium and phosphorylated in situ.. A "defect hypothesiS" could explain the membrane destabilisation which has to take place to allowed fusion processes.

Another study deal with pure lipidic system : we were able to induce stable hemifusion of GUVs by adding lipids 00%) functionalized with either adenosine or thymidine in the membrane. We discuss the role of an extra attractive force, which, by decreasing the intermembrane equilibrium distance, induce a dehydration of the phospholipid polar heads, and a subsequently, force lipid rearrangement of both leaflets in contact.

P227 Evidences for tl~ different location of JminoamJde LOcal Anesthetics in Phosphaddylcboline Ves/ck~

Fracete. LF ~. Schreier, S 2, Sl~mi, A 3, & de Paula, F ~ ( E - m a l l : ~ c a n ~ b r ) . ' ] ~ ofBiocbemi~z~, Inst Biology, U ~ of Ca mp~ , SP, Brazil; "Depertment c~Biochemis~7,

Ira. C h e ~ , University ofSao Paulo, Brazil, Is~uto di Chimica Biologics, UniversRy of Parma, Italy.

High-resolmion NMR is a 8ood tool for the smo'y of the ~ o n of a m l ~ i , c molecules with membranes. Some arlicles in the lim'~mn'e de~n'be the abili W of local anesthetics (LA) to bind to the polar-head group of phos'pbolipid ~ and many of tlmm claim that p(mitioning in the bilayer could be directly related to the anmthctic potency. We investismed the effect of the .=i ,~mkle LA lidocalne Ct.lX~), ctidoca~ (EDC), ~ i t ~ (MVC) and tmpivamine (BVC) upon egg lltOspmm+ "dylcholine (EPC) ~ o ~ , * t l o m Molecular proximities between LA and EPC hydrogcns were examined using HOE and T~ expcrimea~ EPP, spin label allowed the analysis of different bilayer depths while changes in the 31P-NMR spectra monitored LA effec~ upon the polar head-group region. The u n c ~ LA species inm'~c~d with EPC vesicles, ~ the mobiliW of the choline and glycerol EPC hydrogetm. Cyclic aminom~ides (MVC and BYE, with a sulm~mem) caused greater effect tlwn the non.cyclic LDC and EDC, ROESY experiments revealed intermolecular, LAEPC, proximities between all the LA and the choline gout~ of EPC. Membrane o r~aniTntioll was mainly altered for the spin probes (EPR) at the more supcrficial positions of the acyl chai~ These results indicate that uncharg~ LA species, in despite of their fast exchange between membrane and water, present a prefe~e~Jal location at the polar interface of EPC liposomes, also strongly determined by the steric fcatares of the LA molecule. Supported by FAPESP (Proc. 99/4059-0, 98/84-8, 96-1451o9).

P228 T H E R M A L F L U C T U A T I O N S OF T E T H E R E D V E S I C L E S

F. Sev~ek 1,2, G. G o m i ~ e k 1, V. Arrigler 1, S. Svet ina 1, B. Zek~ 1 1Inst i tute of Biophysics, Medical Faculty, 2University College of Heal th Care, University of Ljubljana,

Ljubljana, Slovenia

P O P C phospholipid vesicles were prepared by electro-formation in sucrose solution and observed by optical microscope. Vesicles were allowed to a t t ach to the glass plate of the flow chamber and were subjected to a laminar flow. Depending on the flow velocity, the vesicles were deformed but remained a t t ached by a long thin lipid tube ( tether) which length was controlled by the flow. Non-spherical , flaccid vesicles exhibited shape f luctuat ions which were due to membrane thermal f luctuations between shapes of very similar elastic energy. These f luctuat ions were recorded as the function of te ther length. Large number of digit ized vesicle contours were ana lyzed in terms of Fourier coefficients. From them the average values and distr ibutions of f luctuat ional modes were calculated. Using second order approximat ion membrane tension and bending rigidity were determined. The experimental results were compared to the Monte Carlo calculations of thermal f luctuat ions at cons tan t volume of the vesicle and membrane area. The membrane tension was thus related to the dimensions of the tether .

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P 2 2 9 Continuous Shrinkage of Liposomes and Direct Expulsion of Inner Vesicles

Fumimasa NOMURA, Miki NAGATA, Kingo TAKIGUCHI and Hirokazu HOTANI Department of Molecular Biology, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan

To study the dynamic behav io r of b iomembrane in aqueous so lu t ion , the t ransformat ion of l iposomes caused by surfactants were moni tored by direct, real- t ime observat ion using dark-field optical microscopy. L iposomes are c losed vesicles of l ip id b i l ayer membranes, and have been well s tudied as a s impl i f i ed model o f b io logica l membrane. When Tri ton X-100 was added to a l i posome so lu t ion , spherical l i posomes became smal le r in size with maintaining their spherical shapes. Dur ing this process, the shrinking l iposome exhib i ted two phases a l ternat ively , i .e . , its membrane fluctuated v igorous ly or kept s t i l l .

Some l iposomes encapsula ted a few smal le r l iposomes inside. When Tri ton X.100 was added, the ou te r l i posomes shrank smaller, and surpris ingly, the encapsulated i iposomes escaped when the ou te r i i posomes were v igorous ly f luctuat ing in shape. Since the ou te r l iposomes prepared by our method are uni lamellar , this is conc lus ive evidence that Tri ton X-100 t ransient ly generates holes on a l iposome and thereby excludes inner vesicles directly.

P 2 3 0 EFFECT OF ANNEALING ON THE PHASE TRANSITIONS IN THE MODEL MEMBRANE, DPPC-pHg.3

DOPED WITH SALICYLIC ACID

Lito Pankker and P.S.Parvathanatban, Blud)ha Atomk Res~rch Cemre, Trambay, Mumbui 400 085, INDIA

Calorimetric studies of the Chain Mdting(CM) tnmsiliu b the model membrane DLIPaimitoyl Plmsplmtidyl Choline(DPPC)-pHg.~ doped with Salicylic Acid(SA), a kemtolydr drug, I~ve ~sowm dramatic changes in the tcyl Chain Meldng(CM) chsraetemdcs of the membrane under the influence of tnneoling (at ambient temperature). The duration of annealing, %, was in the range, 0~ %. < 60 days and the molar ratio, It.,, of SA to DPPC was in the range, 0~ Rm-~.$. The interesting fmdinp are the fe41owing: (1) T c M ~ with increasing Rut (2) The limiting volue of 1~ at which the ripple (PIn v ) phase disappears, inere~es with increasing %.. (3) Annmding leads to a smldl but sure inerease in AHrr and Trr (transition eatludpy and temperature). (4) For Ru>0.25, in the range, 8 days < %, < 60 days, there is the evolution of a new high temperature(HT) phase whose strength (enthalpy, AHnT) increases with increasing Ru, at the cost of transition enthalpy, Allc~. Our results seem to strongly indicate the formatiou of two types of regions in the SA-doped membrane: (a) Region I: SA molecules penetrate into the acyl chains, leading to a decreased effective PC-PC interaction. (b) Region I!: SA molecules are located in the lipid-water interface in such a way as to increase the effective PC-PC interactions.

P231 Effect of Choles terol on Opening-up Transformation of Liposome Caused by Ezrin.

_A. Ishino, A. Saitoh, H. Hotani and K. Takiguchi Department of Molecular Biology, School of Science, Nagoya University, Nagoya 464-8602, Japan

Purpose: We studied the dynamic behavior of liposome mused by the interactions between liposome membrane and proteins on real time. We have discovewA that the cytoskeleton-related proteins such as ezrin possessed a novel opening-up activity on a lipid bilayer membz*ane. To investigate the mechanism of this membrane opening-up transformation, we examined the effect of the lipid compositions of liposomal membranes. Resul t s & Conc lus ions : When e2rin, talin or band 4.1 was added to a liposome solution, each liposome opened a stable big hole and subsequently transformed into a cup-shape. The hole became larger with increasing protein concentration, and finally the cup-shaped liposomes transformed into opened lipid bilayer sheets. We demonstrated that protein located mainly along the membrane verges, presumably sealing the exposed hy(kophobic portion at the edge of lipid bilayer. To investigate the opening-up mechanism of membrane, we studied the effect of lipid composition of liposome membrane and found that cholesterol inhibited the opening-up of the membrane. This inhibitory effect of cholesterol suggested that micellisation of lipids of liposomai membrane caused by ezrin or other protein plays an critical role in the opening-up process.

114


P232 SPECTROSCOPIC STUDIES OF LIPID MEMBRANES DURING Ca2+ INDUCED FUSION.

Afonin S. A. V. Pal/edin /nstitute of Biochemistry NAS Ukraine, Kyiv, Ukraine

It was suggested that lipids mediate biomembrane fusion and changes in physical-chemical behaviour of lipid molecules play an essential role in the process. While membranes fuse lipid molecules undergo variety of conformational changes altering bilayer fluidity and local membrane structure. General methodology of fusion studying utilises three major principle approaches: observation of vesicles size change, membrane hydrophobic regions mixing and inner volumes mixing - usually one per unique method. Each method possesses its own advantages and disadvantages. We are evaluating an NMR-based approach to combine different methodological principles in a "one sample" assay. The assay also gives an information regarding conformational behaviour of different parts of lipid molecule. It is based on a double shift reagent application followed by analysis of spectra obtained in terms of chemical shifts, integral intensities and relaxation rates. As a model for NMR-based observation of lipid membrane fusion we have used Ca 2+ induced fusion of phosphatidylcholine liposomes Lipid composition was characterised by TLC and GC/MS. Liposomes size changes were controlled by means of Laser Correlation Spectroscopy. Mobility changes in polar and hydrophobic regions were additionally studied with IR. It was shown that a clear agreement exists between changes in average vesicles diameter comparative to T1, T2 and inner to outer leaflet signal intensities ratio in ~H and 3~p spectra. Also there is a certain correlation between changesin IR bands and 1H NMR data.

P233 CALCULATION OF THE DOUBLET SPLITTING IN 2D NMR OF TIGHTLY BOUND WATER (D20) IN A CHOLINE PHOSPHOLIPID HEAD GROUP AT A LOW WATER CONTENT A.Takahashil, Y.Nalcata2 and T. Takizawal lDept, of Physics, 2Dept of Biophysics, Gunma University, Maebashi 371-8510, Japan

In the 2D NMR study of DzO molecules bound tightly to a choline phospholipid head group, we have calculated the splitting AVQ of the doublet specmun of D20 in the liquid crystalline phase, using the following formula

AVQ - ( 3e2qQ/(4h ))(3eosZfl "- 1)S/2, S = < (3cosZ~l '- 1)/2 >, /1 "= 105"/2, where fl ' is the angle between the bisector of the DOD angle and the bilayer normal (A.Takahashi et al.: J.Phys.Soc. Jpn 65 (1996) 635). In the present work, the value of AVQ is calculated for the DPPC-D20 system at a water content of 4.7 moles per m01e of lipid. The lipid molecules are assumed to be packed in a hexagonal lattice on a bilayer plane. where the surface area per lipid molecule is taken to be 57.8/!~z The calculations were made for two eases; two lightly bound water molecules undone tightly bound water molecule. In each case the favourable sites of the water moleonles were referred to the models l~oposed by Pullman et al. (Chem.phys.Lett.33 (1975) 11). In addition, the energies of the interactions between adjacent lipid head groups were taken into account. These interactions play an important role in the calculation of the average of order parameter S in such a low water content.

The results are in good agreement with experiment.

P234 Protec t ion of D P P C l iposomal vesicles agains t g a m m a rad ia t ion d a m a g e by an t iox idants

Dipti Marathe and K.P. Mish ra

Radiation Biology Division, Bhabha Atomic Research Centre, Mumbai - 400 085

The oxidative damage to sonicated dipalmitoylphosphatidylcholine (DPPC) vesicles suspended in aqueous buffered solutions was investigated following y-irradiation. Radiation induced peroxidative damage was measured by the induced permeability changes revealed by the leakage of pre-loaded carboxyfluorescein (CF) and fluidity changes by the polarization of incorporated DPH fluorescence probe which showed fluidization at low doses but rigidization at higher doses of irradiations (0.3 -6 kGy ). Induced structural changes in vesicles were investigated by DSC studies which showed a dose dependent increase in the main phase transition peak at 40 0 C in irradiated vesicles giving maximum effect at 6 kGy reflecting induced increased rigidity in the structural lipid bilayer by radiation. Experiments also showed that vesicles prepared with incorporated antioxidant, ct- tocopherol, in the bilayer membrane were remarkably protected against damage by radiation exposure. The magnitude of protection was found to increase with the increasing concentration of antioxidant demonstrating involvement of lipid free radical mediated peroxidation reaction mechanism in the damaging process.

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P235 I n v e s t i g a t i o n o f t h e r e l a t i o n s h i p b e t w e e n l i p i d b i l a y e r m a c r o - a n d

m i c r o s t r u c t u r e a n d m e m b r a n e f u n c t i o n Kent J~r~ensen

Department of Chemistry, Technical University of Denmark, DK-2800 Lyngby and Department of Pharmaceutics, The Royal Danish School of Pharmacy DK-2100 Copenhagen [[email protected]]

The dynamical and s t ructural behavior of the lipid bilayer part of biomembranes plays an important role for functional membrane properties such as the activity of membrane associated enzymes and for the passive permeat ion of molecular agents. By means of calorimetry, fluorescence, and Monte Carlo computer simulations the macroscopic phase behavior and microstructure of lipid bilayers composed of different phospholipids and polymerlipids have been investigated. The combined experimental and theoretical investigations suggest that the cooperative behavior of the lipid bilayer and the formation of local lipid structures plays an important role for the functioning of membrane active enzymes such as phospholipase A2 and for the lipid bilayer barrier properties. Such results are of importance for a deeper understanding of the lateral lipid membrane structure-function relationship.

P236 DYNAMIC ASYMMETRY IN MEMBRANE BILAYERS

AND ITS M O D U I ~ T I O N WITH CHOLESTEROL Usha B. blair, R. Rukmini and Amitabha Chattopadhyay

Centre for Cellular & Molecular Biology, Hyderabad 500 007, INDIA

Biological membranes are asymmetric in nature. Compositional asymmetry in membranes is well established. In membranes with high degree of curvature, the packing arrangement of the lipids in the two leaflets could give rise to dynamic asymmelry. We have monitored the dynamic asymmetry in model membranes of DOPC by incorporating the fluorescent lipid NBD-PE selectively in the inner or the outer leaflet. Incorporation of NBD-PE selectively in the inner leaflet was achieved by reducing the outer leaflet fluorescence using the water soluble reducing agent dithionite. Our results show differences in motional restriction experienced by NBD-PE in the two leaflets as measured by the Red Edge Excitation Shift (REES). This is further supported by polarization and fluorescence lifetime measurements of NBD-PE. Since cholesterol is known to modulate the organization and dynamics of membranes, we investigated the effect of cholesterol on dynamic asymmetry. With increasing cholesterol concentration, we observe that the changes in fluorescence polarization and lifetime indicate differential modulation of the individual leaflet dynamics. The results of this study could be relevant in understanding the dynamic asymmetry that could be encountered in membranes with a high degree of curvature such as sorting vesicles, endosomes, the inner mitochondrial membrane or specialized regions of plasma membranes (domains), and its role in modulating the structure and function of livids and proteins residing in these membranes.

P237 INFLUENCE OF CHOLESTEROL ON THE DOMAIN STRUCTURE OF LIPOSOME MEMBRNES

AS MEASURED BY TEMPOL ESTER SPIN PROBES .M.~Pentinrc l, J.~;Irancar l, Z.Stoli~ I, K. Fifipin 2, and S.Pe~t1-1'2

Ij.stefan Institute, 2Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

Electron paramagnetic resonance (EPR) in combination with appropriate spin probes is a convenient method for investigation of heterogeneous cell membrane structtwe. However, to get an adequate information about the membrane domain structure and its alterations, diffemlt types of spin probes with different partitioning between the domains, should be used. For this purpose esters of Tempol (.l-oxyl-2,2,6,6-telramethyl-4-hydroxypiperkline) with aromatic acids (benzoic acid, 1-naphthoic a~id, 2-naphthoic acid, 4-pbenylbenzoic acid and 4-isopropylbenzoic acid) were synthesized.These spin probes were supposed to distribute preferentially to cholesterol reach domains due to the interaction of cholesterol with the phenyi rings. They were enu'apped into DPPC liposomes with ditferent cholesterol concentration. The EPR spectra are superimposition of spectra reflecting different ordering and dynamics of their local environment. Comparing the calculated and experimental EPR spectra and relative portions of their components the distribution of different Tempol esters between different types of domains was estimated. We observed that the EPR spectra line-shspes of these spin probes were sensitive to cholesterol concenlration. Therefore they were found convenient for investigation of real cell membranes as an additional tool to conventionally applied fatty acid spin probes. Advantages and disadvantages of Tempol esters in comparison to fatty acid spin probes will be discusseo.

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P 2 3 8 MEMBRANE ORGANIZATION AND DYNAMICS OF DEHYDROERGOSTEROL, A NATURALLY OCCURING F L U O R E S C E N T ANALOGUE OF CHOLESTEROL

Amitabha Chattopadhyay 1, S.C. Biswas 1, 1~. Rukmini 1, Satyen Saha 2, and Anunay Samanta 2 1 Centre for Cellular & Molecular Biology, Hyderabad 500 007, India,

and 2School of Chemistry, University of Hyderaba~ Hyderabad 500 046, India Cholesterol is an important constituent of eukaryofic membranes and plays a crucial role in membrane

organization, dynamics, function and-sorting. Dehydroergosteroi (DHE) has proved to he a very useful analogue of cholesterol since it is naturally fluorescent, not toxic to cells, and has been found to faithfully mimic cholesterol in both biochemical and cellular assays. Despite the potential usefulness of DHE in membrane studies, detailed characterization of DHE in membranes, specially at low concentrations, is lacking. We have monitored the organization and dynamics of membrane-bound DHE utilizing its intrinsic fluorescence. Red Edge Excitation Shift (REES) of membrane-bound probes provides an estimate of the mofional restriction experienced by the probe. However, DHE does not exhibit any REES both in fluid phase as well as in gel phase membranes. Since the origin of REES lies in the magnitude of change in dipole moment of the fluorophore upon excitation, we calculated the dipole mlament change of DHE by semiempirical quantum chemical calculations. The calculated dipole moment change was found to be 0.61 D which is significantly low and consistent with lack of REES effect in membranes. Membrane penetration depth analysis using the parallax approach points out that the fluorescent part of DHE is located at 12.8 ]k away from the centre of the bilayer i.e., at the membrane interface. These studies could be useful for future studies of cholesterol dynamics in membranes exploitin~ DHE fluorescence.

P 2 3 9 EFFECFS OF INTERACTION FREE ENERGY BETWEEN SOLVENTS AND SURFACE SEGMENTS OF PHOSPHOLIPID MEMBRANES ON THEIR ELASTICITY KojLKinoshi~ Masahito Yamazaki Satellite Venture Business Lab., Shizuoka Univ., Hamamatsu, JAPAN

Recently, we have proposed that the interaction free energy between solvents and the surface segments of biomembranes or phospholipid membranes, plays important roles in their structures and phase behaviors: such as the intermembrane interaction, the H.-L,, and Lp'-Lpl phase transitions [1-4]. To get more information on the interaction between solvents and the membrane surface segments, we have investiga _ted effects of the solvents (especially DMSO and acetone) on elastic properties of the membrane of DOPC.

The area compressibility modulus, K, of DOPC-GUV (giant unilamellar vesicle) in water, in 20%(v/v) DMSO/water and in 20%(v/v) acetone/water at 20~ were 161,179, and 71 mN/m, respectively (Micropipete aspiration method). By the excimer method, the ratio of excimer to monomer fluorescence intensity (E/M) of pyrene-PC in DOPC-MLV (multilamellar vesicle) decreased with an increase in DMSO concentration, whereas it increased with an increase in acetone concentration. To investigate effects of these solvents on the polarity of the membrane interface, the fluorescence intensity of laurdan in DOPC-MLV was measured. With an increase in DMSO concentration, generalized polarization (GP) of the lanrdan increased, whereas it decreased with an increase in acetone concentration. These results can be explained reasonably by the "interaction free energy" concepL [1] Biophys. Chem., 74:237-249,1998. [2] Chem. Phys. Lipids, 85:53-65,1997. [3] BBA, 1330:199-206,1997. [4] BBA, 1284.'233-239,1996.

P 2 4 0 MICROSCOPIC IMAGING OF A CELL USING ENVIROMENT SENSITIVE,DYE, LAURDAN

Tetsuhiko Ohba, Tai Kiuchi,, Yoshitoshi, Kamakura, Akira Goto, Takaaki Kumeta and Kazuo O ~ Department of Physics, Tohoku University Graduate School of Science: Aoba-ku, Sendal, Japan 980-8578

Laurdan, a fluorescent probe for membranes, manifests a large spectral shift depending on polariiy of its environment. In lipid bilayer membranes, emission peak of laurdan changes from 440 nm in gel phase to 490 nm in liquid-crystalline phase. Using this unique property, the authors have constructed an imaging system that is able to observe spatial distribution of membrane polarity under a microscope. The system is composed of a fluorescence microscope, monochromatic filters, a cooled-CCD camera, and an image-analysis system. The image is expressed as generalized polarization (G.P.) [Parasassiet. al Biophys. J. 57 (1990), 1179-1186]. At first, the apparatus was applied to liposomes of synthetic phospholipids and CHO cells. Phase transition was clearly imaged on the liposomes, and a CHO cell was imaged by the difference of membrane polarlity.

This method was further applied to the study on nerve growth factor (NGF)-induced outgrowth of neurites in PC 12D cells, a subline of PC 12 cells. PC 12D cells extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP in the same way as primed PCI2 cells. Observations were performed on the following PCI2D cells stained with 2 mM laurdan in the medium. They were control cells (no NGF added), cells exposed to NGF for 30min, for 6 hours and for 2 days. Increase of membrane fluidity is observed in the cell body region just after the NGF stimulation.

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P241 MONTE CARLO SIMULATION OF TWO-COMPONENT BILAYERS: DMPC/DSPC MIXTURES.

I.P. Sugar*, T.E. Thompson', K.K. Thompson" and R.L. Biltonen" *Departments of Biomathematical Sciences and Physiology/Biophysics, Mount Sinai School of Medicine, New York, NY 10029. "Department of

Biochemistry, University of Virginia School of Medicine, Charlottesville, VA 22908.

In this paper we describe a simple two-state model of a two-component phospholipid bilayer system. Application of Monte Carlo methods to this model permits simulation of the observed excess heat capacity versus temperature curves of dimiristoylphophatidylcholine (DMPC)/distearoylphosphatidycholine (DSPC) mixtures as well as the lateral distributions of the components and properties related to these distributions. A comparison of the thermodynamic properties of DMPC/DSPC mixtures with the configurational properties shows that the temperature characteristic of the configuration properties correlate well with the maxima in the excess heat capacity curves rather than with the onset and completion temperatures of the solid/fluid phase transition. In the two-phase coexistence region we also found excellent agreement between the percolation threshold temperatures at different system compositions detected in FRAP experiments and the midpoint temperatures of the percolation of gel clusters calculated at the same compositions (Supported by grants from the NIH and NSF.)

P 2 4 2 Statistical Mechanical T h e o ~ for the Adsorption of Protein to Liposomal Membranes

Y. Suezaki, H. Ichinose, K. Takiguchi*, and, H. Hotani* Physics Laboratory, Department of General Education, Saga Medical School, Saga, 849-8501 Japan * Division

ol Molecular Biology, Graduate School of Science, Nagoya University, Nagoya 46401, Japan

Saitoh and others observed interesting topology changes of spherical lipid vesicles to coffee cup shaped vesicles, sheet-shaped vesicles, vesicles with more than one hole by adding protein, called talin (Saitoh, A. et al, Proc. Natl. Acad. Sci. USA 95, 1026 (1998)). These shape changes are due to the adsorption of talin to the peripheries of the vesicle. The adsorption isotherm of talin between an aqueous solution and the vesicle membrane was analyzed by taking account of the bending energy of the membrane. The equilibrium is determined by the balance of the energy gain for the adsorption of talin to the periphery of the vesicles and the change of the bending energy of the membrane due to the shape change. The apparent line tension energy changes as a function of the concentration of talin as a result of the competition between the entropy and the adsorption energy. The theory not only revealed clearly the nature of the exciting phenomena of the shape change of lipid vesicles, but also clarified its adsorption isotherm quantitatively. The observed coexistence of coffee cups and sheet-like vesicles were reproduced and the phase diagram was reproduced. Vesicles with two orifices were also analyzed and theoretically reproduced (Y. Suezaki, H. Ichinose, K. Takiguchi, and, H. Hotani, Biophys. Chem. in press).

P 2 4 3 Phase behavior of GM3/DPPC and 3-O-AcGM3/DPPC multilamellar vesicle

M. Akiyama 1, S.Matuoka 1, K.Tsuchihashi 9- and S.Gasa 2 1Dept.Phys. ~Dept.Chem.School of Medicine, Sapporo Medical University, Sapporo Japan

It is known that large amount of ganglioside GM3 is expressed in tumors and induced antibody towards GM3. The experiment on complement-dependent liposome lysis has shown that the reactivity of antibody M2590 with DPPC/cholesterol liposome containing GM3 abruptly increased above certain threshold of GM3 content(about 10%).

In the present work, we found phase separation into GM3-enriched domain and GM3-poor domain by means of x-ray diffraction in GM3/DPPC multflamellar vesicle. The GM3-enriched domain clearly appeared above 6 mol % GM3 content. The appearance of the GM3-enriched domain as a function of GM3 content resembles to dependence of the liposome lysis on concentration of GM3. This result suggests that M2590 recognize the GM3-enriched domain and consequently the threshold in reactivity with GM3 is observed.

Phase behavior of 3-O-acetylated derivative 3-G~AcGM3 in DPPC and 3-O-AcGM3/GM3 in DPPC will also be discussed.

118


P 2 4 4 Protein mediated galactosylceramide transfer from sphingomyelin/phosphatidyleholine mixed bilayer

vesicles - Implications for selective galaactosylceramide lateral partitioning

P. Mattjus*, J.G. Molotkovsky t, ll.M. Pike* and R.E. Brown', *The llormel Institute, University of Minnesota, Austin, MN, U.S.A., *The Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

Glycolipid transfer protein (GLTP), purified from bovine brain and specific for glycosphingolipids, has been studied using a fluorescence resonance energy transfer assay (FRET). Anthrylvinyl labeled lipids (galactosylceramide) and perylenoyi labeled lipids (triglyceride) were used as the FRET pair to follow the rate of GLTP facilitated transfer of AV-labeled galactosylceramide (AV-GalCer, 1 mol%) from donor to acceptor POPC vesicles. We previously showed that the GLTP mediated transfer process is significantly retarded by the presence of negatively charged lipids (5-10 tool%) in the donor vesicle surface. If the donor vesicle carries a positively charged lipid the transfer process is not retarded to the same extent. In this study we prepared donor vesicle composed to both sphingomyelin (SPM) and phosphatidylcholine (PC) at different molar ratios. We found that the AV-GalCer transferred from PC donor vesicles was faster than from SPM vesicles. Gradually increasing the content of PC of mixed PC/SPM vesicles did not however increase the transfer rate in a linear fashion, bul rather showed a considerably slower transfer rate for vesicles containing 5-50 mol% PC, compared to vesicles containing only SPM or PC. It is plausible that AV-GalCer partitioning among the two phospholipids changes at different mixing ratios and diminishes accessibility to GLTP. It is also possible that the transbilayer distribution of AV-GalCer between the inner and outer leaflets in the mixed vesicles is affected.

P245 FOLDING AND MEMRANE INSERTION OF AN OUTER MEMBRANE CHANNEL PROTEIN

Ashish Arora, JOrg H. Kleinschmidt, and Dept. of Molecular Physiology and Biological Physics, University of Virginia

Health Sciences Center, Box 10011, Charlottesville, VA 22906, U.S.A.

Outer membrane protein A (OmpA) of E. coli is shown to form channels with two different single channel conductance levels in planar lipid bilayers. In 1M KCI and at an applied voltage of 100 mV, the most frequent events are small channels of -60 pS conductance, but larger channels of -260 pS are also quite prominent. Both types of channels can be regenerated after unfolding the protein in 8 M urea and refolding it in the presence of detergent micelles or lipid bilayers. OmpA forms an 8-stranded antiparalle113-barrel in lipid bilayers. Refolding expedmefits show that the bilayer-water interface is critical for structure, formation. Several intermediates have been identified in kinetic experiments of the folding pathway of OmpA. Time-resolved distance determination by fluorescence quenching and polarized ATR- FTIR experiments show that the tryptophans of OmpA first move to the polar" headgroup region where most secondary structure is formed, then penetrate more deeply into the upper hydrocarbon region of the lipid bilayer, before they finally cross the bilayer to form the native I~-barrel. Similar experiments performed with single Trp mutants of OmpA indicate that the protein folds and inserts by a concerted rather than a sequential mechanism into lipid bilayers.

P246 EFFECTS OF SYNTHETIC ANT1OXIDANTS ON STRUCTURE OF ERYTHROCYTE MEMBRANE

O.G. Luneva. L.Ya. GendeL K.E. Kruglyakova, V.A. Fedin Biology Faculty. Moscow State University. Moscow. Russia:

Institute of Biochemical Physics of Russian Academy of Sciences, Moscow, Russia.

The influence of synthetic antioxidants from different chemical groups (phenol antioxidant - phenozan-1 (Ph-l) and derivatives of 3- hydroxypyridine (3-HP)) on the structure of the plasmatic membrane and morphology, of e rythrocytes was studied using a spin probe and scanning electron microscopy.

ESR spectra of the spin probe introduced in the intramembrane space demonstrate that antioxidants induced concentration-dependent structural changes in the e .rythroc.~e membrane.

Scanning electron microscopy revealed changes in e rythrocyte morphology as affected of Ph-l. The antioxidant in concentration range 10 -7 - 10 -3 M induced concentration-dependent changes in discocytes content: it decreased as the ceils were transformed to the forms with other surface architectonics by Ph-1.

Ph-I a t l 0 r - 10 -5 M acts as an echinoc3r agent and a t l 0 4 - 1 0 "3 M acts as a stomatocytogenic agent. Effects of the antioxidants are due to its distribution in various regions of the erythrocyte membrane and associated structural changes.

119


P247 Fluorescence Assay in Investigating Enzymatic Lipolysis of Phospholipids and Its Role in Membrane

Lipid Oxidation

O.S.Kuptsova 1, J.W.Borst 2, N.V.Visser z, A.J.W.G.Visser 2 ~Moscow State University, Division of Enzymology, Chemistry Department, Moscow, Russia 2MicroSpectroscopy Centre, Department of Biomolecular Sciences, Wageningen University and Research Centre, The Netherlands.

Phospholipids are important constituents of biological membranes and their physico-chemical properties in membrane arrangements have bccn the subject of extensive studies. The bilayer structure commonly assumed by phospholipids has been shown to be an integral part of biological membranes and a knowledge of biotransforrnations of membrane lipids and their structural behavior is essential for an understanding of membrane fimction. Unilammc}ar vesicles (liposomes) as model membranes have been used in this study to investigate hydrolysis of membrane lipids by phospholipase A2 and the relationship lipid peroxidation and lipolysis via application of fluorescence assay tccniques was estimated.

P248 CELL MEMBRANE CRYODAMAGES ACCORDING TO SPECTROSCOPY OF FLUORESCENT PROBES DATA

T.S.Dyubko Institute for Problems of Cryobiology and Cryomedicine of the Ukrainian National Academy of Sciences, 23 Pereyaslavskaya So'., Kharkov, 310015, Ukraine; Fax: + 380 0572 720084; E-mail: [email protected]~ [email protected] .or ua [email protected]

At this work cell membranes sU'uctural changes caused by fast freezing have been estimated by fluorescent spectroscopy method. Have been analyzed: (1) own protein fluorescence; (2) fluorescence spectra of probes localized at various areas of membrane; (3) effectivenessof energy transfer between donor-acceptor pairs located at the surface and at nonpolar areas of the membrane; (4) DSM probe fluorescence spectra inhomogeneous broadening parameters. The objects of a research were liposomes, proteoliposomes, containing integral protein, natural ewthrocyte and hepatocyte membranes. It is established, that the fast freezing reduces to irreversible conformational changes of biomembranes affecting as a surface, and also core of bilayer. At this case the role of integral protein presence at the bilayer in biomembranes response on cryoaction is essential. The role of membrane proteins and protein-lipid interaction disturbances in a reversibility of biomembrane structural changes caused by freezing is discussed.

P 2 4 9 Membrane molecular order in growing myelin figure

Toshihiko OGII-tARA and Kiyoshi MISHIMA A) Dept. of General Sci., Azabu Univ. ADept. of Phys., Showa Univ.

Myelin figures, which are composed of concentrically stacked multilamellae of lipid bilayers with a lot of water inside the tube, grow from lumps of lipids in excess water at the phase transition temperature. Myelin tubes formations of phosphatidylcholines were observed by optical microscope with cmssed-Nicols and recorded by a video system. The observed transmitted light intensities under crossed-Nicols were measured at a lot of points along the axis of during growth. The intensities measured were almost the same at all points along the axis. However, intensity was weak at an area near the root of tubes. It is said that myelin figure grows by concentration gradient of lipid. However, these results should prove that there is a growing point of myelin figure at an area near a root of a tube and myelin figure grows by swelling from the area.

120


P 2 5 0 The Re-establishment Kinetics of Membrane Premiability during Hydration of Axes Isolated from Triticum

aestivim L. seeds measured by NMR-method A.L. Shvaleva

Dept. of Biophysics, Biology Faculty, Moscow State University, Vorobyevy Gory, Moscow 119899, Russia

Nuclear Magnetic Resonance (NMR) method is one of the not numerous methods for the estimation of re- establishment kinetic of membrane permiability during plant tissue hydration, which allows to arrive information on atohaic-molecular level with no change of controls intact and with minimum action on sample. The aim of our work was the investigation of re-establishment kinetics of membrane permiability in norm during hydration of axes isolated from winter wheat (Triticum aestivum L.) seeds differ in drought tolerance cvs and at different stages of storage by NMR. Spin-spin relaxation times-T 2 examination ,allow us estimate that the re-establishment of axes membrane permiability in normal take place over a period of time. The harnessing of paramagnetic ions (Mn 2§ Cu 2§ Fe 3+) reinforce this fact and suggest the availability of transmembrane water exchange between inside and outside cell space at the first stage of seed hydration. The role of membrane structure in water uptake and their later distribution at seed endosperm discussed.

P 2 5 1 The analysis of membrane potential oscillations and mechanism of transportation processes through intact

excited membrane

N.t~. Radenovi6 I, D.M. Minic 1, M.G. Jeremi61 and t~.N. Radenovi62 Faculty of Physical Chemistry, University of Belgrade, Belgrade, Yugoslavia

Maize Research Institute, Zemun Polje, Laboratory of Biophysics, Belgrade-Zemun, Yugoslavia

The analysis of membrane potential oscillations in intact excited membrane of plant cells and tissues was carried out. Different classes of membrane potential oscillations were encompassed by the analysis. Specific parameters of membrane potential oscillations were identified. Furthermore, the behaviour of certain oscillation classes of membrane potential was determined, first of all, in relation to a mechanism of transportation processes occurring in both directions - in and out of cell, i.e. through its excited membrane. It is shown that these processes appeared according to the combined pattern of a passive, active and oscillatory type of ion transport. This type is caused by adequate conformational changes, i.e. by the type of movements of particular molecular structures in intact excited membrane.

P 2 5 2 THE ROLE OF MEMBRANE STRUCTURE IN ACTIVATION

OF MITOCHONDRIAL PHOSPHOLIPASES

Aripov T.F?, Tadjibaeva E.T. b, Vagina O.N.b,Zamaraeva M.V. b, Salakhutdinov B.A. s

a)Sadykov Institute of Bioorganic Chemistry, Uzbek Academy of Sciences, Tashkent,Uzbekistan, b)Ulugbek Tashkent State University; Tashkent, Uzbekistan

The effect of membrane active peptides on the phospholipase activities and structure of mitochondrial membranes has been studied.

It has been shown that crambin inhibits phospholipase activity whereas thionine increases this activity. These effects are based on the ability ofpeptides to cause different changes in the mitochondrial membrane structure. It has been shown by ESR studies that crambin induces the changes in mobility and orientation of hydrocarbone segments of the phospholipide acyl chain along its length. In contrast, thionine basically affects the lipid structure near the surface of the membranes.

121


P253 Structural Studies of the Epsilon-Toxin from C. Perfringens,

A. Co!r M. Poppofl ~. C. Naylort., IL Titbali 3. & A. K. Basak 1. IDept. of Crystallography, Birkbeck College, UK, 2pasteur Institute, France. 3CBDE, Porton Down, UK.

Epsilon toxin is a potent, 32kDa toxin produced by the bacterium C. perfringens. Of the five types of C. perfringens (A-E) only types B & D produce the 8-toxin. These strains have a limited host range and are mainly isolated from sheep, occasionally from goats and cattle, but never from man. The secreted toxin is activated upon removal of a 13 residue N-terminal peptide by trypsin digestion. The mature toxin is highly toxic with derr,~oneerotic and oedematic activities. The toxic properties appear to be due to its ability to bind to vascular epithelial cells causing an increase in vascular permeability leading to vascular damage and oedema in many organs such as the brain, heart, lung and kidney. Its mode of action is still not clear and no enzymatic activity has yet been established, though through chemical modification experiments it has been possible to ascertain certain residues that are likely to be essential for lethal activity. In order to determine the 3D-structure of this protein and to establish its function the protein has been isolated from the bacterium C. perfrmgens and subsequently purified to homogeneity. X-ray diffraction quality crystals have been grown which diffracts to 2.4A resolution. The crystals exhibit hexagonal symmetry with unit cell dimension a=b=123A, c=125A, r ~ y=120 ~ and belongs to space group P6322. Recently we have collected a native data set to 2.5A resolution and the heavy atom derivative search is in progress. The details of the sm~cture will be presented.

P254 Opening and closing of the active site of gas-gangrene ~-toxin may trigger by membrane binding.

J. T. Eaton t, C. E. Naylor I, N. Justin I, D. S. Moss I, IL W. Titball 2 & A~ K. Basak l. ~Department of Crystallography, Birkbeck College, London, 2DERA, CBD Porton Down, Salisbury, UK

A wide variety of Gram-positive and -negative bacteria produce phospholipases C (PLC) with different properties and specificities. The cL-toxin of C perfringens (CP) was the first bacterial toxin to be shown to be a Zn dependent and Ca +2 activated metallo enzyme: a phospholipase C, is the key virulent determinant in gas-gangrene and a wide variety of other diseases in man and animals. The protein is composed of a single polypeptide chain of 370 amino acid residues with a molecular mass of 42.5 kDa. We have determined the 3D-structures of 0r-toxin in a number ofa , ystal forms, in presence and absence of Ca+2, from two different bacterial strains: NCTC-8237, CER89L43 and a divergent strain isolated from

.. . . . . . , an infeoted swan. All the structures are composed of 2-domains, the N-terminal, (ct-helical) ~ / catalytic, shows an anticipated structural homology to the non-toxic B. cereus PLC while the

~ . _ f /~" 9 C-terminal domain essential for the membrane binding adopts an unexpected eukaryotic ~ ' " ~ , ' ) ~ ' " calcium binding C2 fold. The overall topologies of all the r struotures are very similar.

Conformationai changes are responsible for opening and closing the active site clet~ and could play an important role in allowing the entry of substrate. The details of the structural work together with possible mechanism for membrane binding will be explained.

P255 Direct Observation of Membrane Fusion Process

F. NOMURA, M. NAGATA, S. ISHIKAWA, K. TAKIGUCHI, S. TAKAHASHI and H. HOTANI Department of Molecular Biology, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan

Membrane fusion plays an essential role in cellular activity such as exocytosis, vesicle transport among various cell organelles and release of neurotransmitter at synapses. To investigate the mechanism of membrane fusion, we used liposomes as a model of biological membrane, monitoring the fusion process in real time by using optical daxk-field microscopy. To induce the membrane fusion, we used the following peptides, @ HA (influenza hemagglutinin) peptide: 20 residues peptide derived #om amino-terminal end of HA2 subunit, ~)synthesized analogue peptides of HA; negative charged analogue (ES) or positive charged analogue (KS), (~)bee venom peptide: melittin.

We succeeded to visualize the membrane fusion process induced by melittin or the mixture of E5 and K5 at neutral pH. in ~ t i o n to the fusion phenomenon, various behaviors of membrane vesicles inducing aggregation, shrinkage and opening up of vesicles were observed,.d~ending on the buffer conditions. However, HA or F_.5 alone did not induce the membrane fusion at acidic pH, where previously reported as the optimal conditions for the membrane fusion. The fusion occurred only at the narrow range of the conditions with the peptide concentrations, ionic strength and lipid composition of the membrane. Direct and real time observation by using dark-field microscopy is a powerful method to study the dynamic behavior of membrane vesicle.

122


P 2 5 6 Heat Shock Response in Yeast Cell Membrane

Kaam_0~a~c~i_ National Institute of Bioscience and Human-technology, AIST

Hsp30 is only one plasma membrane integral heat shock protein in Saccharomyces cerevisiae ~. It was shown by

NMR relaxation time measurement of membrane lipids in intact cells and fluorescent anisotropy measurement of

DPH bound to intact cell membrane that the Hsp30 integration suppresses thermal enhancement of membrane

fluidity. However, the heat shock did not affect unsaturated fatty acid ratio in the membrane. The Hsp30

integration also suppresses thermal enhancement o f H+-ATPase activity and promotes recovery from hyperthermic

growth interruption. It is concluded that suppression of plasma membrane fluidity results fromHsp30 integration

and suppresses tC-ATPase activity during the heat shock exposure. As a result, ATP is saved for the following

recovery from the damage.

P 2 5 7 EFFECT OF IONIC STRENGTH ON THE INTERACTION OF PROTEGRINS WITH LIPOSOMES

I I 2 2 I t F . ~ , Manfred Kriechbaum, Robert L Lehrer, Alan J. Waring and Karl Lohner lnstitut for Biophysik und ROntgenstrukturforschung, Osterreichische Akademie der Wissenschaffen, Graz 2 (Austria)and Department of Medicine, UCLA, Los Angeles, California (USA)

Protegrins are small amphiphilic peptides (-~2kDa) with potent antimicrobial activity which does not seem to be correlated to a specific receptor, but rather to perturbation of the barrier function of cell membranes. An understanding of how the peptide distinguishes between bacterial and erythrocyte membranes would allow to design novel peptide antibiotics. Interest in this research area has grown since the number of bacterial strains being resistant to conventional antibiotics has dramatically increased. Therefore, in order to gain insight into the mechanism of specific membrane damage we studied the interaction ofp'rotegrin-I with model membranes.

Although the antimicrobial activity does not seem to be affected by the ionic strength of the media, X-ray and light scattering experiments indicated that depending on the salt concentration protegrin-I can adopt different oligomeric states in phosphate buffer. The radius of gyration, deduced from X-ray particle scattering, suggests that the peptide exists as dimers at low ionic strength (0-50raM NaCI) and as hexa- or octamers at higher ionic strength (130-300 mM NaCI). Microcalorimetry showed that the phase behavior of liposomes composed of phosphatidyl- glycerol, a major phospholipid component of bacterial membranes, was also affected to different extent by the peptide depending on the environmental conditions. Further biophysical studies will help to elucidate if there is any correlation between the ag~re~,ation state of the oentide and the mechanism of membrane damage.

P 2 5 8 Peptide-induced Perturbation of Membrane Organization

Susanne Gangl, Bernd Mayer and Gottfried K0hler Institute of Theoretical Chemistry and Radiation Chemistry, University of Vienna, Austria

The three dynamically discemable aspects of lipid-peptide interactions, firstly, fold towards an a-helical structure, secondly, interaction and incorporation into membranes, and thirdly lateral diffusion and aggregation within a membrane are analysed with two model peptides using fluorescence- and CD spectroscopy and fluorescence imaging microscopy. The investigated peptides are an alanine-based a-helical peptide and a transmembrane peptide derived from human Glycophorin A. The unfolding of the ct-helix of a ! 7 residue alanine-based peptide was characterized by CD spectroscopy. The end-to-end distance distribution of a dansylated derivative of the same peptide was assessed using fluorescence resonance energy transfer measurements. In agreement with theoretical structure predictions both helix content and the average end-to-end distance decreased with temperature. The step of membrane insertion was investigated with a 26-residue transmembrane peptide which carries the dimerization motif derived from Glycophorin A and a basic N-terminal sequence. The role of positively charged amino acids as determinants of peptide topology and the role of different 'lipid chain lengths were investigated. Changes of the physical properties of the lipid bilayer in the presence of membrane peptides were investigated by fluorescence resonance energy transfer, fluorescence anisotropy measurements and time resolved fluorescence spectroscopy.

123


P 2 5 9 INTERACTION OF LEUCINYL-PHENYLALANYL-VALINE WITH LIPOSOME BI LAYER

J.Shobini & A.K.Mishra, Department Of Chemistry Indian Institute Of Technology, Madras.Chennai - 36.

We were interested in studying the effect of an essentially hydrophobic tripeptide LFV on the phase transition behavior of a synthetic DMPC iiposome. In this work we have examined the peptide-lipid interaction by studying the interaction of DMPC liposome bilayer with LFV using fluorescence spectroscopy. Three classes of fluorescence probes like ANS, DPH, and 1-ROH have been used to monitor the interaction. Partition coefficient of these probes decreases markedly in the peptide incorporated liposomes. This shows that the membrane permeability is decreasing. The variable temperature studies exhibit i) a decrease in the ratio of neutral/anionic form of ROH, ii) a red shitt of ANS emission and iii) a decrease in the steady state polarisation vlaue of the DPH in lipid bilayer in the presence of LFV. This clearly shows that the phase transition temperature of the lipid is broadening in the presence of the peptide. These fluorescence behavior of all the three probes shows that the peptide induces copmactness of the bilayer.

P 2 6 0 Model system study for cell adhesion with cyclic RGD peptide

. Z. Guttenberg, B. Lortz, B. Hu, E. Sackmarm Physik Department E22, Tech. Universitat Mllnchen, James Franck Str., 85748 Garching, Germany

The human platelet transmembrane protein O~llbl~lli) is a member of the integrin receptor family and binds to the RGD sequence of extracellular-membrane proteins like fibrinogen or fibronectin. To establish a model system for cell adhesion, we used a cyclic RGD-containing hexapeptide, synthesized with lipid anchor or linked to biotin. The binding kinetics of the peptide protein-bond was measured with surface plasmon resonance and the behavior of the lipo-peptide in lipid membranes was investigated with filmbalance and DSC. For the model system the lipid anchored peptides were embedded into giant DMPC vesicles, that also contained lipids with a polymer headgroup, to prevent unspecific binding. For the experiments the integrin was either reconstitued to a supported lipid bilayer or the solubilized protein was bound directly to the surface. The adhesion area of the vesicles was observed with an interferometric technique, together with a flow chamber, that allowed the measurement of the bending modulu~ ~:, the membrane tension Z and the adhesion energy W. The influence of different peptide and lipo-polymer concentrations was tested and adhesion forces were measured for some special cases. Also differences in cluster formation for fluid and immobilized receptor protein could be observed.

P 2 6 1 Glutathione Depletion of Erythrocytes and Structural - Function State of Membrane Proteins and Lipids

Kozlova N.M., Lukyanenko L.M., Antonovich A.N., Slobozhanina E.I., Chernitsky E.A. Institute of Photobiology, Natl. Acad. Sci. Belarus, Akademicheskaya Str., 27,

Minsk, 220072, Belarus. [email protected].

It is shown that the depletion of intracellular glutathione (GSH) by 80% through the treatment of human erythrocytes by 1 mM l-chloro-2,4-dinitrobenzene leads to the increase of thiobarbituric acid-reactive substances (TBARS) formation and to decrease of intensity of 1,6-diphenyl- ! ,3,5-hexatriene fluorescence, the later correlating with TBARS formation (r=0,92; p<0,05) and with the decrease of GSH (r=-0,82; p<0,05). Judging from parameters of intrinsic membrane fluorescence, fluorescence of membrane-bound 4,4- diisothiocyanostilbene-2,2-disulfonate and from the activity of membrane-bound acetylcholinesterase the structural state of principal membrane proteins in this case did not change but the activity of membrane-bound methemoglobin reductase was reduced. The obtained data suggest that depletion of intracellular glutathione leads to the change of the structural state of membrane lipids but only those which are in contact with methemoglobin reductase (lipids which are in contact with acetylcholinesterase were unchanged).

124

C2 : Membranes :Ion-Channels, Pumps and Carriers

P 2 6 2 Deceleration of O-pyromellitylgramicidin channel kinetics in bilayer lipid membranes

by polylysines of different chain length. Andre~ V. IOvlov l'z, Yuri N. Antonenko ~, Elena ,4. Kotova ~, and Alexander A. Yaroslavov z

i, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia; z Department of Polymer Sciences, School of Chemistry, Moscow State University, Moscow 119899 Russia

This work deals with the effect of polylysines of a different chain length on the kinetics of ionic channels formed in a bilayer lipid membrane (BLM) by O-pyromellitylgramicidin having three negative chArges at the C-terminus. The method o f sensitized photoinactivation is applied to study the channel association-dissociation kinetics. The time course of Xhe flash-induced decrease in the transrnembrane current follows a single exponential decay that is characterized by a time constant ~. According to our previous results (Krylov et al., FEBS Letters 1998), the addition of polylysine to the bathing solutions of BLM leads to the deceleration of the photoinactivation'kinetics. Here we studied this effect with a series of polylysines differing in the~ chain length. The value ofx is shown to increase with the increase in the polylysine molecular weight. It has appeared that the effect essentially depends on the ionic strength, being diminished at medium and completely abolished at high electrolyte concentrations. It is concluded that polylysine causes the segregation of O-pyromellitylgramicidin molecules into domains, which disrupt at high polyelectrolyte concentrations.

P 2 6 3 Evidence for Nonlinear Capacitance in Biomembrane Channel System

Subhendu Ghosh, Areal K. Beta and Sudipto Das Department of Biophysics, University of Delhi, South Campus, New Delhi

The electrophysiological properties of voltage dependent anion channel from mitochondrial membrane have been studied in a bilayer membrane system. It was observed that the probability of opening of the membrane channel depends on externally applied voltage and the plot is a bell shaped curve symmetric around the probability axis. A scheme of conformational energy levels under varying externally applied voltage was formulated. Assuming the probability follows Boltzmann distribution we arrive at an expression of change in energy containing a separate term identical to the energy of a nonlinear capacitor. This fact indicates the existence of an added capacitance due to the channel protein. Further it was shown that the aforesaid channel capacitor could be a function of voltage leading to nonlinearity. We have offered a general method of calculating nonlinear capacitance from experimental data on opening probability of a membrane channel. In case of voltage dependent anion channel the voltage dependence of the capacitor has a power 0.786. The results have been "interpreted in view of the structural organization of the channel protein in the membrane. Our hypothesis is that the phenomenon of capacitor behavior is a general one for membrane channels.

P 2 6 4 Accessibility of Tryptophans in the Single Tryptophan Mutants of Mitochondrial Uncoupling Protein

UCP1 Eva U ~ o v & Mascod Jelokhani-Niaraki, Karl Freeman.and Petr Jezek

Inst. Of Physiology, Czech Aead. Of Sciences, Vidensl~i 1083, 142 20, Prague 4, Czech Republic

Quenching of emission of the two existing tryptophans, W173 and W280, in the uncoupling protein (UCPI) of brown adipose tissue mitochondria in its wild-type and in WI73A and W280A mutants was studied using a wide range of quenchers such as iodide, acrylamide, copper, and pyridine-based quenchers, and monitoring both steady-state and time-resolved fluorescence. Contrary to Viguerra et al. 1992, (Eur. J. Biochem. 210: 893-899). we found that W280 is generally more accessible to most of the quenchers tested, in spite of its supposed location in the middle of the sixth trans membrane cx-helix. Comparing the results from steady-state and time-correlated- single-photon-counting measurements we distinguished between the static and dynamic portions of quenching. For actylamide, iodide, N-methyl picoline and collidine HCI only W280 was quenched statistically, suggesting an existence of a water-accessible-space in its vicinity. This could be compatible with the existence of a water-filled cavity of the purine nucleotide binding site, located between the fourth, fifth and sixth transmembrane a~-helices of UCPI. On the contrary, W173 was less accessible to acrylamide, iodide, N-methyl picoline, and collidine; was quenched only dynamically, all which is consistent with its buried location, most probably shielded by the third or second matrix segment protruding towards the central plane of the fipid bilayer. Moreover, significantly weaker quenclling of W173 by iodide and complete absence of its quenching by nicotinic and pyridine sulfonic acid shows that there is a negatively charged residue near to W173.

125


P 2 6 5 EFFECTS OF ARGIOPE LOBATA SPIDER TOXINS ON N-METHYL- D-ASPARTATE RECEPTOR

P.B.Usmanov, A.Ongarhaev and A.K.Tonkikh

Insti~e of Physiology and Biophysics, Tashkent 700095, Uzbekistan

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor is a important modulator of synaptie activit~ in the central nervons system. The NMDA receptor r site for glutamate (OrNMDA) anda number of distinct binding sites for glycine, Mg 2+ , Zn 2+ and ope~ channel blockers such as phenc3~lidine and MK-80t. The effects of lowmole~ar weight toxins AV-2-t andAV-7-t, isolated t~om Arg/ope lobataspider venom, on the NMDA receptor were studied by using ligand binding assays with [3I-I]-gtutamate and [3I-1]- MK-801. The AV-2-1 ( 0.01 - 100 pM ) markedlyinhibited the binding of [~H]-glutamate to NMDA receptors on membranes prepared from rat brain. In contrast, AV-7-1 have biphasic effects. At low concentrations ( 0.01- 10 ttM) the binding of [3H] -gl, t~mate increased, but at cencontrations higher than 10 tdV[ it was inhibited. Similarly, in binding assays with [3H]-MK-801, AV-2-1 had also only inhibitory action, whereas AV-7-t had both stimulatory (0.01-1. ttM) and inhibitory ( > l ~ M ) e f f e c t s , Our results indicate that Argiope lobata spider toxins may prove useful in distinguishing various sites of NMDA receptor and for studies of the physiological significance of these sites.

P 2 6 6 Microelelectrode measurements of water transport across membrane channels

Peter Pohl, and Sapar M. Saparov Martin-Luther-Universitdit, lnstitut fur Medizinische Physik und Biophysik, 06097 Halle, Germany

The competition of ion and water fluxes across gramicidin channels was assessed from the electrolyte concentration distribution measured in the immediate vicinity of a planar bilayer. The osmotic water flow that passed through the membrane was calculated from the dilution of an impermeable solute within the unstirred layer (USL) at the hypertonic side or from an increase in its concentration at the hypotonic side of the membrane. From the simultaneously measured concentration profile of a permeable solute, the flux of the permeable ion-species was obtained. It decreased when the transmembrane difference in the concentration of the permeable solute was diminished by salt addition to the hypertonic compartment. But even at zero concentration difference, a significant transmembrane ion flux retained. Ufider these conditions pseudo solvent drag is excluded. Sodium and potassium ions are dragged through the gramicidin channel solely by an osmotic volume flow. Their reflection coefficients are equal to 0.26 and 0.43, respectively. From measurements of the transmembrane current that were carried out in parallel to the microelectrode measurements, the hydraulic conductivity per pore was calculated. The method of combined microelectrode and current may be very useful for the characterization of natural water channels (aquaporins). Financial support of the Deutsche Forschungsgemeinschaft (Po 533/2-2) is grateful acknowledged P 2 6 7 COMPARATIVE STUDIES ON THE ACTIVATION OF STORE DEPENDENT CALCIUM CHANNEL IN JURKAT T CELLS BY MONENSIN, OKT-3 AND THAPSIGARGIN. P. Harikumar I and J.P. Reeves z I .FT Division, BARC, Mumbai 40000085; 2. Dept of Pharmacology and Physiology, UMDNJ, NJ 07103, USA.

Monensin, the Na+/H + ionophore caused release of calcium from internal stores and store dependent Ba § uptake in Jurimt T-lymphocytex Moneusin sensitive Ca 2+ store was quantitatively similar to the inositol-l, 4, 5-triphnsphate (IP-3) sensitive store stimulated by the SERCA- ATPmse inhibitor, thapsigargin (1 pM) and by OKT-3 (2 Izg/ml), the monoclonal antibody against CD-3 of the T- rell antigen receptor eompleL The effects of mouensin were dependent on the presence of Na + in the extrnceflutsr medium. Moncasiu did not indnce Ca + release when added after gramicidin (2 pg/ml), t r forming ionophore seler for monovalent cations, suggesting that a Na + gradient aernss the membrane was necessary to elicit monensin indnced Ca + release. Monensin transiently increased r162 pH when added in the presence ofextrar Na*, but not in the presence of gramicidin. Other methods ofcytosolir alkalinizattoL such us adding 20 mM NII4CI, or preloading the cells with 30 mM Na Acetate followed by dilution into acetate free medium also caused Ca 2+ release and store dependent Ba z+ entry. Monensin also exhibited a potentiating influence on store operated channel as evidenced by a marked stimulation of the initial rates of Ba 2+ entry after store depletion by thapeigargin and OKT-& Analysis of the kinetiC data with Hill equation pointed to a two fold decrease of Km for Ba z* from 4 to 1.7 for untreated and monensin treated cells respectively. We r162 that the effects of monensin on Ca 2+ release and stimulation of store dependent channel are due to r162 alladinization, which has been reported to increase the sensitivity of Ca 2+ stores to IP-$. The effects of monemsin on Ca 2+ homeostasis should be borne is mind when interpreting the effects of this agent on the funtioas of Golgi or intrar acidified organelles.

126

C2 : Membranes : Ion-Channels, Pumps and Carriers

P268 Aconitine modification of the cz mbunit of rat brain type II A radium channel

Rao, S and Sikdar, 8.K. Molecular Biophysics Unit, Indian Institute of Science, BanL, alore-12.

Employing the whole-ceil patch clamp technique (I5~ we have gud/ed the effects of aconltine (AC), a site 2 b/rid/rig alkaloid toxin, on the c~ subenit of the rat brain type lI A rod/urn ~lmnne! being e , ~ hetemlogously in chinese lmmxter ovarian (CHO) ceil line., this being devoid of any endogenous elmnneL~ Th~ ~ o f AC modification was ~milar to that descnl~ed by Grischenko ct al (1986) for'the Ha channels of neuroblastoma ceils in their native state. The extent of modification was stimulation dependent. Modification was a c o o ~ e d by about 70% reduction in peak current amplitude, about -40mV shift in activation threshold, a -10mV thi~ in the current peak and a drastic increase in h'I-h* permeability. We studied the trend of AC modification as a function of depolari,in~ pulse number with a two-pulse grotocol, the first pulse to 0mV to record tra~r currents ~ and a second pulse to -50mY to record AC-modified cu.e,~ts (I~0. The progressive decrease in I# and progressive increase in ~ with pulse number correlated well and were exponential in nature. AC (50pM) when added in pipette brought about modification four times as fast as when AC was added in the bath solution. This was obvious from the pulse cons~a-L ~, be/rig around 50 in the former and about 200 in the latter case. 1000 pulses were sufficient for ~ to saturate in the latter case indicating that almost all channels were in modified state. We conclude that the binding site for AC may fie atthe cytoph~'miC end close to the pore and that the presence of 1~ subunit/s is not essential for the binai,~ of AC. (supported by CSIR and DST, India~

P269 Interaction of Tetrapentylammonium with nomal and veratridine modified RIIA Na channel ot-mbuniL Ghatpande,/kS., Rao, S. & .~ikdar, S.K. Molecular Biophysics Umt, Indian Institute of Science, Bangalore-12.

Recently, we showed that the pentapeptide KIFMK, containing the clustered hydrophobic aminoacid residaes I,F&M in the intracellular linker between domains UI and IV of the voltage gated sodium channel, known to restore fast inactivation and block open sodium channels (Eaholtz et al., Neuron, 12: 1041-1048, 1994), decre~_ugl the 'on-rate' of veratridine (VTD) binding without affecting the 'off-rate' (Ghatpande & Sikdar, J. Membr. Biol. 160:177-182, 1997). This suggested that KIFMK and VTD compete for the same binding site in the RIIA Na+ channel, and that VTD reaches the binding site from the cytosolic side. We extended the study to the ion channel blocker, Tetrapentylammonium (TPeA). Experiments were conducted on RIIA Na channel ot-subumt stably expressed in CHO cells (Sarkar, Adhikari R, Sikdar, J1. Physiology (Lon~) 488.'633-645, 1995) in the whole-cell mode of the patch-ciamp technique; TPeA (20-100 p.M) was included in the patch pipette soln. and VTD (100-200 la,M) was added to the bath. Tetrapentylammonium showed use and voltage-dependent block of normal RIIA Na channel However, it did not affect the voltage-dependent gating kinetics of VTD modified RIIA Na channels and the I-V relation, although it affected the inactivation deficient chloramine-T modified channels in a potential and use-dependent manne~, suggesting that loss of TPeA binding was not a-result of removal of inactivation by VTD. The results can be intetlaeted by considering that the TPeA receptor overlaps with the VII) receptor, and modifieation or occlusion of this receptor by VTD prevents TPeA binding, [Sut:Corted by DST & CSIR, India]

P270 ELECTROPHYSIOLOGICAL EFFECTS INDUCED BY AUTO-ANTIBODIES IN RABBIT VENTRICULAR CARDIOMYOCYTES. del Corsso, C', Campos de Carvalho, A.C. and Varanda. W.A. - Dept. of Physiology-University of S~lo Paulo Brazi-i-

It has been shown that auto-antibodies present m sera of patients with Idiopathic Dilated Cardiomyopathy (IDC), like the antibodies against the 13] adrenergic receptor and muscarimc receptor (type MD, can interact with the receptors 131 and M: of cardiomyocytes producing t~chycardia and bradycardia, respectively, when added in preparations of cultured cardiomyoc)aes. In this research we are investigating the electrophysioiogieal effects of the total sera and the IgG fractions of patients with IDC on the whole heart and isolated cardiomyocytes preparations, respectively The patients with IDC are selected in accordance with the criteria of World Health Organization and the cardiomyoc~es are obtained from young rabbits (New Zeland). In experiments using the Langendorff technique, the total sera induced bracb, eardia (dilution 1/50 v/v) in 30% of the eases (N=30). Using the whole cell configuration of the patch clamp technique, we tested purified lgG of 5 patients who were able to induce the above changes in ECG. and we observed that the addition of 300p.g/ml of lgG produced a decrease of 28% in the isoproterenol (IBM) stimulated Ca ++ current. These effects were not completely recovered after 15 minutes of washout with Tyrode solution. Addition of an excess of cAMP to the pipette solution abolish the effect of the IgG. These results show that the sera of our patients with ~ havc antibodies which interact with the musearinic receptor type M2 of rabbit cardiomyoc)~es in an agonist manner. This interaction most probably activate G-coupled-M2 musearimc receptor leading to a decrease in cAMP production. Financial support: FAPESP: CNPq', CAPES

127


P271 OmpAl71 and OprF177, two N-terminal fragments from outer membrane proteins of E. coli and P.

fluorescens induce similar ion channels into planar lipid bilayers. C. EIHamel, E. Dt, N. Saint and G. Molle.

UMR 6522 CNRS, IFRMP23, University of Rouen, 76821 Mont Saint Aignan, France.

The outer membrane proteins OmpA of E.coli and OprF of Pseudomonas fluorescens are able to induce ion channels into planar lipid bilayers under applied voltage. These proteins, in contrast to usual trimeric porins as OmpF or OmpC would be monomeric. Recently a 171 recombinant N-terminal fragment of OmpA was cristallized and its 3 D structure was determined unambiguously as an extented eight-stranded 5-barrel. The reconstitution of OmpAlT1 (1) into lipid bilayers exhibited channel activity and conductance values about 110 pS in IM KCi and 75 pS in IM NaCL were determined. These results indicate that an eight-stranded 6-barrel is able to form ion channels into lipid membranes. In the same way, an 1-177 N-terminal fragment (OprF177), purified and characterized after pronase digestion of native OprF, is still able to form channels with conductance values similar to the ones observed above with OmpAl71. From tiffs homology, we can deduce the OprF 177 fragment would be constituted by an eigllt-stranded g-barrel. It remains to determine the real contribution of the C-terminal part in the formation of the pore.

l-We thank Pr Schulz (University of Freiburg, Gernmny) for the generous gift of OmpAl71 protein.

P 2 7 2 Modulation in Voltage Sensing and Transduetion in Human K + Channels by the N-terminus

Anurag Varshne~/and M.K. Mathew National Centre for Biological Sciences, GKVK Campus, Bangalore 560 065, India

Voltage-gated ion channels are the tetrameric proteins with eacli subunit consisting of six transmembrane segments and contributing a re-entrant loop to the channel-lining pore. Potassium channels are now among the most intensely studied membrane proteins and most salient functions have been mapped on to distinct portions of the protein. The fourth traasmembrane segment ($4) has been shown to move in response to changes in membrane potential and this movement, in turn, has been postulated to result in channel opening or closing. No detailed mechanism for the transduction of voltage sensing to channel opening has been proposed. We have constructed chimeras from our collection of human voltage-gated potassium channels and expressed them in Xenopus ooeytes and characterised the currents elicited in response to voltage pulses delivered using a voltage clamp amplifier. We have previously shown that attaching the N-terminal cytoplasmic domain b_Kvl.4 to the body of hK, l.l results in a slfift of operating range to hyperpolarizing potentials. We now report that the reverse chimera, ie N-terminus of hKvl. 1 attached to rite body hKvl.4 results in dramatic alternations in its voltage sensitivity although it still functions as an outward rectifier. Thus interacdions of the N-terminus with cytoplasmic loops may prove a general mechanism for modulating voltage sensitivity.

P 2 7 3 Fluorescence Scanning of the Shaker K + Channel

E. Loots, E.Y. Isacoff Molecular and Cell Biology, University California at Berkeley

Voltage-gated ion channels open and close in response to changes in membrane potential. A positively charged fourth transmembrane domain is believed to function as a voltage sensor, transducing the change in the electric field into a conformational change in the channel; opening and closing ion clmnnels in an orderly manner. We have examined the conformational changes occurring around this fourth transmembrane domain during channel gating and thus how it may function and interact with file rest of the channel during channel activation (opening) and channel inactivation (a nonconductng state following opening). To obtain information concerning the structural changes occurring during gating we have employed a technique called voltage clamp fluorometry. This technique involves covalently attaching an enviromnentally sensitive fluorescent probe to an area of interest within the channel, and then observing how the fluorescence of this probe changes as the channels are moved from one state to another. By examining multiple sites along the fourth transmembrane domain and around the external section of the pore region we are able to observe how these elements change and apparently interact during the gating processes.

128


P 2 7 4 Regulation of Ryanodine Receptor Calcium Release Channel by DIDS-binding 30 kDa Protein

and NaohiroYamaguchi" Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka

560-8531, Japan

Recently, we have found that DIDS-binding 30 kDa protein of skeletal muscle sarcoplasmic reticulum (SR) regulates excitation-contraction (E-C) coupling, and is very similar to ADP/ATP translocase (AAT), which is mainly present in mitochondria (Biochem. J. 335 (1998) 541-547). Here we investigated the effects of an antibody against AAT and two drugs (DIDS and atractyloside (ATR)) which bind to AAT specifically on the gating property of SR Ca 2§ channel. Consequently, the antibody and DIDS activated the channel, on the other hand ATR inhibited. Especially, the effect of ATR was very unique. At 10 pM of cytoplasmic C.a 7~, ATR decreased the rate constant of macroscopic ion influx through the Ca 2§ channels measured by the light scattering method up to about 60 % and perfectly inhibited about half the population of single C.a 2. channels incorporated into planar bilayers. Furthermore, the inhibition of the Ca 2. channels by ATR was effective at lower Ca 2.. These results support the previous results that AAT exists in the skeletal muscle SR and plays a key role in skeletal muscle E-C coupling, and the number of Ca 2+ channels regulated by AAT is thought to depend on the cytoplasmic Ca 2. concentration, that is degree of excitation of skeletal muscle cells. "Present address: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599-7260, USA.

P 2 7 5 S E L F R E G U L A T I O N O F G A P J U N C T I O N B Y P H O S P H O R Y L A T I O N

P a r a m i t a G h o s h a n d S u b h e n d u G h o s h . D e p a r t m e n t o f B i o p h y s i c s , U n i v e r s i t y o f D e l h i S o u t h C a m p u s , N e w D e l h i - 2 1

Gap junction proteins form cell-to-cell communicating channels in multicellular organisms. We demostrate that rat liver gap junction protein gets phosphorylated by an intrinsic protein kinase. This phosphorylation regulates the permeability of the channel as monitored by spectrophotometric assay. Also, the phosphorylation regulates the functioning of the channel by facilitating its closure at single channel level in planar lipid bilayer as monitored by electrophysiological studies (BLM). Dephosphorylation helps the channel to resume back to its normal functional state. The findings for the first time demonstrate self regulatory mechanisms of rat liver gap junction by phosphorylation.

P 2 7 6 QUATERNARY AMINE LOCAL ANESTHETICS INHIBIT G PROTEIN-COUPLED RECEPTORS

Joseah TiL~vi 13, Gabor Tigyi 23, Karoly Liliom 2 and Ricardo Miledi 3 IBiophysical Inst. University Medical School Pecs, Hungary, 2Dept. of Physiology, University of Tennessee

Memphis; 3Dept. of Psychobiology, University of California Irvine, USA.

Effects and the mechanism of action of quaternary amine local anesthetics were studied in voltage-clamped ovarian follicles and oocytes from Xenopus laev&. 1 mM procaine completely abolished the oscillatory inward currents elicited by acethylcholine and lysophosphatidic acid. In ooeytes expressing mRNA from the rat brain, oscillatory CI r elicited by acetylcholine, serotonin and glutamate were inhibited. In contrast, responses to GABA and kainate were not significantly altered. Procaine caused a fight shift in the dose-response curves and reduced the Imax. IntraceUnlar application of procaine did not inhibit ligand-aaivated responses. Extra-or intracellnlar application of procaine did not alter the To~ current, indicating that neither the endogenous voltage- gated Ca-channels nor the ca-activated CI channels were inhibited. Procaine caused only -5% reduction in currents elicited by pbotolysis of caged InsP~ and did not prevent currents elicited by GTP-T-S-induced direct activation of G proteins. For these receptors, coupling to the phosphoinositide/Ca signal Wansductiun pathway, site of procaine action appears to be on the extracellnlar surface, upstream from the G protein, presumably on the receptor.

129

02 : Membranes :Ion-Channels, Pumps and Carriers

P277 Temperature modulation of Ca2+-dependent K+ channel in Chara: effect of membrane voltage and thermal adaptation Maia R. Diurisic and Pavle R. Andjus, Institute of General and Physical Chemistry, Belgrade, YU

Single-channel properties of Ca2+-dependent K+ channel o f the Characean tonoplast were monitored in inside-out patches, in 5-36~ temperature range (in steps of I~ at membrane potential of 50 rnV. For Chara grown at 20~ single channel conductance, open channel probability and mean open time constant exhibited discontinuities around 15 and 30~ When membrane potential was switched to -50 mV, or after Chara was adapted for three weeks at 10 or 30~ previously observed discontinuities were abolished. Nonlinear temperature dependence of single-channel conductance points to the change in the interaction of channel protein and conducting ions below 15 and above 30~ This change could be imposed by different protein-lipid interactions, since temperatures of discontinuities coincide well with the temperatures of membrane lipid reorganization. This is further substantiated by disappeai'ance of breaks at opposite membrane potential (which directly influences protein-lipid interaction) and after adaptation at extreme temperatures (that induces the change in the lipid composition of membrane).

P 2 7 8 M O L E C ~ . A R DYNAMIC SIMULATIONS OF KCSA:

A POTASSIUM CHANNEL ladira, H Shrivastavq and M.&P. Sansom

Lab. of Molecular Biophysics, University of Oxford

Molecular dynamic simulation ofKcsA, a K + channel was performed, in a fully hydrated POPC bilayer. As the structure of the protein is approximately that o f an inverted cone, particular attention had to be paid to the initial packing o f the lipids around the protein. Simulations with ionizable side chains in different protonation states were carded out to study their effect (if any) on the dynamics of the channel and on the dynamics of pore-water. In the absence o f K + ions, the channel appeared to be closed. The pore radius at either end of the central cavity was less than 1.3 A, the radius o f K + ion. Further more, there is no exchange of water molecules between the central cavity and the surrounding environment over a 1 ns time period, though there is diffusion o f water from opposites ends of the pore into the central cavity. The simulation has also been done in the presence and the absence o f K § ions and the dynamics of the protein in the two systems compared. This work is supported by the Wellcome Trust and the computation was done on OSCAR, a 84 node parallel computer at the Oxford Suporcomputing Centre.

P279 Calcium Channels of the Outer Mantle Epithelium of Anodonta cygnea C. Bamas *+, P.F. Oliveira *+, A.C. Mauricio+and A. M. Rebelo da Costa *+

* IBMC. UP, Portugal; + ICBAS, UP, Portugal

The mantle ofAnodonta cygnea, a freshwater bivalve, is a leaflet that together with the shell circumscribes the extrapallial compartment. It consists of two epithelia: the cavity mantle epithelia which surrounds the body of the animal and the outer mantle epithelium (OME) facing the shell. The mantle controls the composition of the extrapallial fluid and the biomineralization of the shell. It was shown previously that the OME has a high Ca 2+ permeability. Calcium movements are purely diffusional.

The effect of calcium channel blockers on the short circuit current (Isc) generated by the OME was studied. Nifedipine (100 p2Vl) increases the isc, when in contact with haemolymph or shell side of the preparation.

To characterise Ca 2+ channels in this preparation, the outer mantle epithelium was dissected and fragments of the tissue were cleaned of mucus with DTT 3 mM and incubated in a solution containing collagenase 1 mg/ml and hialuronidase 1 mg/ml in order to obtain cell clusters. These were fixed on Petri dishes covered by poli-D-lisine and mounted on an inverted microscope. The patch clamp technique in the cell-attached configuration was used. The voltage protocol applied was performed in steps of 20 mV during 1000 ms. The reverse potential obtained, corrected for an intracellular potential of -30 mV was +31,2 inV. (Supported by projects PECS/P/SAU/216/95, PBIC/C/BIA/2050/95 and fellowships BD/16260/98, BTI/16868/98 and BD/5383/ 95)

130


P280 EFFECT OF DEPOLARIZATION ON ~AMP LEVEL IN RAT BRAIN SYNAPTOSOMES: CALCIUM-DEPENDENT PHENOMENON. S.V. Fedorovich, V.S. Chubanov#, M.V. Sholukh#, S.V. Konev. Institute ofPhotobiology, Minsk, Belarus, #-Belarussian University, Minsk, Belarus It is well established, that cross4alk between Ca 2+ and cAMP - dependent pathways on postsynaptic level of intracellular signalling involves in induction o f synaptic plasticity and formation o f memory engramms, but it is yet unresolved question on this cross-talk on presynaptic level. We studied effects of potassium- and veratrine-induced depolarization (PID and VID, respectively) on cAMP level in synaptosomes (neuronal presynaptic endings), isolated from rat brain hemispheres (SH) and cerebellum (SC). PID (65 mM KCI) decreased cAMP level in SH and SC, as determined by RIA. Aider pretreatment of synaptosomes by 1 mM IBMX (inhibitor of cyclic nucleotide phosphodiesterase, PDE) PID reduced concentration of cAMP in SH and raised cAMP level in SC. Thus, in SH, reduction o f cAMP involved activation of PDE and inhibition of adenylyl cyclase (AC), and only activation o f PDE in SC. ARer preliminary activation of AC by 100 mM forskoline PID decreased AC activity in both SH and SC. The effects of VID were alike. The observed effects disappeared in calcium-free media. Similar effects were induced by 10 mM A-23187 (calcium ionophore). Thus, a cross-talk was found between calcium and cAMP pathways of cellular signal transduction in presynaptic endings, probably involved in some forms of synaptic plasticity.

P 2 8 1 Affinity of Na§247 in secretory cell membrane to divalent cations

N.Fedirko, V.Manko, M.Klevets De F. of Human & Animal Physiology, Lviv State University, 290005, Lviv Ukraine T~ae purpose of this research was testing the ability of Sr 2§ and Ba 2§ cations to substitute Ca 2§ in

transporting cycle of the Na§247 of secretory cell membrane in Chironomusplumosus L. salyvary gland. Inward Na§247 current (IN~(ca)) of isolated cell of salyvary gland was registered by mean of voltage clamp method in conditions of intracellular perfusion in response to membrane hyperpolarization from -20 to -60 mV at physiologycal Na § gradient. Increasing of [Ca2§ from 1.0 to 1.76; 3.0; 5.0 and 10.0 mM evoked significant rise of inward INa(Ca) on 30.1; 34.9; 37.0 and 37.0 % accordingly. This way, dependence of INa(Ca) on [Ca2+]~ is hyperbolic that testify about saturation of exchanger cation binding site by cytosolic Ca 2§ The same character has dependence of inward current amplitude on [Sr2+]c: increasing of Sr 2§ concentration from 1.0 to 1.76; 3.0; 5.0 and 10.0 mM current amplitude slow rised on 7.1; 12.1; 12.5 and 13.6 % accordingly. Similarity in kinetic parameters of concentration dependence of investigated current at availability in external solution Ca 2§ and Sr 2§ testify about binding Sr 2. by exchanger similar to Ca 2§ Increasing in extracellular solution concentation of Ba 2§ cations, which was substituted for Ca 2+, from 1.0 to 1.76; 3.0; 5.0 and 10.0 mM evoked conceatration-dependent increasing of investigated current amplitude on 6.2; 18.5; 28.1 and 35.9 % accordingly. The fact that, at rising of [Ba2§162 to 10.0 mM the current still increased testify on lower affinity of Na+-Ca2§ for this cation.

Therefore Na§ of secretory cell membrane has different affinity to other divalent cations, which increased in sach consequence: Sr2§ z§ P 2 8 2

THE EFFECTS OF GRAMICIDIN AND GRAMICIDIN ANALOGUE ON CATION CONDUCTANCE OF SKELETAL MUSCLE FIBRE. N.Shvinka

Latvian Institute of Experimental and Clinical Medicine, O.Vacietis Str. 4, Riga LV-1004, LATVIA The effects of gramicidin A and synthetic covalently linked gramicidin dimer ( H O C H 2 C H 2 N H - C O ( C H 2 ) 2 C O - NHCH2CH2OH) on potassium conductance of single phasic fibres from m.illeofibularis and m.semitendinosus of Rana esculenta were studied. The experiments were performed in isotonic K2SO4 solution under constant current conditions using a double sucrose gap technique. Both these substances increased the potassium conductance of the muscle cell membrane due to newly formed gramicidin channels. The effect was progressive, continuously increasing with time and reaching steady state during exposure time of 6 rain. At room temperature the channel formation by gramicidin was much faster than that of the head to head covalently linked gramicidin dimer. The value of the steady state conductance induced by gramicidin monomer (10 .6 M) was 33 times higher as compared to that induced by gramicidin dimer at the same concentration. An increase of temperature by 8-10 ~ resulted in a considerable rise of both monomer- and dimer-induced conduetances because of temperature accelerated potassium transport in gramicidin channel and higher rate of channel formation. The activation energy for the potassium conductance in gramicidin monomer- and dimer channels was 0.036 kJ/mol and 0.026 kJ/mol, respectively. The temperature dependence of adsorption was more pronounced than that of desorption. Free energy of channel formation ranged from 0.140 to 0.163 kJ/mol for monomer and from 0.126 to 0.221 kJ/mol for dimer. The effect of temperature on adsorption kinetics was much greater than in the case of bilaylers, indicating a remarkable entropy change in muscle fibre membrane.

131


P283 Mechanisms of Proton / Potassium ion Exchange in Phospholipid Vesicular

Membranes by the Combined Action of Nonactin and CCCP

B. S. Prabhananda and Mamata H. Kombrabail, Department of Chemical Sciences,

Tata Institute of Fundamental Research, Mumbai, 400 005 India.

The proton concentration difference (ApH) across soybean phospholipid vesicular membrane can decay by the combined action of the K + ion carrier.nonaefin and the weak acid carbonyl cyanide m-chlorophenylhydrazone (CCCP). Experimental observations under different concentration conditions suggest that in the rate limiting step of ApH decay K + cation and CCCP- anion are translocated across the membrane by two mechanisms contrary to popular belief: (a) After translocating H + as CCCPH, CCCP- anion is back transported along with the transport of nonactin-K + ions providing the compensating charge flux of K + ions. (b) The translocation of the ternary electroneutral complex nonactin-K+-CCCP - back transports CCCP- and transports the compensating charge flux of K + simultaneously. At higher concentrations of nonactin the mechanism "(b)" dominates. In vesicle solutions of - 0.3 ionic strength, the apparent K + dissociation constant of the nonactin-K + complex in the membrane was estimated to be - 0.15 M. In our experiments, temperature jump was used to create ApH.

P284 ON THE POSSIBILITIES FOR A METHODTO FACILITATE MEMBRANE TRANSPORT

STUDIES BASED ON BULK MAGNETIC SUSCEPTIBILITY DIFFERENCES AND NMR S.Aravamudhan ,Department of Chemistry, North Eastern Hill University

Shillong-793 003, Meghalaya, India. Differences in compartmental bulk magnetic susceptibility can be used to distinguish spins. Large differences in magnetic Suceptibility can arise by selective addition of "Susceptibility reagents". The NMR detection sensitivity depends on the compartmental location of the observed molecule I and, among the several factors, suitable choice of paramagnetic concentration can eliminate external signals. In a method as per the recent report 2 on Demagnetization Factor calculations there seems to be an in-built possibility for mapping the induced-field pattern in uniformly magnetized, and, even, arbitrarily shaped specimens. A consideration for tapping this potential for membrane transport studies by NMR is being envisaged.

1. "NMR Studies on Membrane Transport" by B.P. Hills and P.S. Belton in Annual Reports on NMR Spectroscopy Ed. G.A. Wcbb Vol. 21, p. 140 (1989). 2. "On the possibilities of Calculating Demagnetization Factors by Lattice-Sum Method Consideration" Paper presented by S. Aravamudhan at the 5th NMRS Symposium, IIP, Dehradun (1999).

P285 A m p h o t e r i c i n B c h a n n e l s in t h e b a c t e r i a l m e m b r a n e .

Berenice Venegas-Cotero and Ivs Or tega Blake. Centro de Ciencias F~sicas, UNAM., Cuernavaca, M6xico.

Recent ly (Biochim. et Bipophys. Acta 1375, 43, (1998)) we have r epor t ed tha t it is possible to form channels of the ant imicot ic amphote r ic ine B in the absence of sterols. The idea of the need of sterol as an integral par t of the channel has been a s u m m e d for a long time, and has been a driving mo t i f on the search for derivatives wi th less collateral effects. We advanced the idea tha t cholesterol 's role is in the plastif ication of the membrane . But one quest ion tha t inmedia t ly arises is, why the ant imicot ic does not work on the bacter ial m e m b r a n e ? In this work we in tend to answer this question, showing tha t it is indeed possible to have channels in the bac t e r i a l m e m b r a n e , using t e m p e r a t u r e as an agent for the mofification of the m e m b r a n e phase.

132


P286 The Proton Pump of the Outer Mantle Epithelium of Anodonta cygnea

P.F.. Oliveira*+, I. A. Lopes*, C. B a n ~ *+ and A. M. Rebelo da Costa *+ * IBMC, LIP, Portugal; + ICBAS, UP, Portugal

When mounted in vitro in Ussing type chambers, the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea generates a spontaneous electrical potential and short-circuit currents (I)o) which can be as high as 50 mV and 100 ~tA cm -2, respectively. These currents can be accounted for by a seoetiun of protons towards the shell side and a secretion of bicarbonate towards (or removal of protons from) the hemolymph side. The proton extrusion was attributed to the operation of a proton pump located at the apical side of the epithelium. The presence of an electrogenic proton pump, of the V-pump type, in the apical barrier of the OME was confirmed with the use of its specific inhibitors, Bafilomycin A] and Concanamycin A. They had a dramatic effect on the I,o only when in contact with the apical side producing 90 % of inhibition in respectively 3 and 35 minutes.

Celts of the OME were homogenised and the total ATPase activity was assayed following the release of inorganic phosphorous. The effect of different salts, specific inhibitors, pH, temperature and ATP on V-ATPase activity was studied. The activity of this enzyme presented the highest values at 27~ pH 7.4, [Na2SO3] 200 mM, [Na2ATP] 4.5 raM. The effect of the ATPase inhibitors Concanamycin A, Bafilomycin A], DCCD and NEM on the activity of the V-ATPase was also studied. (Supported by projects PECS/P/SAU/216/95, PBIC/C/BIA/2050/95 and fellowships BD/16260/98, BTI/16868/98 and BD/ 5383/ 95)

P287 Abnomality of Glucose Transporter in grythrocyte Membranes of

Non-Insulin Dependent Diabetes Mellitus Zhi-hong Zhangk Xiao-jian Hu !, Han-qing Zhou !, Wei-ying Cheng 2, Hang-fang Feng ~

LDept. of Physiology and Biophysics, Liren Laboratory, Fudan University, Shanghai, China, -' Xinhua Hospital, Shanghai Second Medical University, Shanghai, China

Non-insulin dependent diabetes mellitus (NIDDM) is characterized with impaired glucose uptake. With a photometric method we obtained that the maximal velocity of glucose trans-zero entry was decreased in patients as compared to the controls. However, the rate of glucose cis-infinite exit exhibited no significantly difference. Using phloretin we observed that the inhibitory constant was increased si~nific, antly after phloretin treatment in the glucose entry kinetics experiment. The measurement of temperature dependency of glucose transport showed that the activation energy for glucose entry in patients' erythrocytes was increased by 30%. Furthermore, we studied the abnormality of fluorescence quenches in NIDDM erythrocyte membranes. The tran.qfer tendency of tryptophan from hydrophilic environment to hydrophobic environment was decreased in patients' eythrocytes as binding with glucose, and the opposite tenciency was observed as cytochalasin B and phloretin instead of glucose. These results indicated that the decrease in glucose entry in the erythrocyte membranes of NIDDM patients was due to the GluT1 change in structure-- mostly the outer domain of the glucose transporter. This research was sul~ported by the National Natural Science Foundation of China.

P288 KINETICAL PARAMETERS OF Na~-DEPENDENTAND Na'-INDEPENDENT TRANSPORT OF Ca 2~"

ACROSS THE MITOCHONDRIAL AND SECRETORY CELLS MEMBRANES L.O.Dubitsky, L.S.Vovkanych, I.A.Zalyvsk'y

Ivan Franko Lviv State University, Gryshevskogo str. 4, Lviv 290005, 13kralne

Ca"- transport in isolated gastric glands of guinea pig and isolated rat liver mitochondria (RLM) have been studied with the use of 45Ca '+. We show that decrease in extracellular [Na +] to 20 mM is accompanied v~ith the increase of Ca 2+ influx to thegastric glands, preloaded with Na +. The hyperbolical concentration dependence of the Na +-dependent Ca 2+ influx into gastric glands means that it is carder-mediated (Na-Ca-exchanger). V,.,, of the Na + - dependent Ca :§ influx is 1.22 nmol Ca~+/min-mg protein, and K~.5 - 1.07 raM. The low levels of the Ca 2+ influx V,.,, and Ko s indicate its minor role in the stimulus-secretion coupling of the secretory cells. Evidently, the role of Na-Ca- exchanger is to maintain the secretory cells calcium homeostasis.

The Na-Ca-exchanger has not been identified in the RLM membrane. However, decrease in medium pH in the range of 7.4-6.5 results in significant increase in Ca -'+ efflux from the Ca -'+ preloaded RLM. The si~oidal dependence of Ca 2+ efflux velocity fi'om the H + concentration in the medium with the Hill coefficient of 2.4 could be assumed as an evidence of the presence of electronentral Ca-'+/2I-I+-exchanger in the RLM inner membrane. The V.,~ of the Na+-independent RLM efflux of Ca -'+ is 6.56 nmol/min-mg protein, and Ko.5 for H + is 52.10 nM (pH 7.28). It means that in physiological conditions this exchanger takes part in the regulation of inlracellular and intramitochondrial calcium concentration_

133


P289 Ca 2+ INFLUX DURING THE RAT HEART EX'I KA~YSTOLE: A MECHANICAL-ENERGETIC STUDY.

Savio-Galimberti E.; Bonazzola P.; Ponce-Hornos J.E. ININCA & Cfit. Bioflsica, Fac. Med. &Fac. Odont., UBA. Buenos Aires ARGENTINA

It has been shown that the Ca 2+ source for a 200 ms extrasystole (ES) is exlracellular, it is accumulated in the sarcoplasmic reticulum and released during the post-extrasystole (post-ES) (Am. J. Physiol. 276 (45): H309- H316, 1999) inducing the post-extrasystolic potentiation (post-ES-pot). To study the routes of Ca2+ influx, (Ca 2+ channels and Na+xCa 2+ exchanger) extrasystolic stimulation was performed in the presence of either verapamil (VER) (a Ca 2+ channel blocker) or Li +', that has been shown to inhibit Ca 2+ uptake (J. Mol. Cell. Cardiol. 12: 1367- 1382, 1980). Miotbermic responses were measured on line with the Pressure-time Integral (PtI). Post-ESPpot was calculated as the difference (post-ES PtI-control PtI). The heat output for each contraction was decomposed into three energy components. VER decreased ES PtI (8.12-~.64 mN.mm'2.s vs. 4.644.0.41 mN.mm2.s, n=18, p<0.01), but did not affect post-ES-pot. Li + decreased ES PtI (-0.754-0.22 mN.mm'.2.s, p<0.05, n=7) and the energy component attributed to Ca 2+ cycling (-0.244.0.08 mW/gww, p<0.05, n=7). Post-ES-pot was decreased by Li + (-0.70 +0.28 mN-mm'2.s, p<0.05, n=7). Then, during the ES at least two fractions of extracellular Ca 2+ can be distinguished. One of them can be affected by VER and one affected by Li + (which would be the responsible for the post-ES-pot). Therefore, while during the ES Ca 2+ would enter the cell via both mechanisms, the fraction affected by Li + seems to be the one associated to the post-ES-pot. Supported by grants OT-018 (UBA), PIP 4564 (CONICET).

P 2 9 0 INTRAMOLECULAR OSMOSIS: A PROPOSED MODEL

Mario Parisi, Claudia Capurro and Roxana Toriano. Lab Biomembrar~, Dto Fisiol., Fac de Med., Univ Buenos Aires, Arg~fina and Service de Biologie Cellldaire, DBCM, CE Saclay, CEA, France.

Water permeability plays a pivotal role in cell physiology, either in animal and plant ligsm~. Cloning, locali7~on and functional characterization of aquaponus has given important information on the mechanism underlying water transfers. Recently water movements (absorptive and/or sectetor) were studied ina recently cloned epithelial cell line derived f ~ m the renal corlica! collecting deft of the rat (RCCD 1). A ~ , normally present in these cells, were not expressed. Uuexpectedly, net water se~i~iion was observed while, on the other side, vasopressin stinmlated isotonic water absorption. These results, put the problem of the water pathway operating in this conditions. 3"84 is a well studied secretory cell line ori~nated in a hmnan colon carcinoma that express an eaterocytic phenotype. Once again aquapofins are not expressed in this cell line Interesting enough water-ions estechiometry was quite different in different secretory condflious. This puts attain the question about ioB-water coupling mechanisms in the absence of aquaporins. A water-ien cotransport model has been recently proposed to understand these and previous results. As an alternative, we propose an << inWamolecular osmotic model >> in which a water channel is associated, in the same mac~omolecular structtae, to a salt transporter. According to this model, ions are transported into a molecular cavity, generating an osmotic gradient Then water will move into this cavity, driven by the osmotic gradient, through the water channel. Finally a salt solution will be hydrostatically transferred outside the stroctme. The theoretical flame of the model will be discussed.

P291 Molecular Dynamics of Ca-ATPase Regulation by Phospholamban OPLB) in Cardiac Sarcoplasmic Reticulnm Laxma G. Reddv,~ Larry It. Jonu% and David D. Thomas. Department of Biochemist, Univermty of Mmnasota, Minneapolis, MN 55455, and Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN 46202.

Pbospholamban (PLB), a 52-amino acid integral membrane protein, reversibly regulates the Ca-ATPase (Ca-pump) function in cardiac sarooplasmic reticulum (SR) through I~-adr~ergic mediated phosphorylation. The pentemeric structire (on SDS-PAGE) of PLB was proposed to be ~.po.~. t for its.regul .at~'y. function. B as~.. ~ ~ . t e ~ . . mutagonesis and co-expression with Ca- ATPa~. (.SEt~.C ..A2a) and on. spin-label detectm.n of oh.g__ojnen, c state m lipid bdayers, it has .t~en proposed that the monomeric form ofPLB .m ~e inld~bito~..specie, .and depolyme~, o.n OfPLB ~s essential for its regul .ato.~ function. However, the oligomeric ~a'ucture OfPLB m Rs native milieu, a lipid bflayer containing the Ca-ATPase, and the physlcal mechanism of regulation is not determined.

We have studied the effeots of the olisomeric state of PLB on its regulatory function by co-reconstituting PLB and its structural mu .e ( .tea e c end monomenc) wi . ed Ca ATP in baayers. B usf (FET) was.u d to study the oli8omenc s~.ture of PLB m lipid bilayers reconstituted m the .absenoe and .pt/esc~.. of Ca-ATPase. Tune-resolved phosphoreacence amsoU'opy (TPA) was used to study the physical mechanism of regulation (molecular dynamics) of Ca-ATPase m cardiac SR as a function ofPLB phosphotylation.

Our results indicate that the extent of inhibition by PLB or its mutants appears to depend not only on PLB's property to dissociate into monomers but also on the relative potency of the particular PLB monon~r whm interacting with the Ca-ATPase. FET ..r~alts indicate that PLB is an olison3er in membranes, rod. the Ca-ATPase binds prefereafially to the monomer and/or .~nalJ oligonm~ suggesting a monomer or an oligomer Of PLB having fewer than 5 subunits as the inhibitory species. TPA results indicate a strong o.a~clalion between inhibition and aggregation of the Ca-ATPase end upon phosphcxylation of PLB, the release of inh~oition correlates with the disaggregation of Ca-ATPase.

134


P292 M E C H A N I S M S O F A N T I A R R H Y T H M I C A C T I O N O F S O M E D I T E R P E N O I D A L K A L O I D S

B .A .Tashmukhamedov , B.T. Sagdullaev and P .B.Usmanov

Institute of Physiology and Biophysics Tashkent 700095, Uzbekistan

In the present w o r k , mechanisms of ant iarrhythmic action of some diterpenoid alkaloids ( D A ) have been studied. It has been shown that 1 -benzoy lnapd l in , zongor in , dihydroathesine, getisine and benzoylheterathesine are potent cardioactive substances with dose - and rate-dependent action on contractile activity of myocardium. Investigation of contractility kinetics using specific blockers of voltage-gated N a + - and Ca2+-channels, Na+/Ca 2+- exchanger , as well as sarcoplasmic reticulum (SERCA I 1) functions allowed us. to reveal T I ' X - and ryanodine-like action of DA. Patch-clamp experiments on isolated rat cardiomyocytes demonstrated that D A markedly blocked TTX-sens i t ive inward N a + - current in a tonic manner. D A reduced SERCA 11 Ca 2+ release channel (RyR) activity in calcium-overloaded rat ventricutar myocytes in experiments using fluorescent probe Quin-2. At the same t ime , D A have no effect on Ca 2+ - A T P -ase activity of myosine. Analysis o f the da ta obtained showed that D A modula ted the cellular ion homeostasis o f cardiomyocytes. Such action o f D A may explain their potent ant iarrhythmic properties.

P293 Differential effects of deoxycholicacid (DOC) on the transport properties of distal colon/n vitro.

Mauricio, A.C. +*; Heitzmann, D.+; Warth, R.§ Bleich, M.§ Greger, R.+; Ferreira, K.T.G. + and Ferreira, H.G. +* +Dep. Qulmic& CQFB, FCT-UNL, Portugal; *ICBAS, UP, Portugal; *Physiologisches Institut, Freiburg, Germany.

It is well known that humans after extensive ileal resection suffer from diarrhoea. The inhibition of absorption or even a stimulation of secretion could be due to increased delivery of DOC to the colon. In A~ and Cac02 cells subcultured directly on Petri dishes and studied in the whole-cell configuration DOC induced an increase in conductance that could be blocked by DIDS 0.5 mM+ DPC 0.5 m.M. In these cells subcultured on permeable supports DOC induced an increase in conductance that could be blocked by amiloride 100 IxM. SDS 0.1 mM added to the bath solution in Cac02 cells studied in cell-attached configuration induced an increase of channel activity and of the baseline conductance like it was reported with 0.5 mM of DOC. Measurements of cytosolic Ca 2+ activity in rabbit colonic crypt cells using Fura-2 fluowscence showed that was no increase in Ca 2+ activity after the addition of DOC 100 ~.M. In Ussing chamber experiments DOC 10-100 paM induced a concentration dependent secretory lumen negative equivalent short circuit current (I~) from the basolateral as well as from the luminal side. The effect was observed in the presence of amiloride 100 p.M and could be inhibited by azosemide 100 pJvl and 293B 10 ~VI. The addition of DOC [00 plVI t o3.he luminal side in the presence of CI secretion blockers induced a further decrease of I~. DOC did not stimulate a Na + current sensitive to amiloride in rabbit distal colon. There was an additive effect of DOC and cAMP or Ca 2+ induced I,o in rabbit distal colon. In basal colonic crypt cells from the rat DOC 100 txlvl stimulated a K + conductance. In colon DOC act as a secretagogue by enhancing inhibitable CI secretion. We purpose that DOC act on ion channels, directly or indirectly through unidentified channel regulating factors associated to the membrane. (Supported by Project PRAXIS XXI PNCA / BIA / 193 / 96)

135

C3 : Membranes :Transmembrane Signalling and Transduction

P 2 9 4 Signal transduction pathways underlying the extracellular ATP induced membrane fluidization

Orna Za2oorv. Essa Alfahel, Abraham I-L Parola and Zvi Pr/el DeparUnent of Chemistry, Ben-Gurion University of the Negev, P. O. Box 653, Beer-Sheva 84105, ISRAEL

The dynamics of biological memlranes has been shown to play a major role in a variety of physiological processes. The role of membrane dynamics in transmembrane signal transudation changes in membrane fluidity was r in ciliary tissue from frog's palate. Ciliary cells are excitable in the smse that they can respond to a variety of stimuli, e.g., hormonal and neuronal, such as extracellular ATP (ATP~, by altering the pattern of their activity, particularly the frequency

It was found that ATP~ (10 o_M) caused strong changes in fluorescence polarization, which may indicate membrane flui~Tation. This effect depends on the presence of extracellular Ca 2*, which could be replaced by Mg 2+. Thapsigargin and ionomycin, both that deplete Ca 2+ stores at low extracellular Ca 2+ concentration, prevent the induced response. The effect of ATP.ex was blocked by the calcium activated potassium ~ n n e l s inhibitors: quinidine, charybdotoxin and apamin, and was reduced considerably by replacement of extraceLlular Na + by K §

These results indicate, that appreciable membrane fluidiT.~tion induced by exwacellular ATP depends both on the rise of intracellular Ca 2§ mainly from its internal stores, and on hyperpolarization of the membrane. Calcium dependent potassium channels couple the two effects. Moreover the relative involvement of various second messengers in membrane fluidization will be presented.

P 2 9 5 Signaling Cascade in Rapid Apoptosis of 3SB, Murine Thymoma Cell lane, Following X-Irradiation

H.Hama-lnaha: B.Wang, K.Choi, T.Nakajima*, K.Haginoya, M.Mori, H.Ohyama, O.Yukawa*, and H-Iayata Division of Radiology. and Biodosimetry and *Division of Biology and Oncology, National Institute of Radiological

Sciences, Chiba 263-8555, Japan

3SB cells are quite sensitive to X-ray and undergo apoptosis shortly (4h) after X-irradiation (2Gy, ~100%). As reported before (Mutant, Res., 401 (1998) 85-94), X-ray-resistant variants isolated from 3SBHS, subclone of 3SB were resistant to apoptosis induced not only by X-irradiation but also by daunorubicine and C2-ceramide. These facts suggested that signaling via membrane played important role in X-ray-induced apoptosis. In this paper, we report the signaling cascade of apoptosis following X-irradiation of 3SBH5 cell elucidated by various inhibitors and activators and by immuno-blotting analysis. PMA (phorbol myristate acetate), activator of PKC, blocked and chelerythrine, inhibitor of PKC ot and ~, enhanced X-ray-induced apoptosis of the cell. MEK inhibitor enhanced apoptosis and decreased the effect of PMA. These results shows that PKC plays a central role in regulation of X- ray induced apoptosis in 3SB cells and ERK pathway is anti-apoptotic. Furthermore, orthovanadate, tyrosine phosphatase inhibitor, protected the cell from apoptosis. This may be originated from the enhancement of growth signal by vanadate. All results suggested that inhibition of growth or anti-apoptotic signals enhanced the X-ray induced apoptosis of 3SB cells. X-ray irradiation might switch growth signals to apoptotic signals in short time via membrane-mediated events such as activation and/or inactivation of some enzymes.

P 2 9 6 Glutamate Induced Ca z+ m n u x and Mitochondria l Funct ion in Rat Brain Slices

Nanda B. Joshi and Sridhar K. Kannmpa~, Department of Biophysics, National Institute of Mentat Health and Neuro Sciences, Bangalore-560029, India.

Excitatory aminoacid neurotransmitter glutamate is involved in neurotransmission in the central nervous system, but it becomes a potent neurotoxin under variety of conditions. Several in vitro and m vivo stmfies have demonstrated toxicity caused by excitatory aminencids in neurons, however, the molecular mechanism of excitotoxicity is not completely understood. The present study was undertaken to understand the role of mitochondria in glutamate induced neuronal cell death. Gh~amate induced Ca 2+ influx, mitochondrial depolarization and energy status was inyestigated in rat brain slices. Our results showed that exposme of gl,aamate to cortical slices increased Ca 2+ influx, depolarized mitochondrial membrane, inhibited phosphorylation potential and increased mitochondrial redox state. Further studies preformed in the presence of protonophore and inhibitors of oxidative phosphorylation and elect~on tran~er chain demonstrated that the Ca 2+ influx activated by glutamate receptor may be modulated by mitochondri& These results suggest a possible involvement of mitochondria in excitotoxicity.

136


P 2 9 7 Glycosphingolipid-enriched microdomains in U-87 M G cells contain protein tyrosine kinase. Preeti G. Joshi and Mau Sinha. Department of Biophysics, National Institute of Mental Health and Neurosciences, Bangalore-560 029. lndict

Earlier we have shown that antibodies raised against glycosphingolipids galactocerebroside (GalC) and ganglioside GM1 induce IP3 formation, increase tyrosine kinase activity and intracellular Ca 2+ concentration in U-87 MG and neuro2a cells respectively. These studies suggested that the GalC and GM1 are possibly associated with proteins involved in transmembrane signalling. Recent studies suggest that many siLmalling proteins partition into the glycosphingolipid and cholesterol enriched microdomains in the cell membranes and form detergent insoluble complexes.

We have isolated and characterized the glycosphingolipid-enriched complexes from U-87 MG cells. Complexes insoluble in nonionic detergent triton x-100 were separated by isopycnic centrifugation on sucrose gradients and 1 ml fractions were collected. Fractions 1-5 contained less than 2 % of the total protein, most o f which was found in the denser fractions 6-10. When tested for the presence of GalC by ELISA, fractions 3-4 displayed a moderate concentration of the lipid but denser fractions were devoid of GalC. The fluorescence anisotropy of 1,(4-trimethylammonium),6-diphenyl-l,3,5-hexatriene (TMA-DPH) in fractions 3-4 was substantially higher as compared to other fractions corroborating the presence of glycosphingolipids. The tyrosine kinase activity was found only in fractions 3 and 4. Thus, our results reveal that protein tyroSine kinase is anchored to GalC-enriched microdomains in the plasma membrane of U-87 MG cells.

P298 The Regulation of the Stimulated Respiratory Burst of Human Neutrophil by IntraceHular Calcium

XuB.Sh~ Tianhui Hu and Ling Bei, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

The role of intracellular free calcium, [Ca2+]i , ill the tMLP- or PMA-stimulated respiratory burst (RB) of h,man neutrophil lenkocytes was studied by looking into both magnitude and kinetics. When [Ca2+]i was elevated by treatment of the cells with thapsigargin frG), the intensity of RB was enhanced ~erJaendonsly, while the onset speed remained unchanged. However, substantial elevation of [Ca2*]i did not significantly enhance the Intensity of PMA- stimulated RB, but markedly accelerated its onset. As [Ca2~]i rises, both enhancement and speeding up of the DiC8 (a synthetic DAG) stimulated RB were observed. At sufficiently high [Ca2+]i level, the RB in DiCS-stimulated cells becomes same fast as in tMLP-stimulated cells, but still much slower in PMA-stimulated cells. The results indicate that the ranch fast response in tMLP-stimulated cells in comparison with PMA-stimulation is not only due to the transient rise of [Ca2+]i, but also due to the higher efficiency of DAG than PMA in activating protein kinase c (PKC). The regulation of [Ca2+]; on the expression of fomyl peptide receptor on cell surface was also studied. When intracellular free Ca ~ was depleted using BAPTA plus EGTA, the actin-microfilament equilibrium was substantially shifted to the direction of assembled mierofilaments. When [Ca2+]i was elevated substantially by TG- treatment, sitmificant inhibition of actin-polymerization was found. Flow cytometric analysis showed that polymerization of aetin reduced expression of the receptor, while depolymerization of actin promoted the expression. The studies reveal that [Ca2+]i plays an important role in either activation of PKC, or promotion of the receptor expression by regulating the cytoskeleton.

P 2 9 9 Racin, a multifunctional rac-regulator in Dictyostelium discoideum.

Menno L.W. Knetsch, Nicole SchMers and Dietmar J. Manstein Dept. of Biophysics, Max Planck Institute for Medical Research, D-69120 Heidelberg, Germany

Dictyostelium contains at least 14 different rac genes, raclA-C and racA-J that regulate a diversity of cellular processes including endocytosis, phagoeytosis, cortical F-actin organization, cell motility and cleavage furrow formation during cytokinesis. Recently we cloned a gene, racin, encoding one GAP (GTPase activating protein) and two Dbl (Dbl- homology; Guanine-nucleotide exchange) domains. Furthermore racin contains sequences encoding an SH3 and a Pleckstrin-homology (PH) region. Sequence comparison clearly places the putative 148 kDa protein in the class of Rho/Rac-regulating proteins. Racin-null cells showed no defects during growth, late development and fruiting-body formation. However the null-mutants displayed increased osmo-sensitivity in hypotonic conditions and morphological alterations of their contractile vacuole system. Racin-null cells have increased sensitivity for the chemoattractant cAMP. Approximately 10-fold lower extra-cellular cAMP concentrations are sufficient to induce normal chemotaxis, F-actin formation and ERK2 activation. In contrast to vegetative cells, irnmunostaining of F-actin in aggregation competent Racin- null cells was altered, showing intracellular cables and aggregates as well as an expansion of the cortical F-actin network. Redistribution of F-actin was unaltered in Racin-null cells upon cAMP treatment. These results indicate that the rat-regulator Racin plays a critical role in the correct organization of the contractile vacuole system and acts as a negative regulator of cAMP-dependent transmembrane signaling..

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P300 E l e c t r o m a g n e t i c F i e l d A b s o r p t i o n i n S t o c h a s t i c C e l l u l a r S y s t e m s : E n h a n c e d S i g n a l D e t e c t i o n i n I o n C h a n n e l s a n d C a l c i u m O s c i l l a t o r s J o h n S a n d b l o m a n d J u r i s G a l v a n o v s k i s . D e p a r t m e n t o f Med ica l B iophys i c s U n i v e r s i t y o f CRiteborg, Box 433, SE 40530 CRiteborg T h e b a s i c p r o b l e m in u n d e r s t a n d i n g t h e m e c h a n i s m s o f low f r e q u e n c y e l e c t r o m a g n e t i c f ie ld i n t e r a c t i o n s w i t h biological s y s t e m s a r i s e s b e c a u s e o f t h e s m a l l s t r e n g t h o f t h e s i g n a l c o m p a r e d to t h e t h e r m a l no i se in t h e i r r a d i a t e d s y s t e m . W e h a v e i n v e s t i g a t e d , e x p e r i m e n t a l l y a n d t h e o r e t i c a l l y , t h e p o s s i b i l i t i e s for s i g n a l a m p l i f i c a t i o n r e l a t i v e to no i s e in t h o s e p a r t s o f t h e c e l l u l a r c a l c i u m s i g n a l l i n g p a t h w a y in cel ls w h i c h c o n s t i t u t e s t o c h a s t i c a n d c h a o t i c p r o c e s s e s . T h e p a t t e r n o f c a l c i u m f l u c t u a t i o n s i n a cel l l i ne o f T l y m p h o c y t e s (J, u r k a t ) c o n s i s t s o f a r e p e a t e d s e q u e n c e o f r a n d o m l y o c c u r r i n g sp ikes . T h e s p e c t r a l p o w e r d e n s i t i e s o f t h e s e sp ikes w e r e d i m i n i s h e d by a p p l i e d 50 Hz e l e c t r o m a g n e t i c f ie lds . We h a v e m a d e s i m u l a t i o n s on m o d e l s o f c a l c i u m osc i l l a t ions w h i c h r e p r o d u c e t h e e x p e r i m e n t a l r e s u l t s , s u g g e s t i n g t h a t t h e s i te o f i n t e r a c t i o n is t h e Ca2+ r e l e a s e f r o m i n t r a c e l l u l a r s tores .

P301 Photoreceptor cells behavior in static and alternative electric fields of different frequencies

Roxana Pologea-Moram, Eugenia Kovacs, Tudor Savopol, Alexandru Dinu "Carol Davila" Medical University, Biophysical Research Department, P. O. Box 35-43, Bucharest, Romania

We studied rods patterns orientation in static and alternative electric field using computerized videomicroscopy analysis. The orientation patterns of rod photoroceptor cells were different in the presence (orientation parallel to the field) and in the absence (no orientation) of static electric fields. In static electric field and in the presence of ouabaine (when Na§ § pump is abolished) we obtained the same statistics as in the absence of the field. This means that the orientation of rod cells in static electric fields is dependent on the cell metabolism. This is why this kind of studies could be a poss~le test for morphological and physiological integrity of rod cells. In alternative electric fields, at low frequencies (below 450 kHz), we saw that rods are orienting along the field direction while at higli frequencies (over 450 kHz) they orient perpendicularly to the field direction. Fitting this data using elongated ellipsoid model shows that this behavior is determined by minimal interaction energy of rods and the alternative electric field.

P302 Studies on effect of alternating electric fields on human erythrocytes in different buffer solutions

S.H. Sanghvi and K.P. Mishra*

Electronics Division and *Radiation Biology Division, Bhabha Atomic Research Centre, Mumbai - 400 085

Human erythrocytes suspended in isotonic solutions of differing conductivities were subjected to alternating electric fields of varying frequencies and amplitudes to understand the basic biophysical mechanisms of interactions and to develop protocols to achieve efficient cell fusion. External fields obtained from pulse generator were applied to cells suspended in various solutions on microscopic glass slides having lining of surface coated gold and observations were recorded on a video system. Cells in isotonic phosphate buffer (2x10~ cells/ml) were found to align into pearl chains when ac field of 200 kHz and 10 V was applied. The length and stability of cell chains was found dependent on the field frequency and amplitude. Moreover, the field required to induce formation of chains was found to strongly depend on the conductivities of the suspension medium. Fields that produced substantial chain of cells in phosphate buffer failed to form chains in isoosrnolar solutions of sucrose or mannitol but displayed rotation indicating modulatory influence of suspension conductivity on the field induced cellular behavior. Furthermore, erythrocytes from diabetic patients were found to yield significantly altered response to ac fields. Implications of these results in the molecular mechanisms of cell fusion are discussed.

138


P303 Magnetically Detected Injury Currents in Bean Plants

Jazbingek, V), Thiel, G. 2, MOiler, W. 3, Wtibeller, G. 3 and Tronteli. Z. t ~Institute of Mathematics, Physics and Mechanics, University of Ljubljana, Ljubljana, Slovenia

2pflanzenphysiologisches Institut, Universit~t G6ttingen, G6ttingen, Germany 3physikalich-Technische Bundesanstalt, Institut Berlin, Berlin, Germany

Electric potential measurements bring the information on the presence of tissue injury in higher plants. One expects that magnetic feld measurement and its time evolution over the whole plant will further elucidate processes following the plant injury. Measurements were performed on several bean plants (Vicia Faba) in magnetically shielded room with multi-channel SQUID magnetometer system. Since the expected injury currents are almost dc, measured signals were modulated by positioning a plant on a moving table which was oscillating with frequency 0.5 Hz and amplitude 3-5 cm. Duration of each measurement was 20-25 minutes with sampling frequency 250 Hz. Within the first 5 minutes the plant was injured by a burning candle placed for a few seconds under one of the lower leafs.

Simulation study and measurements were carefully analyzed. They clearly demonstrate the presence of magnetic response. The depth of current distribution and current density (approx. 10 nA/mm 2) were obtained. In some plants the signal spreading velocity (about 1 cm/min) was determined.

P304 MATHEMATICAL MODELLING OF CYTOSOLIC C~L'IUM OSCILLATIONS: IONIC FIJJXES AND ~.FCIRIC

IK)TENIIAL D[FFERENCF_~ ACROSS THE MEMBRANE OF ENDOPLASMIC RETICULUM

Ale~ Fairnet ~, Marko Marhl ~, lVlilan ~ '~b

"Uniwrsity o f ~ , Faculty of Education, ~ cesta 160, SI-2000 Mimaxr, Slovenia b JosefStefan Institute, Jamova 39, SI-1000 Ljubljem, Slovenia

A theoretical model of inlracellular calcium oscillations taking imo accotmt a new comeption of calcium stores in endoplasrnic reticulum (ER), two types of cytosolic proteins with distinct calcium binding kinetics as well as mitochon&ia has been recently prcsented(Marhletal., Biophys. Chem. 71 (1998) 125-132). We take h,ao accot~ the reoem expedmmtal'results, sbown that a taflaer small portion of ER traticipates in calcium sequestering, lVloreover, we analyse the ~ eonlributions of calcium fluxes across the Ell menlmmv m a consequvrg~ of caleitma binding to cytosolie Imxeins ~ d calcium uptake byth, m i t o c ~ By asstaning the Nemst equilibrium equation with f,~f~et to high permeate monovalent ions across the ER tnmlxa~ the tramrmmbmiie potential of the ER can be predicted up to few millivolts. It is shown that our predictions of the values of the ER ~ potential emerge from the redistribution of calcium iota across the ER ~ . These values ace actually much higher due to involvement of other calcium stores than they v~uld be expected by sole consideration of changes in cytosolic calcium conccntr~on. Since the ER t r a n s ~ potential is not mcasmable by the existing e x p c r ~ l mettmds its prediction might be of great help for experimental and theoretical r~,~,earch of intracellular information processing and t r a m ~ signalling.

P305 INCREASE IN CYTOSOL Ca ̀+ CONCENTRATION AS AN INTERMEDIATE STEP OF INTRA- CELLULAR SIGNALING INDUCED IN PLANT CELL BY LIGHT AND PHYTOHORMONES I.D. Volotovski~ S.G. Sokolovski (Institute of Photobiology, Minsk, Belarus), M.R. Knight (University of

Oxford, Oxford, UK) The release of Car" into cytoplasm was found to be a common feature of many responses which are

registered as a result of action of red light (R) addressed to phytochrome (Phy), phytohormones (H), and putative mediators of intracellular sisma!ling , cAMP and r on plant cell. R-induces Car" released in oat protoplasts from the intracellular Car" stores. Using inhibitors, the principal inner store was found to be endoplasmic reticu- him. The PH-induced increase in [Ca2+]~ is mainly eansed by the release of ion from intracellular stores (auxins, gibberellic and abscisic acids). However, epibmssinolid activates only Ca r" permeability of plasma membrane. It acts on the surface of plant cell. It'was also found that R controls the activity of phospholipase C (PLC) and the breakdown of phosphatidylinositol-4,5-bisphosphate, precursor of inositol-l,4,5-trisphosphate (cellular Car" mobilizer). Phy acts using G-proteins since GTIX/S substitutes the R effect and GDPI~S inhibits stimulation of PLC activity even by R illumination, Combined action of R and GTP'/S did not reveal any additive effect indicating G- proteins are localised in direct chain of phytochrome transduction. Membrane-permeable analogs of cAMP and cGMP result in an increase in [ca2§ ]~ in pr03oplasts of trasgenic tobacco expressing Car" sensitive photoprotein aequorin that indicates close interaction between the systems of two types of mediators. The presented date are discussed from the point of the existence of a crosstalk among different signalling pathwav~ in plant ce l l

139

C3 : Uembranes :Transmembrane Signalling and Transduction

P 3 0 6 b~ve~m Or ~ e ]emeemm u, S~apee Ck~

~d AI~ V. Ch~

De--~ o: ~ Phys~ca, 7"h,~ ~ K~ U~wv~O,, ~,v,

Dept. of B i ~ Kiev Medical ~versify, KJev, Ub~ne Ced-to-ceil conmxmication was muiied by the methods of phase ~ ' o n theory. Such pmpertim of qmtially finit~

size systems as the pair correlation fuactiom and correlation length were investigated. For these purposes the Mumter method

was rued. Fammu~ of this method is concluded in solving of Ormtein-Zemike (OZ) differential equation with the delta-

fimction as the zeroth approrlmatlon for the direct correlation function. Next itemiom for the pair and direct correlation

functions were found with the help of integral OZ equation. To inveslisate the cooperative processes in i~jnaptic deft some

speculative approximations (namely plane-parallel geometry of the deft, location of receptors on the boundary surface inside

the layer, etc.) wee used. Symptic ~ o n was studied using the hypothesis ofisomorpldm for the sym~n "mediator- receptor" in symptic clefts and binary liquid systems near the critical mixing-consolute point.

P 3 0 7 STRUCTURAL BASIS FOR KINETIC MECHANISM OF CYCLIN DEPENDENT KINASE-5

P. Sharma 1, P.J. S ~ h 2, ~ Shatrma 1 , N.D. Amin ], J. Barchir Jr?, R.W. Albers 1 and H.C. Pant 1 . ~Laborato~ of NeurochemJst~, National Institute of Neurological Disorders and Stroke; Centre for Molecular

Modelling, Centre for Informati'on Technology; 3Lab of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD-20892.

Cyclin Dependent Kinase-5 (CDK-5) unlike other CDKs is active only in neuronal cells. However it phospho~ylates serines/thrconines in (S/T)PX(K/R) type motifs like othe~ CDKs. CDK-5 specifically phosphorylates S/T only in KSPXK motifs in the tail portion of Neurofi~ment-H confirming the highly specific substrate recognition mteraction~ We probed the structural basis for its substrate specificity by analyzing the conformations of peptides containing either KSPXK or KSPXXX repeats. The conformation of these peptides in 50% TFE was very different: the KSPXXX repeat peptides folded into helical conformations, however KSPXKmotifintroducedal~-bendinthesubstmtepei~delmckbone. Dueto lackofacxysml mucmreof a CDK/subsWate complex the structural basis of this interaction ( e n z y m e ~ ) is unknown. We ideatified the substrate binding site of CDK5 by modeling peptides derived from Histone HI and NF-H. Mutation to Ala of certain residues (D86 and D91) in cdk5 resulted in substantial loss of affinity to the subsWate peptide. These data supported the binding site ptopesed by our model. Recent results have indicated that CDK-5 phosphorylates its peptide substmtes through an ordered hi-hi mechanism in which pep~de substrate is the first substrate to bind followed by ATP binding, whereas most other protein kinases bind ATP first followed by pep~de substrate.

P 3 0 8 EVIDENCE FOR NA§ 2~ EXCHANGE IN CERTAIN NON-EXCITABLE CELL LINES

M.Balasubramanyam*, M.Condrescu, J.P.Reeves and J.P.Gardner~ UMDNJ, New Jersey Medical School, Newark, NJ 07103 USA (*Center for Biotechnology, Anna University, Chennr~ 600025, India) Recently the Na+/Ca 2+ exchanger (NCX) has claimed a wider tissue distribution and many tissue-

specific isoforms have been identified. We examined putative 'forward' and 'reverse' mode NCX activities using specific 4SCa and fura-2 based cyto~lic calcium (Ca0 measurements in cultured iymphocytes (Jurkat, Molt, YAC-I and EBV-transformed B cells), the promyelocytir cell line HL-60, THP-I monocytes and Jurkat cells transfected with the bovine cardiac NCX (17-3J cells). Of cells treated with thapsigargin (Tg, 100 nM) and subsequently exposed to Ca~-free media, only THP-I and 17-3J cells showed rapid extrusion of C~ (NCX-mediated) in Na-containing solution. Ouabain-treated cells exposed to +/- Na-solution demonstrated the reverse mode NCX activities, 4~Ca uptake was significantly (29-57%) enhanced in low Na-solution m HL- 60, TI-IP-I and 17-3J cells. When LaCls and SKF 96365 inhibited the store-operated Ca 2+ channels, Tg- mediated Ca 2+ entry still showed enhanced rates (0.4-2.9 fold) of Cai in low Na-solution only in the above cells. 5(6) carbuxyensin (plasma membrane calcium pump inhibitor) treatment did increase NCX-mediated 4~Ca uptake and Tg-evoked increases in Cai in HL-60, THP-I and 17-3J cells and was additive with ouabain. These results suggest the Na+/Ca 2+ exchanger contributes to Ca 2+ homeosiasis in non-excitable cells and this transport becomes apparent when the parallel system (PMCA pump) is compromised or inhibited. The lack of demonstrable Na+ICa 2+ exchange activity'in lymphocyte cell lines needs further investigation.

140


P309 Membrane modifications of photoreceptor cell during micromanipulation by optical tweezer

Shamci Monajembashi t, Gotz Pilarczyk I, K.O. Greulich I and Eugenia Kovacs' ISingle Cell-Single Molecule Department, Institute of Molecular Bioteehnology, Jena, Germany 2Biophysics

Research Department, "Carol Davila" Medical University, Bucharest, Romania

Intact rod photoreceptors from freshly isolated frog retina were used for testing micromanipulation by optical tweezers (1064 nm, 2mW Nd-Yag laser). Optimal power densities able to rotate the cell by 90 and 180 o without producing membrane lesions after 5 minutes of continuous manipulation were selected. The optimal power density, able to rotate the cell by 90* without producing membrane lesions after 5 minutes of continuous manipulation was found to be 44mW/m 2. The tempereture modification within the cell suspension was monitored during manipulation and did not exceed 1.7 ~ C after 10 minutes of beam application.

Cell membrane modifications at long time manipulation (more than 10 rain) were followed up using DIOG fluorescence. Cell bendings at long distance from beam application point were observed as well as local membrane breakdown regions which repaired in several minutes after stopping irradiation. Optical tweezers appear thus to be a possible benign technique for manipulating visual photoreeeptors.

P310 Retinal Intracellular Records for the Rabbits After Exposure to I~-particle Doses

EI-Refaei F.M), Talaat M.M. 2, EI-Awadi A.I.land Ali F.M. 3 I Biophysics Unit, Research Institute ofOphthal, Giza Egypt

2 Biophysics Dept., Faculty of Science, Ain Shams Univ. 3 Biophysics Dept., Faculty of Science, Cairo Univ.

This study aimed to investigate the effect of [3-particles on the intracellnlar recording of local ERG at different depth for the New Zealand rabbit's retina at the doses of 40-100 Gy. The study included direct effects (after 24h) and delay effects (after 30 days and 90 days) for the treated and scattered eyes. The obtained data showed dose dependent changes and photoreceptors were the most radiosensitive cells in the retina. The significant changes were observed above 60 Gy. The inner retina (ganglion cells) showed shght changes at the dose of 100 Gy only. The results are confirmed by the histopathological examinations. We conclude from this study that there is a need to protect the scattered eye and to use the radioprotector.

Key Words: j3-particles, retina, intracellular records, histopathology.

P311 KINETICS OF CALCIUM CURRENT WITH RELEASE-DEPENDENT INACTIVATION

IN RAT VENTRICULAR MYOCYTES Jana Pavelkov~ Alexandra Zahradnfkov~

Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia The L-type calcium current in whole-cell patch-clamped rat ventricular myocytes has a prominent fast component of inactivation, which is suppressed by conditions that reduce calcium release from the sarcoplasmic reticulum. We have developed a phenomenological kinetic equation, assuming independent pathways of channel activation and inactivation mechanisms, which quantitatively described this release-dependent inactivation (RDI) by means of 4 parameters. RDI was delayed behind activation of calcium current. Within a single cell, the delay was stochastically distributed around the mean h with a standard deviation w d. Both parameters decreased with test pulse voltage. The rate ofRDI (1/x~) had a bell-shaped voltage dependence. RDI affected -65% of L-type Ca channels on average. To study the relation of RDI parameters to spatiotemporal Ca 2. dynamics in the tubulo-reticular junction, we have constructed a single-channel Markovian model of the excitation-contraction coupling unit (ECCU), consisting of one L-type calcium channel and a group of 5 ryanodine receptors (RyRs) with defined geometry. The interaction between the channels in the ECCU was implemented by accounting for the formation of instantaneous Ca b gradients during channel openings. The characteristics of RDI in the model were similar to those experimentally observed, providing evidence that RDI reflects calcium signaling during excitation-contraction coupling. Supported by VEGA 5155 and 5156, and by HHMI 75195-547801.

141


P 3 1 2 C ompu ta t i ona l Models o f L igand-Biad ing Sites in l oa Channels and G-prote in Coupled Receptors

Boris S. Zhorov and Vettai S. Ananthanarayanan Dcpmhxient o f Biochemistry, McMaster University, Hamilton, Ontario, Canada

Numerous experimental data indicate that metal ions such as Na +, Ca 2+, Mg 2+ which are present in mM concentrations in the extracellular environment modulate binding of ligands to ion channels and G-protein coupled receptors. Experiments performed in our laboratory and by other groups demonstrate that various drugs, peptides, and hormones bind metal ions, in particular Ca 2+, and translucate Ca 2+ vial lipid bilayers. However, detailed mechanisms of modulation of ligand-receptor interactions by metal ions remain unknown. Using the crystallographic structure of a K + channel (Doyle et al., t998) and electron cryomicroscopy structure of rhodopsin (Baldwin et al., 1997), we have built homology models of L-

2+ type Ca channel and mu-opioid receptor and performed ducking of specific ligands in these models. The energetically optimal structmes of the proteins with and without ligands were obtained using the Monte Carlo miniroizafion method. For both proteins, inclusion of metal ions to form ternary complexes with the ligands and their binding sites was necessary to explain abundant and often paradoxical a~te on structure-activity relationships of ligands and mutation experiments. Based on the computational models, we have proposed mechanisms'of activation and blockade of L-type Ca 2+ channels and G- protein coupled receptors by their corresponding ligands. Supported by MRC, Canada.

P 3 1 3 PHYSIOLOGICAL PROPERTIES OF MYOCYTES IN CONTEXT OF" AN IIqTEGRATIVE BIOPHYSICS Michailov~ M.Ch., Neu, E., Seidenbusch, W., Gomik, E., Martin, D., Welscher, U., Weiss, D.G. Inst. Exp. Physics, Univ. Innsbruck and TU Vienna, Austria; Inst. Zellphysiol., Univ. Rostock, FRG An integrative biophysics should consider molecular properties of the living system in their multidimensional interdependence and in their unity, i.e..in a holistic approach. On the example of vesical myocytes some results will be givea. A. Motor activity. Whole urinary bladder (guinea pig, rat) in vitro and in vivo showed a phasic ( l-5/min) and, at a critical intravesical pressure, a rhythmic tonic (burst-like; 0. l-0.5/min) activity (isovolumetric cystotonometry), whereby different kinds of phasic motor patterns of detrusor strips and tonic ones of vesical trigone (possible pacemaker for micturiton) appear (n=30). B. Electrical activity. Vesical myocytes generate spikes (S), bursts 03) and burst-plateaus (BP) with more than 10 subtypes (intracellular recording). After a critical stretch (>50 raN) S were transformed into BP and rate of rise and of fall of S increased (0.4+0.2 to 3.3:t:0.7 and 0.4+0.3 to 2.3 + 1.6 V/cm resp.) (probably Ca + +-activated K + stretch channels) (n--101). Open questions ate the correlation between different patterns and the role of mesomorphic state of biomolecules (nematic, smectic, cholesteric) as well as ferro-, piezo-, pyrcolectricity for S, B, PB activities. C. Extra- and intracellular regulation. Intra- and extracellular factors change electrical and motor activities: cAMP decreased, cGMP (0.1-t raM) increased the frequency of BP; procaine (10-100/tM) and cypermethrin (1-10 nM) transformed S into a BP activity (n= 10). In context of an integrative biophysics the physiological and pathological vesical properties from (neuro-) reflex up to molecular level have to be investigated. Lit.: Michailov, Neu et al., Proc. 1UPS 16, 118, 1986; 17, 529, 1989; I8, 216, 1993; Eur. J. Physiot. 419, R98, 1991; 420, R99, 1992; 430, R49, 1995.

P 3 1 4 G A B A c r e c e p t o r s in m a m m a l i a n c o n e p h o t o r e c e p t o r s

B. R. Pattnaik, A. Jellali, V. Forster, D. Hicks, J. Sahel, H. Dreyfus and S. Picand Laboratoire de Physiopathologie Retinienne, Strasbourg, France

The recently classified GABAc receptors activate a chloride current with novel pharmacology. These receptors have attracted significant interest since their discovery in the retina. In mammalian retina these were shown only in bipolar cells postsynaptic to photoreceptors. We have recently found the expression of these GABAc receptors in mammalian photoreceptor using a photoreceptor culture model on a feeder layer of retinal glial cells. While the rods did not respond to GABA puff the cone GABA response showed both GABAA and GABAc receptors mediating chloride current. The GABAc receptors are isolated by the GABAA receptor antagonists bicuculline and SR95531 and blocked by GABAc receptor antagonists I4AA and TPMPA. They are nondesensitising, not activated by anesthetic pentobarbital but are modulated by divalent cations like Zn 2+. Consistent to these results we have confirmed these receptor expression in vivo by recording cones from intact retina. These results provide evidence for a GABA feedback from horizontal cells to cones with multiple modulation which is mediated through GABAA and GABAc receptors.

142

D1 : Molecular Assemblies, Recognition and Metabolic Regulation : Structure of Macromolecular Assemblies

P315 Two Dimensional Crystallization of ArsA Protein on Lipid Monolayers

Hong-Wei Wang and Sen-fang Sui Biophysics Group, State Key Laboratory of Biomembranes

Department of Biological Science and Biatechnology Tsinghua University, 100084, P. R China

ArsA protein is the soluble subunit with ATPase activity of an anion pump in the cell membranes which extrudes the arsenite or antimonlte from the cytoplasm. The protein has been determined in dimeric form in the presence of its allosteric ligand, arsenite, but in monomeric form in the absence of arsenlte. Recently, we have crystallized this protein on two- dimensional plane. We found that the soluble protein was non-specifically adsorbed onto negatively charged monolayers composed of DMPS and DOPC by electrostatic force. At optimum conditions, two-dimensional crystals of ArsA protein were obtained. The crystals' cell unit parameters were a=b=8.2 nm, y---60 ~ From the projection map, one ring composed of two monomers per unit cell was obviously observed. Further study on the conditions for 2D crystals' formation revealed that 2D crystals composed of dimeric rings can be obtained both in the presence and absence of the allosteric ligand, arsenlte. Comparison of the projection maps of crystals in different conditions indicated that the two monomers in ArsAs bound with allosteric ligand were more tightly conjugated than those in ligand-free ArsAs. This phenomenon was also supported by the gel filtration experiments. Further analysis of two-dimensional assembly of ArsAs on monolayers also hinted that monomers of ArsA may form dimers on membranes due to the protein-protein and protein-lipid intei'action.

P316 General model for 3D structure of wide and narrow Z-bands in striated muscle

Pradeep K. Luther, John Barry, Ed Morris & John Squire, Blackett Laboratory, Imperial College, London, UK

The Z-band in vertebrate striated muscle is an intriguing tetragonal network that enmeshes actin filaments from adjacent sarcomeres and so allows the tension generated by the contractile activity to be transmitted along the myofibril. In longitudinal sections the Z-bend resembles a zigzag assembly linking the opposite sets of actin filaments. Narrow Z-bands with one or two zigzag layers occur in fast muscles, and wide Z-bands with three or four zigzag layers occur in slow and cardiac muscles. We have recently carried out 3D reconstruction of the Z-band in fish body and fin muscles (narrow Z- bands), bovine slow muscle (wide Z-bands) and nemaline rods (extended Z-bands). These reconstructions have enabled us to identify different axial levels of links between anti-parallel actin filaments. The number of axial levels for the Z-bands in fish body, fish fin and bovine slow muscle is 2, 3 and 6 respectively. The axial periodicity of the Z-links is close to that of the underlying actin filaments. On the basis of these results we propose a new general 3D model for the vertebrate Z- band which relates the different types found. On this model, the "simple Z-band" that comprises a single zigzag structure is formed by two levels of links, the minimum number required to form a fully networked structure. Wider Z-hands are obtained by successive addition of extra layers of links. Any single layer has links running in one direction only. The symmetry of the Z-band for even number of links is p21 and for odd number it is c2.

Supported by the British Heart Foundation and The Wellcome Trust.

P317 H Y D R O P H O B I C SURFACES OF POLYSACCHARIDES:

T H E I R USE AS S O L U B I L I Z E R S A N D C H A P E R O N E S C. Sivakama Sundari* and D. Balasubramanian**

*Centre for Cellular and Molecular Biology, Hydera~ad 500007 **Hyderabad Eye Research Foundation**, L. V. Prasad Eye Institute, Hyderabad 500034, India.

Stereoelectronic and epimeric considerations impose conformational constraints on oljgomeric saccharide chains, a particularly surprising and useful example being the development of ribbon like structures in r linked glucosides called the dextrins, leading to one side of the ribbon being hydrophilic (with all the OH groups) and the other side offering a relatively nonpolar face. Consequently, we show that dextrins can solubilize liphophiles in water, bind to the minor groove of DNA, bind to surfactants and membrane-active compounds, and inhibit the aggregation and precipitation of denatured globular protein chains, and help chaperone them fold back to their native forms. Such amphiphilicity is absent in cellulosics ([~-l,4-glucosides) and dextrans (r This hydrophobic face of dextrins (and some other saccharides) has been used by us to advantage in a variety of biophysical instances.

143


P318 MOLECULAR MODELLING OF DISACCHARIDE FRAGMENTS OF SIALYLOLIGOSACCHARIDES

INTO THE ACTIVE SITE OF INFLUENZA A SUBTYPE N9 NEURAMINIDASE ENZYME. K. Veluraia, T. Hema Thanka Christlet and M. Xavier Suresh

Department of Physics, Manonmaniam Sundaranar University, Tirunelveli -627 012, Tamilnadu, INDIA.

Neuraminidases are family of enzymes which cleave sialic acid from sialyloligosaccharides irrespective of the type of linkages with some specificity difference between families. Molecular fit studies of sialic acid in the active site of influenza A subtype N9 neuraminidase indicate a minimal allowed orientation ( <1% ) in the Eulearian space. By fixing the sialic acid (NeuNAc) at the active site in the allowed orientation, molecular modelling of disaccharide fragments of sialyloligosaccharide was carried out by changing all the permissible conformational parameters such as glycosidic torsion and the free rotation of exocyclic bonds. In the best fit orientation the modelled compounds namely, NeuNAc a(2-3)Gal, NeuNAc a(2-6)Gal, NeuNAc 0.(2-8) NeuNAc and NeuNAc a(2-9)NeuNAc exhibit the similar glycosidic torsion angles. This enzyme can accommodate the above disaccharide fragments without any stereochemical clash but with good number of hydrogen bonding interactions. An in depth analysis on the conformational preference of sialyloligosaccharide at the active site indicates a structural similarity between these disaccharide fragments which orientate few of the hydroxyl groups of second residues to the identical positions. This finding shows that this enzyme can accommodate a minimum of two sugar residues at the binding site and can cleave the sialic acid containing different glycosidic linkases due to the structural similarity caused by the conformational parameters.

P319 Enzyme-Catalyzed Gel-Sol Transition of Protein Gels: Proteinase versus "Gel-Solase" Actitity H. Berry t, J.Pelta ], D. Lairez 2, V. Laretta-G-arde~ l D~artement de Biologie, Universit~ de Cergy-Pontoise, France 2 Laboratoire L~n Brillouin, CEA-Saclay, France

The extraeellular matrix can be considered as a protein gel. During metastasis dissemination, invading cells use proteinases to disrupt this insoluble network Such protein gels may undergo gel-sol transition when the peptide bonds between monomers a r e broken. Proteinases, catalysing peptide bond hydrolysis, are able to induce this transition. This work concerns the experimental study of proteinase-catalysed degradation of fibronectin and extraceUular matrix gels. We report measurements (280 nm absorbance) of the sol fraction increase as a function of time. Results are discussed in terms of gelation theory used in polymer physics. We investigate the relationships between enzyme kinetics (enzyme concentration or specificity, kinetic parameters) and supramolecular parameters (gel fraction, distance to the threshold). Using the classical reduced variables for gelation critical phenomenon, the proteinase activity disappears in favour of an universal gel-sol transition-catalysing activity ("gel-solase" activity) independent of enzyme specificity, of the gelified protein and of the gel type. We suggest that the universal behaviour of gel-sol critical transition may shed a new light on some biological implications. It provides new insights about the extracellular matrix disruption during metastasis dissemination. It can also be discussed in terms of pro-enzyme cascade activation and of extrac~llular'matrix proteinase specificity.

P320 PROBABILISTIC KINETIC MODEL OF SLOW PEROXIDATION OF LOW-DENSITY LIPOPROTEIN

Dubra~,ka _Kri_loy_ ~ and Nata~a Stqianovi~2and Janko N. Herak 2 1 School of Medicine and 2Faculty of Pharmacy. and Biochemistry, University of Zagreb, Zagreb, Croatia

The microscopic probabilistic model has been introduced to explain the kinetics of very slow peroxidation of low- density lipoprotein (LDL) from human plasma. The LDL oxidation, carried out in very unfavorable conditions, is assumed to be initiated by the traces of the transition-metal ions associated with the iipoprotein. The substrates for the metal-ion attack are (x-tocopherol and the pre-formed lipid hydroperoxide. According to the model, the entire oxidation process consists of rare bursts of events in individual LDL particles. The reactions within the particles are treated in terms of probabilities of individual active species to participate in a specified reaction. The circular flow of the radical reactions could be visualized as cirodar flow of microscopic probabilities. The en-~pirical, macroscopic quantities are quantitatively related with the microscopic probabilities, determined by a set of five adjustable parameters. The differential equations describing the radical generation rate and the rates of change of oxygen, hydroperoxide, co-antioxidant and trapped radicals are numerically solved in a finite difference approach. The model was successfuly tested in the experiments with the controlled addition of small amounts of cupric ions to the LDL solutions, to satisfy the conditions of slow pcroxidation. This model is applicable to other heterogeneous systems of compartmental structure.

144


P321 CROWDING IN FIBRILOGENESIS: EXPERIMENI"AND THEORY Ravi Jasuia. Maria Ivanova, Rossen Mirchev* and Frank A. Fen'one

Department of Phy sics, Drexel University, Philadelphia, PA 19104, USA

The polymerization of sickle hemoglobin is one of the best characterized protein assembly reactions, and is based on a double nucleation mechanism in which fibers can nucleate from pure solution or nucleate on the surface of other polymers. Once nucleated, the fibers grow linearly and are thereafter indistinguishable. This conceptual model has been given a thermodynamic formulation that describes equilibrium constants for nucleation in fundamental terms (i.e. contact energy and entropic loss and recovery). To complete the thermodynamic picture required an accurate description of the crowded milieu in which polymerization occurs. The original description invoked scaled particle theory for the homogeneous nucleation step, and assumed exact cancellation o f nonideality in the heterogeneous step. Recent experiments performed in this laboratory have challenged these assumptions and we are evolving the next generation of description. We will present the results o f our experiments as well as the effectiveness of more elaborate descriptions of vo lume exclusion. Because this description is formulated in fundamental terms, it should apply to any fibril formation process t~at occurs in a crowded medium. *present address, Harvard University Medical School

P322 Membrane assisted bacteriophage M13 disassembly

David Stopar ], Ruud B. Sprnijt 2, Cor J.A.M Wolfs 2, Marcus A. Hemminga 2 University of Ljubljana, Department of Microbiology, Ve~na pot 111, 1000 Ljubljana, Slovenia, 2University of

Wageningen and Research Center, Department of Biomolecular Sciences, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands

During the infection of Escherichia coli, the phage coat is dissolved in the host cytoplasmic membrane, while viral DNA enters the cytoplasm. The process of bacteriophage M13 disassembly was followed by using site-specific cysteine mutants of the bacteriophage M13 coat proteirL Intact phage mutants were labeled with either fluorescence or spin label for the purpose of fluorescence and electron spin resonance spectroscopy. The phage particles were then disrupted in different membrane model systems (lipids, ionic and non-ionic detergents). In conjunction with CD spectroscopy, local and overall structural changes were monitored during protein coat solubilisation. Only strong detergents, such as SDS and CTAB were able to disrupt the tight phage structure. No phage disruption was observed below detergent critical micellar concentration. Based on this finding and the linear dependence of phage disruption on detergent concentration it is suggested that the solubilization of the phage coat protein starts with an attachment to and penetration of detergent monomers into the phage particle. The detergent concentration in the phage increases in a proportion the detergent concentration in the aqueous phase. When the concentration of the detergent in the phage is equal to cmc of the detergent a massive solubilization of the coat proteins occurs.

P 3 2 3 Role of Ca ++ and CI" on Fibrin Networks Structure and Dynamics

G. Ar~ovito ~, M. De Spifito ~ Istituto di Fis., and INFM, Fac. Med. Chit., Universitd Cattolica S.C., Ego F. Vitu, 1 -00168 - Roma, Italy

Fibrin,networks are biological gels playing a key role in blood coagulation processes and in many other emorhenlogy problems. They are three-dimensional networks formed by branched polymers grown in solutions of fibrinogen macromolecules (Mw = 3.4x105) activated by the thrombin enzyme. In this work we present Static Light Scattering (SLS) and Dynamic Light Scattering (DLS) study of the fibrin get dynmnics. The gels were grown at room temperature from fibrinogen solutions in a variety of both CI (0A M - 0.5 M) and fibrinogen concentration. Wavevector q values were ranging between 3 . 0 x 1 0 2 - 3.3x10 s cm ~. Our data show that the gel is a three dimensional network of thick fibers entangled together with an almost linear self-similar structure. This structure is characterized by a mass fractal dimension Dm that persists for length scales smaller than the gel mesh size ~1 and larger than the average fiber diameter ~2. Fractal dimension, mesh size and average fiber size have been found strictly dependent on and correlated to the physico-cbemical parameter of the gelling solution. By increasing the CF t concentration an ionic screening of monomer binding sites responsible for the lateral aggregation is allowed so that ~, decreases. This mechanism however has a non trivial secondary effect in an increasing of Din, being the latter dependent on the persistence length lp which'is expected to decrease with the fiber size. The elastic modulus G obtained in different experimental conditions from DLS measurements has been correlated to persistence length values. Finally, the major role played by Ca ++ on fibrin networks porosity has also been investigated.

145

D1 : Molecular Assemblies, Recognition and Metabolic Regulation : Structure of Macromolecular ASsemblies

P324 How Pentamers Pack on Two-Dimensional Plane?

- Two-Dimensional Crystallization of C-Reactive Protein Sen-fang SUl and Hong-Wei Wang

Biophysics Group, State Key Laboratory of Biomembranes Dept. Biol. Sci. & Biotech., Tsinghua Univ., 100084, P. R China

The packing of regular pentagons on two-dimensional (2D) plane is always a difficult problem to both mathematicians and physicists, for the pentamedc symmetry's unsuitability for 2D lattice. Since Many biological molecules show pentamedc structures and the types of their 2D arrangement may reflect the interaction between molecules, it is also significant for biologists to study on the packing of pentagons on 2D plane. During the study of C-reactive protein's 2D crystallization, we were concerned by such a problem. C-reactive protein is composed of five identical subunits thus to be a classical pentameric protein. By monolayer technique, we obtained two types of CRP's 2D crystals and found their coexistence on monolayers. By analysis on the projection map of the two types of 2D crystals and the spatial relationship between them, we built models on the packing of these pentamers on 2D plane. Comparison with the CRP's 3D atomic model built by X-ray crystallography leads us to the hypothesis that the strong interaction among the proteins in 2D crystals may be due to the electrostatic force between contacting subunits in adjacent pentamers. Models based on the electrostatic interaction between adjacent pentamers were built to interpret the mechanism of the 2D crystals' formation. It also gave hints on the proteins' dissociation on membranes, which made fta'ther study of the phenomenon possible.

P325 Three-dimensional (3D) Mapping of Elongation Factor-G (EF-G) on the E. coil 70S

Ribosome: The Mechanism of Translocation

Rajendra K. Agrawal I, Amy B. Heagle 1.2, Pawel Penczek 1,3, Robert Grassucci 1,2, and Joachim Frank ~,2.3 ~Wadsworth Center, 2Howard Hughes Medical Institute and 3Department of Biomedical Sciences, State

University of New York at Albany, Empire State Plaza, Albany, NY 12201-0509

3D cryo-electron microscopy was used to visualize EF-G on the 70S ribosome in GDP and GTP states. The tip of domain IV of EF-G and the anticodon arm of A-site tRNA, in the pre-translocational state, interact with the same site on the ribosome. Results show that GTP hydrolyis is necessary (i) for the binding of EF-G to the pre- translocational complex, and (ii) for the completion of translocation. We are able to locate large conformational changes in the ribosome that are associated with EF-G binding and with EF-G-dependent GTP hydrolysis. In the GDP state, the L7/L12 stalk is elongated and an arc-like connection is seen between its base and G' domain of EF-G, whereas in the GTP state, the stalk splits into two subdomains and the arc-like connection is absent. In addition, upon EF-G binding, the head of the 30S subunit moves with respect to the 50S subunit. Based on these results, a two-step mechanism oftranslocation is proposed.

P326 Three-Dimensional Reconstruction of Ryan~dine Receptor Isoform Three in Different Conformational

States As Visualized by Cryo-Electron Microscopy Maniuli R. Sharma*, Loice H. Jeyakumar, Sidney Fleischer and Terence Wagenknecht.*

*Wadsworth Center for Laboratories and Research, NY State Dept. of Health, Albany. NY 12201. U.S.A., Dept. of Molecular Medicine, Vanderbilt University, Nashville, "IN 37235, U.S.A.

Ryanodine receptors are intracellular calcium release channels that play a key role in excitation-contraction coupling. The third isoform of the ryanodine receptor (RyR3) is the least understood among all the three known isoform. Using cryo-electron microscopy and single particle image processing technique, we present the first three-dimensional reconstructions of RyR3 in two different functional states. A difference map between the open and close states of the RyR3 reveals significant conformational changes in the transmembrane region, and the cytoplasmic region of the RyR3. Due to -70% sequence homology with RyR1 and RyR2, RyR3 appears similar in its three-dimensional architecture to other two isoforms of the ryanodine receptor. This study presents results important in understanding the structure-function relationship of ryanodine receptors during excitation-contraction coupling.

146


P327 Structure of Abnormal Molecular Assemblies (Collagen VI?) Associated with Human Full

Thickness Macular Holes

Carlo Knupp', Peter M.G. Munro § Pradeep K. Luther', Eric Ezra + & John M. Squire .("

" Biophysics Section, Blackett Laboratory, Imperial College, SW7 2BZ London, UK; § Department of Pathology, Institute of Ophthalmology, ECIV 9EL London UK.

Transversely banded deposits with an approximately 100-nm periodicity have been seen in association with a number of eye pathologies (e.g. age related macular degeneration). Recently such aggregates have been discovered in the cortical vitreous of a patient suffering from full thickness macular holes. The aggregates in the vitreous were of sufficient size and regularity for us to attempt ultrastructural studies in the electron microscope. The molecules forming this aggregate pack in a centred tetragonal unit cell of dimensions approximately 26 x 26 x 180 nm. The aggregate has been discussed in terms of its possible protein constituents and collagen VI has been singled out as the most likely protein to form the aggregate. Two alternative models for the molecular packing have been proposed. Understanding the structure of these banded deposits in the eye may help to throw light on the pathophysiological mechanisms of the diseases to which they are correlated.

P328 VISUALIZATION OF THE CEASES OF MICROTUBULE DEPOLYMERIZATION THAT OCCURS AT THE SITES OF MAP2 CLUSTER. Koji Ichihara, Hidefumi Kitazawa, Yusuke Iguchi, Hirokazu Hotani and Tomohiko J. Itoh. Division of Biological Sciences, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.

Individual microtubules repeat alternating phases of polymerization and depolymerization both in vitro and m living cells, a process known as "dynamic instability." In cell free systems, MAPs regulate the dynamic instability especially by increasing the frequency of rescue. To investigate the role of MAPs on microtubules dynamics, we attempted the simultaneous observation of both the phase changes and the distribution of MAP2 on individual microtubule. MAP2 was modified selectively on its projection region by x-rhodamine iodoacetamide, an SH-directed fluorescent dye, and 2.4 mole of the dye was incorporated into one mole of MAP2 molecule. The activity of microtubule binding did not change significantly upon modification. When 0.027 laM of the fluorescent MAP2 was added to microtubules which had polymerized from 14 plVl of pure tnbulin, individual microtubules became visible under fluorescence microscopy. Fluorescent clusters in various sizes were distributed inhomogeneously along the microtubul~s. When dynamics of microtubules was observed by dark-field microscopy after determining MAP2 locations under fluoregcence microscopy, depolymerization of the microtubules often ceased at the positions of those clusters formed. Every cluster did not always cease depolymerization, but depolymerization did cease at only the cluster sites, indicating that the MAP2 cluster on microtubule lattice could cease microtubule depolymerization.

P329 The spin labeling of thiol groups in human plasma LDL

Greta Pifat', Marina Kveder', Slavko Pe~ar § Milan Schara § "Ruder Bo~kovi~ Institute, Zagreb, Croatia, § Stefan Institute, Ljubljana, Slovenia

AFaculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

In this study the possibility of more specific spin labeling of the protein component in LDL has been investigated. We have used the methanethiosulfonate spin label in order to probe the reduced cysteine residues in apoB. The covalent modification of protein was studied using electron paramagnetic resonance (EPR) spectroscopy. The results indicate that the spin labeled sites are predominantly buried in the LDL particle. However, the EPR spectra reveal also the contribution of cysteine residues assigned to the surface of apoB which was independently proved by Elhnan procedure. That thiol specific labeling can be useful in studying stability/conformation of apoB was demonstrated in the experiments with denaturing agents. These findings are discussed in the framework of the linear model of apoB wrapping around of the LDL particle. The approach presented in this work offers a new possibility to match data from the EPR method with the primary structure of apoB which still cannot be handled with modern spectroscopic techniques providing the structural information at the atomic resolution.

147

D2 : Molecular Assemblies, Recognition and Metabolic Regulation : Biophysics of Immune Systems

P 3 3 0 CRYSTALLOGRAPHIC STUDIES OF ANTIGEN-ANTIBODY INTERACTIONS INVOLVING A

HBV SURFACE ANTIGEN PEPTIDE Deevak Nair I, Kavita Singh I, Kanury V. S. Rao 2 and Dinakar M. Salunke ~, 1National Institute of Immunology, New Delhi and "Immunology Group, International Center of Genetic Engineering and

Biotcchnology, New Delhi Elucidation of the structural principles of molecular recognition involving biological macromolecules

will enable understanding the code of molecular communication. A set of genetically distinct monoclonal antibodies, recognize a common epitope defined by sequence DPAF on a peptide PS1 (HQLDPAFGANSTNPD) derived from hepatitis.B virus surface antigen. These set or antibodies thus provide a model system for defining the invariant structural properties associated with molecular recognition. Crystals of the first of these antibodies PC283 Fab-peptide complex were obtained and the X-ray intensity data were collected upto 2.9 A resolution. The complex crystallized in the space group P2,2~2~ and the structure was determined using the molecular replacement method. The structure of Fab fragment from the moneclonal antibody against 2,2,6,6 - tetramethyl - I -PiperidinyloxT -2 Dinitropbenyl (PDB code: IBAF) was used as an initial model for molecular replacement beta_ .use IBAF l~s 86% sequence homology with PC283. The structure is presently being refined using XPLOR add the model for the entire Fab molecule as well as the peptide have been built rote the electron density map. The pre.sent I ~ . and Rrr~ ~ 24 ~/~ and 27 %,o ~spe~'tivcly~ The sst~ct~re sh0.ows 7 that.the binding ~cu/s pnmanty mrougn a large numoer ot mseracuons oetween Asp, t'ro~ Ala ana rne ot r b t pepuae ann me hypervaiiable loops of the antibody. Asp" forms a salt bridge wtth Lys "~ of the heavy chain. Phe~is inserted in a h~,drophobic _pocket formed by the residues of different CDRs. These obseryations are consistent with the earlier findings that the PSI peptide binds to the PC283 antibody through the DPAF moiety.

P 3 3 1 Immunological Implications of Structural Mimicry between Peptide and Carbohydrate

Kanwal/eet gaur, Deepti,lain, B. Sundaravadivel, Manisha Goel and D. M. Salunke National Institute of lmmunology, New Delhi-l10067

The two chemically dissimilar molecules, a peptide and a carbohydrate moiety, sharing a common receptor, Concanavalin A, exhibit molecular mimicry. The differential binding of various analogs of the peptide and the sugar with the polyclonal antibodies raised against each of them enabled establishing the topological equivalence between these molecules. The humoral response analysed in terms of primary (IgM and IgG) and secondary (IgG) antibody titers, shows subtle differences in their profiles and cross-reactivities. The antigenic immunodominance during maturation may be invoked to explain these differences. The matured polyclonal antibody response against any ligand need not necessarily represent a complete topological map of the antigen. Therefore, the extent of mimicry may depend on the extent of overlap between the immunodominant epitope and the mimicking epitope. It was observed that the mimicry as seen by the immune system depends on the extent and nature of the T-cell help provided either in terms of a carder protein or the single peptide as the T-cell epitope. Thus, the.carbehydrate-peptide mimicry, which was originally defined as a static topological equivalence between two chemically distinct molecular species in terms of polyclonal antibody erossreactivity is influenced by the kinetic immunological factors. This observation may provide important basis for analyzing, mechanisms responsible for autoimmune disorders.

P 3 3 2 ACTIVATION OF NEUTROPH1LS AND THEIR REGULATION BY GRANULOCYTE COLONY STIMULATING

FACTOR IN NORM AND AT NEUTROPENIA E.I. Kovalenk0, G.N. Semenkova, S.N. Cherenkevich

Department of Biophysics, Belarus State University, Minsk, Belarus

Neutrophil phagocytes provide the basic defence of organism against of pathogenous microbes invasion. Regulation of neutrophil's activation is necessary at occurrence of a number pathological states and can be carried out with participation of cytokines, in particular, granulocyte colony stimulating factor (G-CSF). Functional activation of human bio~l neutrophils in norm and at chemotherapy-induced neutropania (in children with non-Hodgkin's lymphomas and acute lymphoblastic leukemias) and the molecular and cellular mechanisms of the G-CSF influence on cell's function were studied by chemiluminescence methods. It was revealed that G-CSF acts synergistically with latex particles, lectin phytohemagglutinin on the processes of active oxygen forms (AOF) production at adhesion of the cells to glass and on secretory degranulation of the azurophilic granules. The significant decreases of the AOF yield, myeloperoxidase secretion from the cellular granules and also content of myeloperoxidase inside the cells occur at neutmpenia. Possibility of correction of this disturbances with the use of G-CSF was shown. Hydrolysis of'phospholipids by PLA2 and subsequent methabolism of arachidonic acid play essential role in development of the neutmphils changes at neutropenia and in signal transduction in this cells at action of G- CSF.

148


P333 Crystal structure of N-Glycoprotein Linkage Region Models and Related Saccharides

T. Lakshmanan ~, D. Sriram b, S. Srinivasan b and D. Loganathan ~ a Department of Chemistry, b Department of Physics, Indian Institute of Technology Madras, Chennai 600 036

The oligosaccharide parts of glycoconjugates play vital roles in numerous biological recognition processes that are mediated by carbohydrate-protein interactions. Elucidation of the structure and conformation of both the linkage region and terminal ( non-reducing end ) glycan portion of glycoproteins is fundamental to a better understanding of the inter- as well as the intramolecular carbohydrate-protein interactions. A major research program of our laboratory is focused on the enzymatic synthesis and biophysical aspects of glycoprotein-derived oligosaccharides and glycopcptides. Our initial efforts led to the crystal structure of a N-glycoprotein model, l~-l-N-acetamido-D- glucopyranose (1), and the benzamido analog (2). The molecular assembly of the hydrates of 1 and 2 in is driven by seven hydrogen bonds representing interesting patterns. In addition, further stabilization of packing due to ~-~ stacking is observed in 2. Encouraged by the success in obtaining hydrates of such compounds, the crystal structure of the corresponding disaccharide amide (3) prepared from lactose was also investigated. The structural analysis of the hydrated 3 revealed several hydrogen bonds including an inter residual one between 0-3 of glucose and 0-5 of the galactose residues. The structural investigation has also been extended to 13-l-N-acetamidochitobiose(4), a disaccharide model of the linkage region. Details of the synthesis, crystal structures and the molecular packing will be presented.

P334 Trafficking of the hemochrom atosis protein HFE with transferrin receptor in intestinal epithelial cells

Ramalingam T.S., Lebr6n, J.A & Bjorkman F.J. Div. of Biology and Howard []ughes Medical Institute, California Institute of Technolgy, Pasadena CA 91125. USA. lIFE is a class 1 M l-lC-related protein that is mutated in patients with the iron overload d~isease hereditary hemochromatosis . A majority of hereditary hemochromatosis patients are homozygous for a mutation that changes Cys-260 of the mature protein to a tyrosine (C260Y). Recently, HFE was shown to bind to the transferrin receptor (TfR) ( Feder et al., 1998, PNAS 95, 1472-1477) , reducing its affinity for iron-loaded transferrin .HFE has been found in transferrin-positive intracellular vesicles (Gross et al., 1998, J. Biol. Chem. 273, 22068-22074) , imply ing that HFE trafficks to acidic compartments together with TfR. In order to determine i fTfR binding is required for HFE localization in endosom es, we studied HFE trafficking in a transfected human intestinal epithelial cell line (HuTu-80). HuTu-80 cells were transfected with constructs encoding various forms HFE (wild type HFE, C260Y HFE, and a mutant HFE that shows greatly reduced affinity for TfR; WSIA HFE; Lebr6n & Bjorkman, J. Mol. Biol., in press) attached to cytosolic side of green fluorescent protein (GFP). Localization and transferrin mediated iron transport of HFE-TfR complexes were followed by real-time dual-color confocal fluorescence microscopy and by biochemical experiments. C260Y HFE showed an intracellular distribution, most likely corresponding to the endoplasmic reticulum or Golgi, consistent with previous studies that demonstrated that this mutation disrupts light chain association and cell surface expression (Feder et al., 1997, J. Biol. Chem. 272, 14025-14028). Whereas wild type HFE was found mainly in transferrin-positive intracellular compartments, W g l A HFE was found mainly at the cell surface, thus TfR binding is required for trafficking of l iFE into endosomes. The effects of the three forms of l iFE on ferritin expression will also be discussed.

P 3 3 5 P H O T O A C T I V A T I O N OF I M M U N O P R O T E I N S

A. K. Sinp_h n and T. N. Gayatri

Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai - 400 076, India

The ability to photoactivate biological macromolecules remotely, at specific locations and times, allows manipulations of a wide variety of cellular activities and gives rise to many practical applications of biochemical and medical significance. In this work, 2-nitroaryi chromophores have been homobifunctionaily cross-linked to Fe region of immunoglobulin (IgG) and the resulting bioconjugate has been subjected to UV-A irradiation. Photoactivation and controlled release of IgG has been revealed by irradiation time dependent Protein-?, binding levels and SDS page analysis. Binding values from irradiated conjugate could exceed those of irradiated controls because the cbromophore coating protects underlying IgG from radiation.

149


P 3 3 6 Peptide-Carbohydrate mimicry: Interaction of ConA with peptide and carbohydrate ligands.

Deepti Join, Kanwaljeet Kaur, B. Sundaravadivel and Dinakar M. Salunke National Institute of hnmunology, New Delhi-I 1006Z

The topological correlation of poptide and sugar mimicry established by the crossreactivity of polyclonal antibodies was further investigated by X-ray crysatallography. The structure of concanavalin A (ConA) in complex with a carbohydrate-mimicking peptide, DVFYPYPYASGS has been refined upto 2.75 A to the final R factors of 22 % (Rc~t) and 27~ (R~). The peptide binds to ConA at a site, which is largely defined by hydrophobic residues. It binds in two different modes and in two slightly different conformations demonstrating structural adaptability in ConA-peptide recognition. However, neither of these sites overlaps with the structurally characterized sugar binding site. Nevertheless, the peptide inhibits ConA-induced T cell proliferation in a dose-dependent manner suggesting that it occupies functionally relevant emended carbohydrate binding site of ConA. Comparison of the structures and the hydrophobicity features of trimannose, and the YPY region of the peptide show excellent correspondence. The effect of designed analogs of the carbohydrate-mimicking peptide on Con A-induced T cell proliferation and on anti-sugar pAbs crossreactivity also support the topographical correlation between the sugar and the peptide. The substitution of Tyr by Pbe in the YPY motif shmvs marginal effect on the inhibition of ConA induced T cell proliferation and no effect on cross-reactivity with anti-sugar pAbs. On the other hand, a Ser substitution shows significant loss in both these activities. Thus, it can be concluded that the aromatic residues contribute to, both carbohydrate mimicry and ConA binding. The structural mimicry established here and reflected in the antibody responses against either of the molecules provides an excellent model for exploring the finer aspects of immune mechanisms that may be responsible for discrimination of quasi-equivalent antigens.

150

D3 : Molecular Assemblies, Recognition and Metabolic Regulation : Cell Surface Interactions

P 3 3 7 In terac t ion between Agglutinat ing Proteins and Oligosaccbarides by Activated Molecular Dynamics

Emesto R. Caffarena ], J. Raid Grigera 2 and Paulo. M. Bisch 1 l lns t i tu to de Biofisica "Carlos Chagas FUho" (UFRJ) - 2I f lys ib - (UNLP) CCS - Bloeo G - Cidade UniversitAria - 21.949.900 - Rio de Janeiro. Brasil

Several biologic processes o f signaling and recognition are related with interactions between sugar molecules and proteins. Lectins are multivalent proteins that bind cell surface carbohydrates with high specificity for a wide variety o f cells. T-antigen Gall3(l-3)GalNAe) is a well defined tumor-associated antigen causing a severe malignancy in man. This structure is generally expressed in cells as O-linked glycans prominently in more than 85% of human carcinoms. Among the proteins that recognise T-antigen, peanut agglutinin (PNA) is most widely used in experiments. We present here a molecular dynamics-approach using the stochastic boundary method in the time scale o f tens o f nanoseconds. The structure, dynamics and energetics o f the solvated site o f the peanut agglutinin (PNA) interacting with the T-antigen have been studied. Next, this methodology can be Used to study the interaction between the PNA lectin and other carbohydrates that present special interest because they are possibly involved in Chagas disease.

P 3 3 8 2D Cable Properties of Cell Adhesion

Probed by Linear Array of 100 Transistors and Extracdlular AC-Voltages

V. Kiessling and P. Fmmherz MPI for Biochemistry, Dept. Membrane and Neurophysics, Am KlOl~erspitz 18a, 82152 Martin~ried,

Germany

In cell adhesion a thin sheet of electrolyte is insulated laterally from the cytoplasm by the cell membrane and is kept on bath potential at its periphery. The system is a core-coat conductor or 2D cable. Ionic and capacitive currents through the membrane give rise to a voltage drop in the narrow extracellular space. The electrical characterization of this cleft is important for the interpretation and application of neuron-silicon junction& To study the physics of the 2D-cable in detail a chip with a linear array of 96 field-effect- transistors with a lattice constant of 3.6pro was devclopecL Giant eqahrocytes, made by electrofnsinn, and neurons of snails were ~t~r .bed with polylysine. The beth was moo~__dat~! by small AC-voltages in a range from 10Hz to 100kHz. The response of 36 tran.~i.,aors was recorded simultaneously in amplitude and pha~. The extracellular vOltage-profile was fitted with solutions of the 2D-cable equatio~ We obtained the specific membrane conductivity and the sheet resistance of the extracellular deft. With its width as determined by fluorescence interference-contrast microscopy, we calculated the specific iesistan~. It was by one to two orders of magnitude highex than in the bulk electrolyte.

P 3 3 9 Crystal Structure of a lectin from the seeds ofArtocarpus hirsuta

K. N. Rao, M. M. Gurjar, S. M. Gaikwad, M. I. Khan and C. G. Suresh Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.

A lectin isolated from the seeds of Artocarpus hirsuta, with specificity for methyl et-galactopyranoside, has been purified and crystallized. Biochemical characterisation showed that the lectin is a stable tetramer made up of two pairs of non-identical subunits. Different forms of the crystals were grown from phosphate buffer in the pH range 6.0-7.0 using 35% saturated ammonium sulphate as precipitant. The crystals belonged to two orthorhombic and to two hexagonal forms. The crystals diffract well upto 3.0 A resolution. X-ray data was collected on MAR image plate mounted on Rigaku rotating anode X-ray generator. One of the two hexagonal forms, grown in the presence of glycerol was found to be unsuitable for X-ray crystallographic studies. Crystal structure of the lectin could be solved by molecular replacemgnt method making use of the model coordinates of a dimer of jacalin, the galactose binding lectin from Artocarpus integrifolia. The molecular replacement solution showed that the A. hirsuta lectin molecules formed tetramers in the crystal structure. In the hexagonal form the tetramer was formed by two dimers related by crystallographic 2-fold axis, whereas in the two orthorhombic forms the tetramers constituted their respective asymmetric units. Presently work is in progress to refine the structure using high-resolution data and to determine the amino acid sequence using independent methods.

151


P340 SOLVENT ACCESSIBILITY OF CARBOHYDRATF_~

P. Kaliannan, M. Michael Gromiha, M. Elanthiraiyan Department of Physics, Bharathidasan University, Tiruchirapalli - 620024, INDIA.

The solvent accessible surface area (ASA) of a few polysaccharides, namely i) carrageenan (1CAR), ii) agarose (1AGA), iii) capsular polysaccharide (1CAP), iv) guaran, and v) hyaluronan (1HUA) have been computed using the solvent accessibility technique of Lee and Richards. The results show that the average variation of ASA for the various atoms in the molecules lie in the range 1 to 30/~2. The average ASAs for carbon (10.54/~2 ) and oxygen (22.39 ~2) are relatively very high when compared to that for sulfur and nitrogen. Irrespective of position of sulfation either at 2 or at 4 (in 1CAR), the charged groups interact almost equally with the solvent. In 1CAP the ring carbons have very less ASA values and the glycosidic oxygens are partially buried. The ASA values for the chains A and B in 1AGA and 1CAR indicate that there are not much interchain interactions and the chains in both the molecules interact equally with the solvent. Residuewise analysis indicate that the ASAs of residues vary alternately, similar to that of the hydrophobic behaviour of 13-strands in proteins. The results also suggest that in these polysaccharides D-configuration residues have higher ASA values than L-configuration residues.

P341 "Mild Hyperthermia Moldulates Biological Activities of Interferons" K. Chadha t, J. Payne I, J.L. Ambrus ~, and ~I.P.N. Nair 2. tDepartment of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo New York 14263 USA; and 2Department of Medicine, Kaleida Health, State University of New York at Buffalo, Buffalo New York USA

We observe a significant enhancement of antiviral activity of human IFN-~ -~ and -y and murine IFN-~ and -u when cells are subjected to mild hyperthermia ((39~ during assays. Hyperthermic treatment (39 ~ of primary human fibroblast cells for 4 and 24 hours (pre-assay hyperthermia) prior to interferons antiviral assay further enhances their antiviral activity. An enhancement in the level of interferon induced enzyme, 2,5- oligoadenylate synthetase, is also observed subsequent to the treatment of cells with interferon and hyperthermia. This increase in 2,5-oligoadenylate synthetase level is perhaps, in part, responsible for the observed increase in interferon antiviral activity due to hyperthermia. On the contrary, the hyperthermic treatment significantly decreases interferon synthesis when cells are challenged with a viral (Sendai) or non-viral (Poly rIrC) inducer. Such a decrease in interferon synthesis is seen both in human and murine cells. Mild hyperthermia suppresses interferon-mediated enhancement of natural killer cell activity of both human and murine cells. Finally, hyperthermia considerably increases nHuIFN-ct antiproliferative activity on different human tumor cells as compared to the normal physiological temperature.

P342 COCAINE UPREGULATES HIV-1 ENTRY CORECEPTOR, CCR5 AND DOWNREGULATES HIV-1

SUPPRESSING CHEMOKINE, MIP-l[3 GENE EXPRESSION Nair. Madhavan.P.N..* Mahajan S*., Chadha, K.C.+, Hewitt, R.,* and Schwartz, S.A.* *State University of

New York at Buffalo, Roswell Park Cancer Institute, Buffalo, NY, USA. Earlier studies suggest that cocaine has a significant role in the susceptibility to and progression of HIV-1 infection and CNS dysfunctions. Although cocaine has been linked to immunopathogenesis of HIV-1 infection, the molecular mechanism(s) of cocaine induced immunodeficiency in HIV infected subjects leading to AIDS and associated encephalopathy have not been clearly elucidated. We hypothesize that cocaine may mediate these effects through down regulation of HIV-I suppressing chemokines and/or upregulafion of H1V-1 entry receptors in HIV-1 infected subjects, resulting in disease progression to AIDS and associated encephalopathy. Peripheral blood mononuclear cells (PBMC) were cultured with different concenu'atioas of cocaine (10 "~ to 10"riM ) and total RNA was extracted, reverse transcribed and cDNA was amplified by PCR using CCR5 and MIP-113 primers. Culture supematant was quantitated for MIP-I~ protein by ELISA using specifc antibodies. Our results showed that cocaine significantly downregulates the secretion as well as the expression of HIV-I suppressing chemokine gene, MIP-I3, and upregulates the expression of HIV- 1 entry coreceptor, CCR5 by normal PBMC. These studies support a role for cocaine as a cofactor in the pathogenesis of HIV infection and suggest that the mechanisms of cocaine induced susceptibility to and progression of HIV-I infection and CNS dysfunctions may be mediated through the inhibition of H1V-1 suppressing chemokines and upregulation of HIV-1 entry coreceptors.

152


P343 The lipoamide arm in the glycine decarboxylase complex: a structural approach of the enzymatic mechanism

J. Bourmti~non ], M. Faure 2, C. Cohen-Addad 2, M. Neuburger I , R. Ober ~, L. Sieker 4, D. Macherel ] , R. Douce ] IPhysiologie Cellulaire V~g~tale, CEA/CNRS, 17 rue des Martyrs, 38054 Grenoble Cedex, France; 2Institut de Biologie

Structttrale J.P. Ebel, CEA / CNRS, Grenoble, France, 3College de France, Paris, France; 4University of Seattle, USA. The mitochondrial glycine deear~xylase complex consists of four different component enzymes (P-, H-, T- and L-proteins) that carry out the oxidative decarboxylation of glycine produced during the photorespiration cycle in plants and glycine catabolism in animals. During the reaction, the lipoyl-lysine residue of the H component undergoes a cycle of reductive methylamination, methylaminr transfer and electron transfer. The X-ray crystal structures of the three forms of the H-protein (lipoamide oxidized, reduced and loaded with methylamine) show a unique conformation of the core protein which presents striking structural similarities with lipoate or biotin domains of other systems. The lipoamide arm is freely swinging in the solvent when oxidized or reduced, but when loaded with methylamine, it rotates about 90 ~ around the lysine linkage to hide into a cleft at the protein surface. This results in the formation of a stable complex between the [4- and the T-protein during the methylamine transfer step. On the other hand, the reduced lipoamide arm is released from the cleft before interacting with the, L-protein. The crystal structure of the L-protein reveals, as in other dihydrolipoamide dehydrogenases, an active site with a narrow and long cavity. As the reduced lipoamide arm is free to move into the solvent, it can bind into the active site of the L-protein without any steric hindrance and is reoxidized by L- protein without any specific interaction between the H- and the L-components.

153

D4 : Molecular Assemblies, Recognition and Metabolic Regulation : Metabolic Regulatory and Control Networks

P 3 4 4 CLONING OF THE EXTRAGENIC SUPPRESSOR LOCUS OF pdc2 MUTATION IN YEAST

D.S. Gunmmnhy ,S. Velmumgan and Z. Lobo Department of Biological Sciences, Tara Institute of Fundamental Research,

Mumbai 400 005

PDC2 is a tcg-I~ory gone required for the synthesis of the glycolytic enzyme pyruvate decarboxylase. pdc2 mutants cannot grow on glucose. A dominam exaagenic suppressor XSP18, ofpdc2 restores pyruvate decarboxylase activity. However, it affects the glucose inducible enolase 2. A genomic fibra~ has been constmOed from the DNA isolated fromXSPl8 suppressor bearing strain ofpdc2 m~dant This h~raty has been used to clone theXSPl8by complementing the glucose growth pheaotype ofpdc2 mutant. Few clones have been isolate~ Identification of the ASPI8 locus amongst these complementing clones is in progress.

P 3 4 5 Protective effect of arginine against lipid peroxidation in intact spermatozoa

Sudha Srivastava, Ratna S. Phadke and Girjesh Govil* Tata Institute of Fuadamental Research, Mumbai-400005

Prashant Desai and Evans Coutinho Bombay College of Pharmacy, Mumbai-400098

We have shown earlier the beneficial roles of arginine in the efficacy and preservation of spermatozoa. For example, it stimulates sperm motility and glycolytic activity. The influence of arginine in reversing the irnpairment caused by glycolytic inhibitors, iodoacetamide and iodoacetic acid has been assessed. We report here the effect of arginine in the control of autoperoxidation in goat spermatozoa. Arginine is found to inlu'bit the lipid peroxidation in a concentration dependent manner. Effect was also observed in the cases of peroxidation induced by chemical method, l~eezing and UV exposure of the cells. Co-incubation of (z-tocopherol with arginine is a more effective antioxidant than (x-tocopherol alone.

154

E1 : Bioenergetics : Photosynthetic Systems and Primary Processes

P346 SPECTRAL CHARACTERISTICS OF PIGMENTS IN WHEAT CHLOROPLASTS UNDER INTERMITTENT AND CONTINUOUS ILLUMINATION I.M. Guseinova, S.Yu.Suleimanov, I.S.Zulfugarov, S.N.Novruzova, J.A.Aliev Institute of Botany, Baku, Azerbaijan Spectra/characteristics of functional components of chloroplast thylakoid membrane was studied in etiolated wheat leaves greened in intermittent light (IML) and then transferred to continuous light (CL). Low temperature fluorescence spectra (77 K) of chloroplasts isolated from IML leaves show main emission band at 687 nm and a small peak at 727 rim, characteristic for CC II and CC I, respectively. In seedlings then transferred to CL the fluorescence spectnan is characterized with bands at 687, 696 and 740 nm (main). As greening proceeds the 727 nm peak gradually shifts to longer wavelength and this intensity increases in relation to the 687 nm emission. In the absorption spec~a (A) and their fourth derivatives (A TM) at 77 K of the chloroplasts for IML seedlings peaks of Chl a appeared at 660, 669, 676, 682, 690, 696, 704 and small peaks of Chl a at 710 nm and of Chl b at 648 nm. On transfer of the seedlings from IML to CL in the spectrum A rc the intensity of the Chl b peak at 648 nm is considerably increased, obviously corresponding to the synthesis under these conditions of LHC I and LHC II. The peak intensity at 682 nm decreases in relation to the first spectrum. At the same time a sharp rise takes place in the fluorescence intensity at 740 nm due to restoration of the energy runoff to the Chl a form at 710-712 nm from LHC I synthesized during CL.

P347 Spectral and photochemical properties ofDunalieila salina cells grown under various salt Concentration

J.A. Aliev, M.A. Ismyilov, LS. Zulfugarov, D.IL Alieva, 1". V. Savcheako Institute of Botany, Patamdar Shosse 40, 370073 Balm, Azerbaijan (E-mail: [email protected])

In the present work salt induced changes in pigment and protein contents as well as activities of photosystems in a halotolerant Dunaliella salina cells grown under various salinities (0.5 - 4 M NaCI) have been examined. To compare capacities of photosynthetic activities in the cells grown under various salt concentrations, the photosynthetic 02 evolution activity as well as chlorophyll fluorescence induction curves and 77K fluorescence emission spectra were measure& It was found that in lhmaliella salina cells the photosystem II (PS I1) activity inhibited while the photosystem I (PS I) activity was increased with increasing salinity. In Dunaliella salina cells cultivated at low salt media, the maximum fluorescence emission peak at 77K was observed at 686 nm, whereas in high salt media the maximum emission peak shifted to 710 nm. The peak at 686 nm results from emission of chlorophyll a associated with PS II, whereas the emission peak at 710 nm associated with PSI . When the Dunaliella salina cells grown at low salt media (0.5 M NaCI) were transferred to high salt media (4 M NaCI), the intensity of emission peakat 686 ran decreased with the increase of emission peak at 710 rim. The activation ofPS I mediated electron flow upon acclimation to high salt stress was suggested.

P348 High-temperature chlorophyll fluorescence rise detected on barley leaves with different LHC content Petr I l ~ Roman Koufil, Hana Barto~kov6, Jan Nau~ Department of Experim~tal Physics, Palacl~ University, Olomouc, Czech Republic

The dependence of relative chlorophyll fluorescence intensity on temperature within 25 -75 ~ - Fluorescence Temperature Curve (FTC) - is frequently used for detection of thermostability of thylakoid membranes in chloroplasts. It is believed that chlorophyll fluorescence ori~nates from antennae of photosystem H (PSH) to at least 55 ~ In leaves with reduced content of light-harvesting complexes (LHC) (intermittent light grown leaves or Chl b-less mutants) a pronounced fluorescence increase within 55 - 65 ~ (M2 maximum or region) was detected. We have found that this increase is irreversible and is mammal at emission wavelength of about 675 urn. Interestingly, when the samples preheated to M2 were immediately frozen to 77 K this emission maximum was situated to 688 - 690 nm. Concomitantly, the 77K emission bands F730 and F740 disappeared and F720 dominated in the long-wavelength photosystem I (PSI) spectral region. Within the increase to the M2 region the PSI complex separates into PSI core and LHC of PSI (LHCI) as was deduced from nondenaturating green native Deriphate-PAGE. Spectral analysis of the aggregates remaining on the top of the gel indicates that the aggregates are formed probably by the separated LHCL (This work was supported by grants No. 204/98/1)205 from GA (~R and No. 3150 3010 from Pa/acky University)

155


P 3 4 9 Modifications in the photosynthetic apparatus of grape (vms vbdfera L.) leaves due to individual and

combined application of an insecticide, Endnsuifan and fungicide, Metalaxyl ,Iyoti U.Gaikwad, Sarah Thomas and P.B.Vidyasagar

Biophysics Lab., Dept. of Physics, Univ. of Pune. Pune 411 007, India

lndiscrimin~e application of pesticides is a common practice especially in case of a ~ h crop such as grape (Yitis vinifera L). Since the biochemical processes of the cellular and subcellular levels of the target organisms and the host are often quite similar, the pns~oility of direct effects of protective chemicals on the host plant are great. Hence, unscientific application of these pesticides m y create stress in the planL They may also ~ plant metabolism inviting different diseases and physiological disorders like pink bony whose origin i$ unlmown. It has been observed that the occurrence of pink berries were more wliere application of pesticides was unscientific and heavy. Since the ripening beny is a strong sink for solute from photosynthesis and reserve organs, it would be interesting to study modifications in file photosynthetic apparatus of leaves due to heavy doses of pesticides.

In the present work, we have compared the effects of an insecticide. Endos-lfan ~ a fungicide Metalaxyl when appfied individually and also in combination using thermoluminsesconce (TL) and fluorescence (FL) teclmique. In case of individual application of Endosulfan, decrease in TL intensity for the peak II (-3~ was obsezved, while in case of ~ I , alongwith the conWol peak, emergence of an additional narrow TL band at around -15~ was observed. The FL results showed effect of both the pesticides on the electron accep, tor side of photosystem H. The results'of their combined application would be d~'ussed..

P 3 5 0 INTERACTION OF ISOLATED CHLOROPHYLL A/B LIGHT-HARVESTING COMPLEXES WITH PURIFIED THYLAKOID LIPIDS,

FORMATION AND STRUCTURAL FLEXIBILTY OF LAMELLAR AGGREGATES G. Garab ], I. Simidjiev I, S. Rajagopal l, Zs. V~rkonyi l, S. Stoylova 2, Z. Cseh t'3, E. Papp 3, L. MustArdy I and A. Holzenburg 2 ~lnst. Plant Biol., Biological Research Center, Hung. Acacl Sci., Szeged, Hungary; 2Dept. Biochem. Mot,. Biol., Univ. Leeds,

Leeds, UK; 3Dept. Biol. Physics, EOtvOs l_, Univ., Budapest, Hungary We investigated the interaction of the two main components of chloroplast thylakoid membranes of higher plants, the monogalactosyldiacylglycerol, MGDG, and the chlorophyll a/b light harvesting complexes of photosystem H, which constitute about 50-50 % of the total lipid and protein contents of the membranes. By using elecu'on microscopy, circular dichxoism and fluorescence spectroscopy we have shown that (i) upon the association of the two components large lamellar aggregates are formed despite the fact that MGDG is a 'non-bilayer lipid'; (ii) the proteins are embedded in the artificial membrane in a way to possess a long range hexagonal order and long-range chirality in their chromophore arrangement; (iii) lamellar aggregates .of LHCII are capable of undergoing light-induced reversible structural changes affecting the long-range chiral order of the chromophores, and the fluorescence yield of chlorophyll a molecules; (iv) these reversible structural changes are facilitated by the addition of purified lipids, with MGDG causing the largest effect; (v) the structural rearrangements can be trapped by lowering the temperature below 10 ~ It is proposed that the stntctural changes are induced by thermal fluctuations due to a statistical dissipation of absorbed photon energies; the rapid thermal fluctuations can induce structural changes via a release of bound cations which can be reversed only in 1-2 mm. These structural changes, via wansiently opening dissipative pathways, are thought to play a role in the light adaptation and photoprotection of green plants.

P 3 5 1 Protein-Ch_romophore Intercommunication during the Light Cycle of a Photosensor

R.Bruder , U.K.Genick l, T.T. Woo x , D.P. Millar I , K. Gerwert 2, E.D. Getzoff t ~Department of Molecular Biology and Skaggs Institute for Chemical biology, The Scripps Research Institute,

Ida Jolla CA 92037. 2De~aftnlent of Biophysics, Ruhr University Bochum, D-44780 Bochum, Germany.

In order to understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we study Photoactive yellow protein (PYP) with slxclxoscopic techniques and X-ray crystallography. PYP is a 14 kDa blue-light receptor in the cytoplasm of purple bacteria which presumably mediates negative phototaxis and undergoes a photocycle upon absoqXion of a photon by its anionic p- hydroxycinnamoyl chromophore. We monitored the photocycle reactions of the protein and the chromophore simultaneously by step scan FTIR difference ~ with a 30 ns lime resolution. Band assignments were performed by site-directed mutants and FT Raman spectroscopy. The results show that the hydrogen bond network around the chromophore rema~ intact in the early photocycle intermediate I~, accompanies by larger movements of the protein backbone. This implies that the chromophore isomerizes around two bonds. Co-isomerization allows the reaction to take place on a fs time scale without collisions within a demely packed protein environmenL Our results on the active site mutant Y42F show that Y42 is a crucial residue for stabili7~tion of the native chromophore configumlion and for enhancement of PYP's quantum efficiency of fight absorption. The fluorescence cp _~O~un yield of Y42F is increased by an order of magnitude when compared to the wild-type. Mutation of tyrosine for phenylalanine results in a second PYP population with an altered chromophore configuration.

156


P352 Reinvestigation of the triplet-minus-singlet spectrum of chloroplasts

Ta.~s J/tvorfi u , Gy6z6 Garab I and K. Razi Naqvi 2 ;Biological Research Center, Institute of Plant Biology, H-6701 Szeged, PO Box 521, HUNGARY

'Department of Physics, Norwegian University of Science and Technology, N-7034 Trondheim, NOR WAY

The triplet-minus-singlet (TmS) spectTa of Chla/b LHCII (light-harvesting complexes, isolated from photosystem II of higher plants) have been reported to contain a negative signal in the Qy region of chlorophyll a (Chla). At first sight surprising - - since the entire spectrum originates from carotenoids, which do not absorb in the Qy region - - the signal has been attributed to a change, m the absorption spectrum of a Chla molecule, brought about by the arrival of triplet excitation on a neighbouring carotenoid (Car). On the other hand, the published TInS spectrum of spinach chloroplasts is devoid of a similar signal, and its decay time considerably shorter than that of the TInS spectrum of antenna complexes; if authentic, the absence would imply that the TmS spectrum of chloroplasts is contributed by a pool of Car's which do not influence the absorption spectra of their Chla neighbours. The TmS spectrum of spinach chloroplasts was reinvestigated and found to contain a prominent bleaching signal centered at 681 nm.

P353 Effects of Gamma irradiation on the photosynthetic apparatus of spinach leaves using physical techniques

Md.Kalimullah l, Jyoti Craikwad t, Sarah Thomas I, Manoj Semwal 2 and P.B.Vidyasagar I IBiophysics Lab., Dept. of Physics, Univ. ofPune, Pune 411 007, India

2 Conunand Hospital, Dept. of Radiotherapy, Pune, India

Studies of gamma radiation on micrusomal membranes, tissues, plants and humans have been studied to varying extents. It was observed thai irradiated chara cells lose their capacity of cell division and fail to elongate. However, they showed a tendency to restore the effect of irradiation by developing large number of branches. Development of photosynthesis in an irradiated etiolated plant resulted in a delay in greening process. In case of higher plants, effects of gmmna irradiation on plasma and vacuolar membranes of cultured spinach cells have been reported. But practically no reports exist on the stu~es related to ganuna ir~i:~tion on photusynthetic electron transport chain in lugher plants.

In the present work, we have studied the effects of gamma irradiation on the photosynthetic apparatus of spinach leaves using fluorescence (FI.,) and'thermoluminescence (TL) techniques. The leaf discs we.re irradiated with various doses ranging from 0.05 Gy to around 10kGy. The results obtained from the TL and FL ~ x m a recorded from unirradiated and irradiated leaf samples, for different doses of gamma irradiation would be discussed.

P354 On the origin of heat-induced decrease of chlorophyll flouorescence emitted from photosystem H

Roman Kouril, Petr llik & Man Naus Department of Experimental Physics, Faculty of Science, Palack~ University, 771 46 Olomouc, Czech Republic

Changes of chlorophyll fluorescence emission and excitation spectra at 77 K of barley leaves (Hordeum vulgare L. cv. Akcent) induced by heat treatment were studied. To avoid the effect of fluorescence reabsorption the fluorescence spectra were measured with diluted leaf particles" (Weis, Photosynth Res 6: 73-86, 1985) that were prepared after the heat trealment. The obtained 4a_ta indicate that after healing of leaves to 35-45~ the heat- induced decrease of chlorophyll fluorescence emitted from internal antennae of photosystem II (PSI1) is accompanied by the increase of chlorophyll fluorescence ori~nated from photosystem I. This is in the agreement with previously published dat~ However, excitation spectra at 77 K show that this decrease of PSII emission is not connected with detachment of peripheral pool of light harvesting complexes LHCII from PSI1. This finding will be discussed with respect to state I - state 2 transitions before the heat ~reatment.

157


P355 Perception of Colours of Flowers and Leaves by a Polarization- and colour-sensitive retinea

How do the pollinators solve the problem of the Polarization-induced False-colours of Plant Surfaces Istv;in Pomozi, C-dbor Horv~th, Rfldiger Welmer and Gary D. Bernard

Department of Biological Physics, Eotvos University, Budapest, Hungary

Using ~ideopolarimetry, the reflection-polarization characteristics of different plant surfaces were measured. The polarizational false colours of flower petals and plant leaves induced in polarization- and colour-sensitivc retinae were computed and visualized. These reflection-polarization-induced flase colours were investigated for plant surfaces with different spectral characteristics and surface microsculpturing as functions of the viewing angle, the polarization sensitivity and microvillar orientation of the photoreceptors. We demonstrate that homogeneously coloured and highly polarized plant surfaces would appear in variable iridescent false oclours due to their curvation, and their colour distribution would strongly depend on the viewing direction, orientation of the head. polarization sensitivity and microvillar alignment. Presenting an alternative receptor-physiological solution, we show that receptor twist is not the only possible resolution to the false-colour problem. It is also demonstrated that ffthe bee's colour vision system would'be polarization sensitive, then a homogeneously green meadow would be perceived as a kaleidoscopical field in all the colours of the rainbow due to the polariT~lional false colours. In such a variegated, multicoloured optical environment bees could very difficult find visually the flowers. Finally, on the basis of the receptor twist in bee eyes we propose arguments for the hypothesis that polarization vision may be more ancient than perception of colour.

P356 L I G H T - H A R V E S T I N G BY C A R O T E N O I D S IN P H O T O S Y N T H E S I S

Ana Damjanovi~, Thorsten Ritz and Klans Schulten Department of Physics and Beckman Institute, University of Illinois at Urbana-Champaign

In photosynthetic light-harvesting carotenoids and chlorophylls act jointly to absorb sun-light. The absorbed light energy is transfered in less than a picosecond into electronic singlet excitations of the chlorophylls only and fimneled further into the photosynthetic reaction center. In this study, the pathways of singlet excitation transfer between carotenoids and chlorophylls and especially the role of the optically forbidden carotenoid 2Ag state in the transfer have been investigated computationally on the basis of the x-ray structures of three proteins, the light-harvesting complex II of purple bacteria Rs. moHschianum and Rps. acidophila and the peridinin-chlorophyll- protein of dinoflagellate A. carterae. The electronic states of chlorophylls and carotenoids have been described by means of semi-empirical PPP-SC~'-CI calculations. Various asymmetries in carotenoids have been accounted for in our study, resulting in an improved description of the 2Ag state of carotenoids. The couplings and transfer rates between different carotenoid and chlorophyll electronic states have been determined.

P357 Light induced chlorophyll a destruction and protein conformationai change of CP43 and CP47

Wang Jushuo I Shan Jixiu 2 Gong Yandao I Kuang Tingyun 2 Zhao Nanming I i Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane

Biotechnology, Tsinghua University, Beijing 100084, China 2 Photosynthesis Research Center, Institute of Botany, Chinese Academy of Sciences, Beijmg 100084, China

Light induced denaturation of the core antenna complexes CP43 and CP47, purified from spinach, was investigated using the absorption, fluorescence and circular dichroism (CD) spectroscopy. For both of the two chlorophyll-binding proteins, the chlorophyll a molecular structure was destroyed during illumination, which resulted in the decreased absorbance, fluorcscence and CD activity of chlorophyll a, while the protein secondary structure was not apparently changed as indicated by far-ultraviolet CD spectrum. Light was found to bring about a larger decrease of the chlorophyll a fluorescence and the CD activity of CP47 than that of CP43, suggesting that the native stateof chlorophyll a of CP47 was more sensitive.to light than that of CP43. During the incubation at room tcmperature in the dark following illumination, the chlorophyll a fluorescence intensity was found to continuously increase for both CP43 and CP47, but little change in the chlorophyll a absorbance and the protein secondary structure was observed. The further fluorescence increase may be due to the change in the protein tertiary structure. The results suggcsted that light induced not only the destruction of chlorophyll a molecule, but also a slow change in the protein conformation during the following dark incubation period.

158


P 3 5 8 EXCITONS IN LIGHT HARVESTING

Arvi Freiberfl 1' =, K6u Timpmann 1' = Rein Ruus 2, and Neal W. Woodbury ~ 1Department of Chemistry and Biochemistry and Center for the Study of Early Events in Photos3,nthesis, AFizona

State University, Tempe, AZ 85287, USA and Zlnstitute of Physics, University of Tartu, 51014 Tartu, Estonia The main purpose of this work is a better understanding of the optical spectroscopy of photosynthetic light- harvesting pigment-protein complexes, particularly LH1 and LH2 bacteriochlorophyll-protein antennas. A deeper knowledge of the spectroscopy allows drawing more qualified conclusions about the nature of light excitations in the antennas and about the couplings governing their dynamics. The effects of static diagonal disorder on linear and non-linear absorption spectra of excitons in circular molecular aggregates are studied by computer modeling. We demonstrate that this simplified model successfully reproduces main features of the experimental steady-state ground state absorption and prompt pump-probe absorption difference spectra of LH1 and LH2 antenna proteins of photosynthetic bacteria measured under selective population of excitons at low temperature. The nearest-neighbor coupling energy in the B850 ring aggregate of LH2 revealed by simultaneous modeling of the linear and non-linear absorption spectra is V=375+25 cm "=. Our study reinforces the presence of two independent types of spectral disorder governing the inhomogeneously broadened B850 exciton spectra. In the Gaussian approximation, the distribution of transition energies within the B850 ring is described by the standard deviation ffi=210 cm "l, while the 0isorder between different rings is characterized by ae-53 cm 1

P 3 5 9 ELECTRON-EXCITED STATES OF BACTERIAL BIOLUMINESCENT EMITTER: SPECTROSCOPY STUDY

Nemtseva E.V.t, Kudryasbeva N.S.t, Sizykh A.G. 2 'Institute of Biophysics SB RAS, Krasnoyarsk, Russia 2Krasnoyarsk State University, Krasnoyarsk, Russia

Activity of the upper electron-excited states of hacteriai bioluminescence emitter (4A-hydroxiflavin) has been studied with excitation energy accepting molecules. Fluorescent aromatic compounds anthracene and 1.4-bis(5-phenil-2-oxiasolile)benzene (or POPOP) have been chosen. Energies of their lowest excited singlet states are higher than the energy of the analogous state of bioluminescence emitter; spectra of their absorption and bioluminescence do not overlap. Hence, the excitation of these molecules by singlet-singlet energy transfer or by light absorption is excluded.

Anthracene and POPOP were added into the coupled enzyme system NADH:FMN-oxidoreductase-luciferase. Sensitized fluore,~cence of these compounds in the bioluminescence system has been recorded. Data obtained have been discussed as a direct proof of involvement of emitter's upper electron-excited states in the bioluminescent process.

The proposed mechanism of generation of excitation involving upper electron-excited states of emitter can characterize nol only bacterial, but other types ofbioluminescence, too.

P 3 6 0 THERMAL INDUCTION PARAMETERS IN CHLOROPHYLL FLUORESCENCE TO DIAGNOSE STATUS

OF HIGHER PLANT'S PHOTOSYNTHETIC APPARATUS UNDER STRESS

Shikhov V. N., Nesterenko T.V., Tikhomirov A,A. Institute of Biophysics (Russian Academy of Sciences, Siberian Branch)

Effect of elevated air temperature and difference PAR irradiance was evaluated by thermally induced variations in zero level of chlorophyll fluorescence recording structural changes in plants' photosynthetic apparatus. Experimental data have formed the basis to take relative value of low-temperature fluorescence maximum specifying photosynthetic activity of photosystem 2 as a major index of stress effect on plants' photosynthetic apparatus. This parameter has been shown to be indicative of damaging effects for vegetative organisms. However frequently modifications caused relatively small stresses, affect rather the high-temperature part of the curve than the first fluorescence maximum. The work introduces new parameter - normalized area under the thermal induction curve. Experimental data demonstrated that response of new parameter to a stress value is as adequate as that of first, meanwhile its indication of the physiological status of plants is more integral.

159

E2 : Bioenergetics : ElectrordProtonTransport

P361 Kinetics of electron transport and ATP formation in state I and state 2 in Chlmnydomonns reinhardal-

Gior~io Forti, Giovanni F i n a l , Alberto Furla and Romina Barbagallo. Ce~Iro CNR Biologia Cellu]are e Molecolare della Piante, Dip. di Biologia, V'm Celoria 26, Mil~o, Italy.

In Odamydcmouas reinlmrdtii, cytochrome f t t~over was unaffected by state tnmsitions: under low light excitation, the rate constant for reduction was 4.8 ms in state 1 and state 2. Cyt.f reduction was unaffected by DCMU in state 2 but strongly inhibited by it in statel.DBMIB inhibited completely the reduction in both conditions. These observations demonstrate that cyclic electron transport atcxmd PS 1 is respon~ble for cyt f tmnovex in state 2, while the linesx system is active in state 1.02 concentration and the fluorescence of cells incubated in the dark were continuously monitored by a Clark electrode and a Pam fluorometet. As soon as the 02 was used up, Fin initiated a decline which was complete in 12-15 min. ATP/ADP ratio also drqpped from 12-15 to ca. 3 as soon as ana~obiosis was attained. Upon illumination of dark-anaerobic cells, the following events occured, in the sequence: (a) the increase of ATP/ADP ratio to the initial level, in less than 5 s. This ATP synthesis is therefore coupled to cyclic electron flow, measm'ed through the turnover of cyt f. Co) the increase of Fro, indicating the state 2-state 1 wansition and, (c), the onset of 02 evolution, which required several minutes. Based on the sequence and time-course of these events, and on the effects of uaeonpiers and of 02 on state 2 to 1 transition and the onset of photosynthesis, we conclude that ATP, though obviously required for photosynthesis, is not a limiting factor in the reactivation of photosynthetic 02 evolution.The latter depends strictly on the transition to state 1. P 3 6 2

Electron transfer on the donor side ofPhotosystem I

S. Iskenderova, R. Agalarov, 1~ Gasanov. Biophysics laboratory, Institute o f Botany, Acad. o f Sci., Patamdart sh. 40, 370073 Baku,

Azerbaijan

The electron transfer from Plastocyanin to Photosystem I under steady-state illumination has been studied in comparison with the same reaction with cytochrome C6 from green algae Cladophora glomerata. The dependence of the 02 consumption rate on Plastocyanin and cytochrome C6 concentration, electrostatic effect in presence of mono- and divalent cations and pH-dependence were investigated. Comparative investigation revealed the similar sigrnoidal kinetics for both redox proteins. As a regulator feedback in activating complex formation between redox proteins and PSI electrostatic interaction has been suggested. The mechanism of activating complexes formation between Plastocyanin and Photosystem I in frame of trimeric structure o f Photosystem I has been proposed.

P 3 6 3 P r e s s u r e D e p e n d e n c e o f P r o t e i n E l e c t r o n T r a n s f e r R e a c t i o n s

MIYASHITA Osamu and GO Nobuhiro Kyoto University, Graduate school of Science

Pressure dependence of protein electron transfer rate constant is discussed. The rate constant is given as a product of two factors; electron tunneling matrix and nuclear factor. Pressure affects the rate through both of these two factors.

We analyzed pressure effects of these two factors individually The pressure dependence of the electronic factor is discussed by considering the dependence of through-space gaps in the tunneling pathway model. We carried out normal mode simulation on cytochrome c and obtained a quantitative picture.

The effect of pressure on the nuclear factor is

discussed in terms of the Marcus expression of Frank- Condon factor and the pressure dependence of the two parameters, reorganization energy ~. and reaction free energy AG, in it. We show that the dependence of ~ is generally smaller than that of AG.

As the results, we obtained a simple relation to understand the pressure dependence of protein electron transfer reactions; (i) it is closely related to the total reaction volume and to other two factors Z and AG, (ii) the pressure dependence of electronic factor has small contributions to the total pressure dependence, excepting two special cases.

160

E2 : Bioenergetics : Electron/ProtonTransport

P364 EFFECT OF ELECTRON AND PROTON BEAMS ON MICROHARDNESS OF DRY BONES

ILK. Soni and M. Ramrakhiani Department of Physics, Rani Durgavati University. Jabalpur (India)

The irradiation of bones by electron and proton beams are found to change their mechanical properties. The embalmed dry bone samples were irradiated with proton beams of 100 to 300 KeV and electron beam of 2 MeV at different doses. The passage of charged particles through bone preduces ionization and hence free radicals in the protein molecules of bone. Depending on availability of free charges and density of free radicals, rupture and cross-linking occurs, which changes the strength or hardness in that part of the specimen.

The irradiated samples were subjeeted to mica'o-hardness testing by Vickers indentation and Vickers hardness number Hv is computed. It is observed that low energy protons increase Hv where as hardness is reduced by high-energy protons. Due to electron beam also, Hv is reduced at low doses but when dose is increased, Hv increases above the original value. This shows that low energy protons cause cross-links in the upper layers of bone up to 10-20 pm increasing the micro-hardness whereas high energy protons cause rupture in the same layers; these may produce cross-links and hence hardening at greater depth. Electron beam also cause rupture at superficial layers at low doses and at higher doses cross-linking is obtained as the flee radical density increases.

P 3 6 5 Existence of two pockets in the Qo site of cytochrome b~complex

Giovanni Finazzi,, Romina Paola Barbagallo, Giorgio Forti Centro CNR Biologia Cellulare e Molecolare delle Piante, Dip. di Biologia, Via Celoria 26, Milano,Italy.

Cytochrome bd" complex mediates electron transfer between Photosystem 2 and Photosystem 1 in oxygenic photosynthesis. It catalyses the oxidation of plastoquinol at the lumenal (Qo) side of the membrane and the reduction of plastocyanin through the Fe-S cluster of the Rieske subunit and the c-heine of cytochrome f In parallel, electrons are injected into two b heroes (cytochrome b6), which reduce a quinone molecule at the stromal side of the membrane (Qi).We have analysed the effects of two inhibitors (DBMIB and DNP-INT) of the Qo site on the activity of cyt b 4 complex in cells of the green alga C. reinhardtii. We have found that electron transfer to the iron sulphur and the b hemes can be uncoupled by DBMIB, the accessibility of which is strongly enhanced by one complex turnover. On the contrary, DNP-INT behaved as a classical competitive inhibitor. The effects of the inhibitQrs were also analysed in the cyt boCmutant FUD2, where the alTmity for plastoquinol is strongly perturbed. Taken all together, our results suggest a model for cyt bo c activity, where the existence of different binding pockets for quinol and semiquinol explain the occurrence of the concerted oxidation of plastoquinol at the Qo site.

P 3 6 6 Functional Conformation Changes in the TFt-ATPase ~ Subunit Probed by Twelve Tyrosine Residues

I-Iiromasa Yagi, Kacko Tozawa, Nobuaki Sekino, Tomoyuki Iwabuclfi. Masasuke Yoshida ~, and Hideo Akutsu Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University,

*Research Laboratory of Resources Utilization, R-1, Tokyo Institute of Technology

The effect of nucleotide binding on the slructure of the F~-ATPase fl snbunit from thermophilic Bacillus PS-3 (TFI~ was investigated by monitoring the NMR signals of the twelve tyrosine residues. The 3,5-proton resonances of 12 tyrosine residues could be observed for the specifically deuterated fl snbunit. The assignment of 3,5-proton resonances of all the tyrosine residues was accomplished using fourteen mutant proteins, in each of which one or two tyrosine residues were substituted by phenylalanine. Binding of Mg.ATP induced an upfield shift of Tyr-341 resonance, suggesting that their aromatic rings ~ stacked to each other. Besides Tyr-341, the signal shift observed on Mg.ATP binding was restricted to the resonances of Tyr-148, Tyr-199, Tyr-238 and Tyr- 307, suggesting that Mg.ATP induces a eonformational change in the hinge region. This can be correlated to the change from the open to closed conformations as implicated in the crystal structure. Mg.ADP induced a similar but distinctly different conformational change. Therefore, the intrinsic conformational change in the fl subunit induced by the nucleotide binding is proposed to be one of the essential driving forces for the F1 rotation. Reconstitution experiments showed that Tyr-277, one of the four conserved tvrosines, is essential to form the a ~ y complex.

161


P 3 6 7 Photophosphorylat ion in Alkalophilic Halobacteriai Cells Natronobacterim Pharaonis

A.V. Avetisyan, A.D. Kanien, V.P. Skulacbev, and B.A. Feniouk Dept. of Bioenergetics, A.N. Belozersky Institute of Phisico-Chemical Biology

Moscow State University. Moscow. 119899. Russia

Light-driven ATP synthesis is found in cells of the alkaloplfilic l~obacterial cells Natronobacterium pharaonis containing halorhodopsin. Halorhodopsin is a light-driven pmnp. which transports CI from outer medium into the cytoplasm. Photophosphorylation occurs with cyanide-inlfibited respiratory chain or under anaerobic conditions. Thus, halorhodopsin is able to support the photophosphorylation in cells. Protonophoric uncoupler FCCP as well as electroneutral CI-/OH antiporter TPT inhibits the light-driven phosphorylation. ATP hydrolysis in subbacterial particles depends on CI concentration in the reaction medium. Decrease-in CI concentration from 3M upto 0.1M (ionic strength is kept constant by addition of 1.5M Na2SO4) leads to inhibition of ATP hydrolysis. The half- maximal effect is observed at 0.5M CI. DCCD inlfibits the ATP synthesis as well as ATP hydrolysis. These data can be explained by functioning of CI transporting ATPase in lhalobacterial cell membrane. Halorhodopsin pumps CI- into the cells whereas CI ,anions release from the cells through CI ATP-syntlmse is. coupled with the ATP synthesis.

P 3 6 8 LARGE CONFORMATIONAL CHANGES INDUCED BY INHIBITOR BINDING

IN THE CYTOCHROME b6fCOMPLEX C~cile Breyton and Werner KUhlbrandt

Max-Planck-Institut fllr Biophysik, H. Hoffmann strasse, 7 60528 Germany

Binding of stigmatellin, a well known inhibitor of the Qo site of the cytochrome bc- type complexes (the photosynthetic b6f and respiratory bc~ complexes), has been shown to induce large conformational changes of the Rieske subunit in the bcu complex [Zhang et al., 1998, Nature, 392, 677-84, Iwata et al., [998, Science, 281, 64-71, Kim et al., 1998, PNAS, 95, 8026-33]. Such a movement appears necessary to shuttle electrons from the membrane soluble quinol to the extramembrane heme of cytochrome c~. Besides their function of electron transfer coupled to proton pumping, b6f and bc~ complexes share also the 3 high molecular weight subunits. We have studied the effect of the binding of stigmatellin on the b6f complex of Chlamydomonas reinhardtii by electron microscopy. A comparison of projection maps of negatively stained thin 3 dimensional crystals prepared with or without stigmatellin, shows a similar type of movement to that observed in bc~ complexes. A projection map of the b6f complex embedded into glucose was calculated to 9 A resolution, allowing a comparison with a calculated projection map of the bc~ complex.

P 3 6 9 Formation of the iron center in the R2 mutant E238A from E.coli ribonucleotide reductase

Maria Assarsson and Astrid Graslund Department of Biophysics, Stockholm University, S-106 91 Stockholm, Sweden

The ribonucleotide reductase (RNR) is vital for all organisms as it catalyses the reduction of ribonucleotides to the corresponding deox'yribonucleotides, necessary for the synthesis of DNA.

RNR from E.coli consists of two non-identical subunits, denoted proteins R1 and R2, and has an CXz132 structure.

The small R2 protein contains a bt-oxo bridged diferric iron center. Upon activation of the diferrous form of the protein with oxygen, the oxygen molecule is cleaved to generate iron intermediates that oxidize a nearby tyrosine residue, producing the essential free radical needed for the catalytic reaction performed by this enzyme.

The iron center has six amino acid ligands; D84, E115, H118, E204, E238 alad H241 (E.coli numbering). The E238 residue is considered to be important for the ability of the protein tb control the oxygen activation reaction.

In this study the E238 residue was mutated to an alanine. The oxygen activation reaction and formation of the iron center in the mutated protein has been studied by UV/VIS absorption and EPR spectroscopy.

162


P370 Effect of Cd 2+ on the photosynthetic apparatus in Synechocys~s PCC 6803

Zsiros 0., Horv;Ith G., M ~ L., Libisch B., Gombos Z. BiologicaI Research Center, Hungarian Academy Sciences Institute of Plant Biology Cd > usually damages plant metabolism ff its concentration is high enough. The most obvious damage appears m the

I n 2 + 9 9 " photosynthetic apparatus, the present w~rk the short-term effect of Cd was mvesUgated on the growth, Chl content and photosynthetic activity of a cyanobacterial strain, Synechocystis PCC 6803. In the presence of 40 Ixmole Cd 2+ the growth of the cells was blocked and the chlorophyll-a synthesis was strongly inhibited. The Cd2*-treated culture turned into yellowish color within a day. Exposure of cells to Cd 2+ for 10 minutes induced a dramatic decrease in the photosynthetic activity (from I'I20 to CO9 and we co~d not detect any oxygen evolving after I hour. The Cd 2§ stress decreased the PSII activity (from H20 to pBQ as an electron acceptor). A l-h, 2-h, 4-h and 8-h incubation of the Cd 2+ treated cells showed the percentages of origin photosynthetic activity 85%, 70e , 67~ 55% 50%, r e ~ v e l y . To measure the PSI activity we used the light induced P700 absorption changes, but there was no sis difference in the activity between the control and Cd2+-treated cells. Furthermore we monitored other PSI reactions, from DAD to MV and from DCIP to MV. DAD and DCIP (as electron donors) donate the electrons before cytb/f complex and to the cytb/f, respectively. While the PSI reaction center was not affected by the Cd ~ treatment the photosynthetic activity around cyt b/f comlex decreased to 50*/, of the control activity. Our results suggest that the main target of Cd 2+ toxicity is the cyt b/f complex.

P371 ON THE INVOLVEMENT OF ANTIOXIDANTS IN REGULATION AND PROTECTION OF ENERGY

TRANSFORMATION SYSTEMS IN CHLOROPLASTS UNDER NORMAL AND STRESS CONDITIONS N.V.Budagovskaya

.Institute of Plant Physiology of the Russian Academy of Sciences, Botanicheskaya 35, Moscow 127276, Russia ATP formation, ATP-ase reaction, electron and proton transport in chloroplasts of plants differing in tolerance to unfavourable environmental factors have been studied. Various experimental conditions in viva and in vitro were provided which changed the functional state of chloroplasts and the level of free radical oxidation (FRO). Natural antioxidants (cysteine, ascorbate) were used in experiments. It has been demonstrated that these substances may possess both activating and inhibiting effect on chloroplasts depending on their initial functional state. These compounds increase low activity of proton transport and ATP formation and decrease high activity of these processes. The level of activity of energy transformation systems depends on concentration of free SH-groups and on the level of FRO in chloroplasts. The content of SH-groups at different states of chloroplasts has been determined and this index may be used for functional diagnostics. It was noted that susceptible plants in contrast to tolerant plants have low concentrations of SH-groups in tissues and chloroplasts and low activity of energy transformation systems in chloroplasts under normal conditions and especially under stress and senescence. Under stress the change of the sign of energy transformation processes is possible. Addition of antioxidants containing or not SH-groups and alkalization increase the activity of proton transport and ATP formatioh in chloroplasts under action of stress factors and under senescence. The scheme of regulation of ATP formation, including antioxidants and FRO, is proposed.

P372 INFLUENCE OF XENOBIOTICS ON PRIMARY PHYSICO-CHEMICAL PROCESSES IN ENZYME SYSTEMS

.N.Kudryasheva. Institute of Biophysics, Krasnoyai'sk, 660036, Russia

Effects of xenobiotic molecules on enzyme systems were discussed in terms of efficiency of the primary physieo-chemical processes (energy, electron and hydrogen transduction). Bacterial bioluminescent enzyme system Luciferase-NADH:FMN- oxidoreductase was chosen as an example of an enzymatic system, since (1) it features all types processes mentioned above, and (2) bioluminescence response is suitable for monitoring the rate of the enzyn'te process in the presence of xenobiotics. Contribution of three types of transfers to inhibition of the enzyme system was evaluated. Energy transfer processes in the bioluminescent system were studied using fluorescent dyes of various spectral-luminescent properties (energy of electron- excited states, quantum yield, etc) as xenobiotics. Efficiency of sensitized fluorescence of the dyes in the bioluminescent system was compared. Electron transfer processes were studied using series of metal cations. Changes of bioluminescence response integrate effects of the cations in the enzyme system: on the enzyme groups, on the substrates, on the chemical process, on the electron-excited states formation and evolution. The changes of the integral bioluminescence response were shown to depend on redox characteristics of the cations - redox potential and energy of electron acceptation. Hydrogen (e- ~H +) transfer processes were studied using series of quinones. Kinetic changes of the bioluminescence response were found to connect with redox potentials of the quinones, he main contribution to inhibition of the bioluminescent system in this case is due to interaction of quinones and NADH. Influence of the quinones on the enzymes groups was evaluated.

163

E2 : Bioenergetics : Electmn/ProtonTransport

P373 Structure Analysis of Cytochrome r from Sulfate Reducing Bacteria by NMR

~ ' ) , Yuki Fukuoka ~), Tomoaki Ohmura 2), Arima Fukunishi ~), Gota Kawal ~), Kimitsuna Watanabe 4), and Hideo Akutsu l),

'~t'okohama National Univ., 2~Mitsubishi Heavy Industries, LTD., 3~'hiba Inst. ofTech., 4~niv. of Tokyo. 15N labeled cytochrome c3 was prepared. The sequential assienment of the both oxidation states, ferri and

ferrocytochrome c3 signals has been carried out by homonuclear and IH-"N heteronuclear correlation NMR. All backbone signals except N-terminus has been assigned. NOE patterns, chemical shiit index and ~Jtm show that the secondary structures of the reduced protein in solution are not significantly different from these in crystal structure in the fully oxidized state. And all heme ligated histidine imidazole NII protons could be assigned in the reduced state. Also, the chemical shift enisotropy (CSA) of the imidazole protons were determined. CSA of the imidazole NH protons are larger than those of the peptide protons. It suggests the existence of the hydrogen bonds involving imidazole NTI protons. We have also measured IHJSN relaxation and NOE. In the reduced state, these parameters could be determined for 87% of signals, in the oxidized state, however, these parameters could be obtained for 79% of signals. The T~ values in the oxidized state is larger than those in the reduced state for some residues. It suggests the paramagnetic contribution does not affect significantly TI relaxation for certain residues. NOE values in the oxidized state is larger than those in the reduced state. Small values of NOE were observed for the amides in the N- terminus and some loop region in both oxidation states. The localized internal motion in the heme 4 region was found to be larger in the oxidized state than in the reduced state.

164

E3 : Bioenergetics : Biomechanics

P374 Stability Analysis of Micropipette Aspiration of Neutrophiis

Jure Derganc, Bojan Bo~it, Sa~a Svetina and Bo[tjan ~.ek[

Institute of Biophysics, Medical Faculty, University of Ljubljana, Slovenia

During the micropipette aspiration of neutrophils, it is commonly observed that at a certain aspiration pressure the so- called critical point is attained at which neutrophils could no longer be held in an equilibrium and start to flow continuously in the pipette. In our work we present a theoretical analysis of the equilibrium behavior and stability of neutrophils during the micropipette aspiration. We used the basic "liquid-drop model" of neutrophil elasticity extended by including the membrane Hookean elasticity. In this model, the only free parameter of neutrophil membrane mechanical properties is the ratio between the area expansion modulus K and the membrane surface tension V. Our analysis shows that depending on the value of the ratio K/V and the pipette radius two different regimes of neutrophil behavior are expected. At small ratios K/y the neutrophil would behave like a nonelastic liquid drop with the critical point attained when its projection length in the pipette reaches the value of the pipette radius. On the other hand, at larger K/V the Hookean elasticity prevails and the position of the critical point shift.r further in the pipette. We found that the limiting ratio K/7 between the two regimes of the behavior increases with the pipette radius.

P375 Mechanical properties of single rabbit extraocular and limb muscle fibres

JFY Hoh, I ZB Li ] & GH Rosmnmlith 2, IDepamnent of Physiology & Institute of Biomedical Research, University of Sydney, NSW 2006, Australia and 2Division of Information and Communication Sciences,

Macquarie University - Sydney, NSW 2109, Australia Ex~aocular muscles (EOMs) contain a complex mixture of myosin isoforms, some of which are not found in limb muscles. The purpose of this investigation is to explore the mechanical consequences of this diversity of myosin isoforms. Fibre bundles from rabbit psoas, vastus lateralis, and EOMs were glycerinated and single fibres dissected for dynamic stiffness analysis during partial activation. The stiffness minimum frequency, frnin~ which is sensitive to the kinetics of attached cross-bridges, was measured at 15~ for 75 EOM fibres and 61 limb fibres. Limb fibres, known to express myosin heavy chain (MyHC) is6forms HA, IIX and lIB, showed three peaks of frni, values: 10-14 Hz, 14-18 Hz and 21-28 Hz. Fast EOM fibres, known to express developmental and EOM-specific MyHC isoforms in addition to those in limb fast fibres, showed an additional fmin peak around 8 Hz. Preliminary immunochemical analysis revealed that these fibres contain developmental isoforms of MyHC. For 26 fibres, fmin was measured at tempecatures ranging from 13"(2 to 28*(2. An~enius plots of fmin values for limb and EOM fibres all show a breakpoint around 22*(2 except those containing developmental MyHC. We propose that the complex myosin composition of EOMs enable them to produce motor units with a wide range of fmely graded mechanical characteristics.

P376 UNSTRAINED RIGOR CROSS BRIDGES CANNOT SUSTAIN TENSION, ALTHOUGH THEY STAY

ATTACHED. E.L. DE BEER j, B.W.. TREIJTEL 2, P.L.T.M. FREDERIX 1'2, T. BLANGE 2. ZDEPT " MED. PHYSIOL. UU, UTRECHT AND 2DEPT. PHYSIOL., AMC, UVA, THE NETHERlANDS.

A comparison of experiments on single molecules and on intact muscle fibres, leads to a discrepancy about the fraction of cross bridges involved in muscle contraction. This discrepancy can be resolved by the existence of unstrained non-pushing rigor bridges (Howard, Nature, 1998; Huxley, Nature, 1998). To investigate this last question we measured the tension and the in-phase and out-of-phase components of the static and dynamic stiffness of rigor bridges of skinned frog single muscle fibres as a function of strain. The single fibre was brought in rigor by removal of ATP. Initial sarcomere length was set at 2.15 lain. Sarcomere length was followed during the experiment. For the determination of static stiffness strain was put on the fibre by consecutive shortenings of 0.1% to 0.7% of the initial length. Static stiffness was measured by means of a 0.05% step three minutes after the conditioning length change. Time resolved dynamic stiffness at a fixed frequency of 1.00 kHz was measured by sine waves with amplitudes of 0.01%, superimposed on steps with amplitudes between -0.1 and -0.7%. Both static and dynamic stiffness decreased to about the relaxed value at a strain of-0.6% If stiffness originates from the cross bridge, this means that cross bridge stiffness then disappears. After relengthening, stiffness is immediately restored to its original value, showing that cross bridges indeed stay attached. While both tension and stiffness exhibit an undershoot after a shortening, stiffness was immediately restored to its final value after a lengthening. Oppositely, tension showed an overshoot. These experiments confirm that stiffness originates from the cross bridge as will be discussed.

165

E3 : Bioenergetics : Biomechanics

P 3 7 7 Mechanical response of adherent Hying cells assessed by magnetocytometry and optical tweezers

S. H~nun, F. Gallet, L.B.H.P., Univcrsit~ Paris 7, 75251 Paris cedex 5, France V. Laurent, E. Planns, D. Isabey, INSERM 12492, Facult~ de m6decine, 94010 Cr~teil, France

We performed micromanipulation experiments on pulmonary epithelial cells, by two techniques : a magnetic tO~lUe, in magnetocytomett3r (MTC), or a force, in optical tweezers experiments (OPT), is applied to microbeads bound to the integrin mechanomceptor, which is c o ~ to the cytoskr When the force applied with OPT is tangential to the membrane, the bead rotates around the point where it is anchored to the membrane. The measured relationship between the angle of rotation and the momentum of F is linear (about 1.5 ~ per 100 pN.ttm). These experimental conditions are considered to be close to the one encountered in MTC. Nevertheless in MTC the measured angles 0 are higher, and i ~ more rapidly with the applied torque (0,ffi~35 ~ for C,==140 pN.pm). From time to time in OPT experiments, the bead can be pulled away, at a distance d (up to 50 lan), from the membrane and several successive regimes are otr, erved : first d grows linearly with the applied force F (several tens of pN/pm), then F relaxes as the tether begj'~ to form, and finally reaches one or several successive plateaus (40 to 80 pN, for a growth velocity of the tether of about 0.1pro/s). When the OPT are switched off~ the bead relaxes to its Ori~nal position in a few seconds. The formation of these tethers may explain the difference between MTC and OPT experiments. A MTC measurement is the result of an average over 150000 beads, and probably over very different events beads highly anchored to the membrane, and beads almost free to rotate, which give tethers with OPT.

166

G : Modeling,Theory and Bioinformatics

P 3 7 8 A COMPUTER MODELING OF METAL BINDING SITE IN DI PROTEIN OF TttE

PHOTOSYNTHETIC REACTION CENTER II

L.S.Rath 1, EK.Dash', M.K.Raval 1, C.Ramakdshnan 2 and EBalaram 2 1. EG.Departrnent of Chemistry, Rajendra College, Bolangir, 767002, Orissa, India

2. Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India

The calcium binding sites in calmodulin, troponin C, intestinal calcium binding protein ( icb ) and parvalbumin show sequential as well as conformational homology. Structure of Mn substituted troponin ( 1NCY ) and icb ( 6ICB ) also exhibit conformational homology at the metal binding site. Survey of sequence of D1 protein of photosynthetic reaction center II ( RC II ) reveals that the segment 298 - 310 is having sequence homology with the Helix - Loop - Helix type calcium binding sites. A computer aided model is built after the homologous calcium binding sites for 298 - 310 segment of D1 protein which can serve as one of the Ca (II) or Mn (II) binding sites in the RC II.

P 3 7 9 Design of High Quality Structure-Property Regressions

Milan Randic and Subhash C. Basak Natural Resources Research Institute, University of Minnesota, Duluth

5013 Miller Trunk Highway, Duluth MN 55811, USA

We illustrate the use of molecular descriptors that involve variable parameters x, y which differentiate atoms of different kind. The optimal values of the variables (x, y) are determined by minimizing the standard error of the regression. With the values of the variable x = 0 and y = -1.2 we obtain regression for the boiling points of ethers with the standard error of 1.3 ~ (n = 20, from C3H80 to C6H~40). This result is better than hitherto reported regression on the same set of compounds using several descriptors. Small standard error allows not only a reliable identification of outliers but also may point to novel and alternative descriptors for a particular application. Hence, flexible molecular descriptors deserve wider use.

* On leave from: Department of Mathematics and Computer Science, Drake University, Des Moines, IA 50311, USA

P 3 8 0 On Numerical Characterization of DNA Primary Sequences

Milan Randic, Marjan Vracko, Ashesh Nandy, and Subhash C. Basak Natural Resources Research Institute, University of Minnesota at Duluth

5013 Miller Trunk Highway, Duluth MN 55811, USA We outline numerical characterization of primary sequences of DNA that offer a basis for quantitative comparisons of such sequences. The approach is based on 3D graphical representation of DNA in which to the four nucleic acids are assigned four tetrahedral directions. The characterization is derived from eigenvalues of the so called D/D matrices, in which the elements are defined as the quotients of the Euclidean and the graph theoretical distances between points of the graphical picture of DNA. We discuss the convergence and size dependence of the derived numerical characterization of such sequences and their sensitivity to minor changes in the primary DNA sequences. Additional invariants are derived from D~/D k matrices (where k is the exponent of ltae elements of the DJD k matrix). The upper bound for the leading eigenvalue of as k tends to infinity was also determined.

*MR on leave from Department of Mathematics and Computer Science, Drake University, Des Moines, IA 50311, USA; MV on leave from National Institute of Chemistry, Ljubljana, Slovenia, A. N. on leave from Computer Division, Indian Institute of Chemical Biology, Calcutta 700032, India.

167


P381 Modeling the Solubility of Aliphatic Alcohols in Water.

Graph Connectivity Indices versus Line Graph Connectivity Indices Dragan Amic, a Subhash C. Basak, b Drago Beslo, a Sonja Nikolic c and Nenad Trinajstic c

aFaculty of Agriculture, The Josip Juraj Strossmayer University, HR-31001 Osijek, The Republic of Croatia, bNatural Resources Research Institute, University of Minnesota, Duluth, MN 55811, USA and CThe Rugjer

Boskovic Institute, HR-10001 Zagreb, The Republic of Croatia The acqueous solubility of aliphatic alcohols was modeled using valence vertex- and edge-connectivity indices. AUphatic alcohols were represented by weighted trees and the corresponding weighted line graphs. We also searched for the optimum exponent that is used for computing connectivity indices. It is found that the optimum exponents are -0.425 and -0.675 for the valence vertex- and edge-connectivity indices, respectively, when the weighted trees represented alcohols. The best structure-water solubility model for aliphatic alcohols based on a single topological index is the model with the valence edge-connectivity index when the exponent is -0.675. The model based on the weighted line graphs and the valence edge-connectivity index with optimum exponent (-0.85) is poorer (Rffi0.981, S=0.674, 1=--1392) than the models based on the weighted a'ees and the valence vertex- or edge-connectivity indices with standard (R--0.989, S---0.531, F=2279; R=0.982, S=0.680, F=1368) and optimized exponents (R=0.989, S---0.526, F=2320; R=0.992, S=0.43, F=3426).

P 3 8 2 Rules for Protein-structure Prediction-and Design: a Comprehensive Analysis of Coiled.coil Structures

J. Wals~w and D.N. Weolfson Centre for Biomolecular Design & Drug Development, School of Biological Sciences,University of Sussex,BN1 9QG,UK

At the tertiary and quatemmy levels, the coiled (:off is a~guably the simplest protein-structure motif. The regularity of coiied-coil slructures is underpinned by a conserved sequence pattern, known as the heptad repeat. In theory, this n~kes its prediction from sequence relatively straightforward. Nevertheless, there is disagreement between the various coiled-coil prediction methods. Here we present a thorough analysis of known coiled-coil slmctures, which we identified from the Brookhaven Protein Data Bank, and which we used as the foundation of genomic-scale coiled-coil prediction. To find the chamoeristic knobs-into-holes packing of coiled coils, we used a simple assessment of side chain-geometry. This was sufficient to distinguish all classic coiled-coil domain~ from most globular helix-packin 8 arrangements. Analysis of the identified coiled coils revealed 1) that the register of the coiled-coil s~ucttues could be assigned antomalically, using either the spacing of knob residues or the geometry of individual knob-into-hole intemctious; 2) new statistical amino-acid profiles for dimefic, trimefic and tetrameric strtlctur~; 3 that these results reinforce the roles previously established on the basis of the leucine zipper GCN4 core mutants, with mino~ exceptions; 4) that antilmraliel coiled coils tend to be less ideally packed th~n parallel stmctores. Finally, the databases of known ~oiled-coil and negative-control structures enabled us ~recalibrate existing coiled-coil prediction algorithms, and we have used these to estimate the numbe~ of coiled coils in completed genomes.

P 3 8 3 Non-adiabatic Proton Transfer in ]Penicillin Acydase

and Hern~n $.C. Berendsen BISON Research Institute, Depmtment of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747

AG Groningen, The Netherlands.

We present the calculation of .the rate constant of the rate-determining step in the breakdown of penicillin-G by Penicillin Acylase, a two-chain protein consisting of over 750 residues, using a combination of quantum and classical dynamics. This step takes place on a time scale of seconds, and involves a proton transfer concerted with a covalent binding of the substrate to the enzyme. A method of biased sampling was used, the Gibbs free energy of the biased configuration from which the proton transfer is likely to occur was deterrnined by a combination of semi-empirical quantum ca~ulations, thermodynamic integration, and molecular dynamics simulation. The proton dynamics is driven by a fluctuating environment, and was modeled a posteriori, using the proton potentials derived from classical molecular dynamics simulation, by the quantum-dynamical density matrix evolution method, thus including non-adiabatic pathways, i.e. pathways involving excited vibrational states of the proton.

168


P384 Prediction of position specific key residues and properties to form similar structural

subdomains from dissimilar subsequences in protein space.

Boojala V. B. Redd~, llya N. Shindyalov and Philip E. Bourne San Diego Supercomputer Center, Untvenity of California San Diego CA 92093-0537, USA.

An all against all protein structure comparison using the Combinatorial Extension (CE) algorithm [ 1] revealed an interesting gallery of about 75 most preferred subdomain structures [http://cl.sdse.edu /gal._dc4/gal_dc.html] in known protein space [2]. Homologous subdomain structuresin this gallery are observed to be formed even by many nonhomologous subsequences (<25% sequence identity in subsequences of >60 residues). Here~ we present an analysis of these subdomain structural homologs formed by nonhomologous subsequences. Our studies indicate that a few key position-specific residue properties appear to be guiding the formation of each of these preferred subdomain structures. We further discuss a possible sequence based prediction method for these subdomain structures and its usefulness in de novo protein design. References: [l] Shindyalov, I.N., Bourne,P.E., Protein Engng. 9:739-747. [2] Shindyalov, I.N., Bourne, P.E. (Submitted).

P385 MOLECULAR DYNAMICS SIMULATIONS OF RAFFINOSE FLEXIBILITY

M.C. Dormamaria, J. de Xammar Oro and LR. Grigera, Instimto de Fisica de Liquidos y Sistemas Biol6gico (IPLYSIB),CONICET,CIC,UNLP,C.C.565,1900 La Plata, Argentina, [email protected]

The purpose of this work is to present some new data of mobility of raffinose (ct-D-galp(1-6)-ct-D-Glucp-(1-2)- J3-D fruf), in vacuum and aqueous solution studied by molecular dynamics simulation MDS, which has proved to be a powerful technique to predict properties of carbohydrates. The interest in studying the structural and solvation aspects of a biological molecule is because such aspects are intrinsically correlated with its biochemical activity. In particular, raffmose is a naturally occurring trisaccharide isolated from a variety of plants including beet sugar molasses, cottonseed meal, and the seeds of various food legumes. The MD simulations were performed using the GROMOS package. In the solvation case, a raffinose molecule was located in a cubic box of a box length of 2.5 nm surrounded by 487 molecules of water.The analysis in vaccum reveals poor flexibilty of the first giicosidic linkage (1-6), and a restricted mobility of the second linkage (1-2). These results are in agreement with the experimental picture of sucrose as a relatively rigid molecule. In aquoeus solution, from the dihedral angles trajectories more flexibility of the molecule is observed. MCD is-Member of Comision de Investuigaciones Cientificas de la Provincia de Buenos Aires CIC, JXO and JRG are Members od Consejo National de Investigaciones Cientificas y Tecnicas de la Argentina. CONICET P386

On shape changes of vesicles with actin cortex

Monica Neagu and Adrian Neagu

University of Medicine and Pharmaoj Timisoara, Department of Biophysics and Medical Informatics, P-ta Eftimie Murgu Nr 2, 1900 Timisoara, Romania

ABSTRACT Shape equations for phospholipid vesicles including a spherical actin cortex are derived in 2 and 3 spatial dimensions. They arise from the condition of stationarity of the membrane free energy at fixed area and volume'. Numerical solutions are presented in the 2 dimensional case, while, in the more involved context of 3 dimensional shapes, the free energies of different, experimentally observed, shapes are compared. The relevance of the 2 dimensional study for the realistic case is also discussed. The theoretical results are analysed in the view of known experiments concerning shape changes of serf-assembled actin bilayer composite membranes.

169


P 3 8 7 MD Integration by Split Integration Symplectic Method

Matej Praprotnik and Du$anka Janegi~ National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia

A Split Integration Symplectic Method (SISM) for long time step MD simulations is described. The SISM involves splitting of the total Hamiltonian of the system into the harmonic part and the remaining part in such a way that both parts can be efficiently computed. The Hamilton equations of motion are then solved using the second order generalized leap-frog integration scheme in which~the high-frequency motions are treated analytically by the normal mode analysis which is carried out only once, at the beginning of the calculation. SISM requires only one force evaluation per integration step, the computation cost per integration step is approximately the same as that of the standard leap-frog-Verlet method, and it allows an integration time step up to an order of magnitude larger than can be used by other methods of the same order and complexity. The simulation results of MD simulations of tl~e model system of linear chain molecules show that the SISM posses long term stability and the ability to use long time steps. The approach for MD simulations described here is general and applicable to any complex system.

P388 MOLECULAR DYNAMICS SIMULATIONS OF DIPEPTIDE (ALA-PRO) IN WATER:

CONFORMATIONS OF CIS AND TRANS ISOMERS USING DIFFERENT WATER MODELS.

Pekka Mark and Lennart Nilsson

Karolinska Institutet, Department of Biosciences, S-14157 Huddinge, Sweden

Abstract: Molecular dynamics simulations of a single dipeptide (Ala-Pro) molecule in water using different water models, the TIP3P (transferable intermolecular potential 3P), SPC (simple point charge) and SPC/E (extended simple point charge) were performed using the CHARMM molecular mechanics program. Both cis and trans isomers were simulated and conformations were compared when different water model were used. Analysis shows that both isomers have several conformations, but the total conformational space is limited. The stucture of dipeptide (Ala-Pro) in aqueous solution has been studied by nuclear magnetic resonafice spectroscopy and our results are in good agreement with these reported NMR data.

P389 Sweet apolar~onic hydration

V. Martorana, D. Bulone, L. La Fata, M. Manno, P.L. San Biagio CNR IAIF, viaLa Malfa I53, 90146 Paka'mo, Italy.

Sucrose is commonly thought to have a deep influence on the functionality of many biomblccules. In particular it has been found that high concentration of sucrose is necessmy to start gelation of high metoxyl pectin solutions. The physical basis for such phenomena has been traced back to water-s~,~,m- hydration competition effects, hydrogen bond network modifications, dynamics slowing-down, etc. These effects and their relevance are studied at the molecular level, using Molecular Dynamics and Free Energy ~ o n . Our attention has been focused on sucrose-rich solutions containing simple apolar or ionic solutes, taken as mod~fls of analogous biomolecular groups. Long and accurate Poterdlal of Mean Force calculations of Na +-CI" and Methane-Methane in concemmed mcrme ~ a t i o m am lxs4onmcd and compered with the pmr water solvent case. In l~qicular ~ in Jtmic md d b ~ fmtmm of ~ are amlysed in detail This atlows to morn the nmtecdar mecl~aimn f q m ~ i b l e f~r lh8 mzxlifl~tion ~'a~e solute..eolute po~mtial ~ 'face as cmaed by the ~ of

170

G : Model ing,Theory and Bioinformatics

P390 A Theoretical Study on the Eleelrmtadr Properties in the Active Site of Palmia. Lament E, Dardem~, Araken S. Werneck s, Marfal de 0. Nero b and Paulo ~ Bisch a.

*Instituto de Bio/qsica Carlos Chagas Filho - Universidade Federal do Rio de Janeiro/Brazil ~ de Quimica Universidade de BraMlia/Brazil

Papain is a cysteine ~oteinase closely related, both slrocturally and functionally, with other important proteinases found in parasites, which are recognized as potential targets for chemotherapy against Chages' disease and Malaria. In this study we calculated the electrostatic potential in the region of the active site of papain with and without the solvent and counter ion's conlributions. We calculate also the interaction energy between the peptide inhibitor leupeptin (Ac-Leu-Leu- Arginal) and various important amino acids belonging to the enzyme. The electrostatic potential was computed using a procedure based in a multiconter multipolar expansion (up to quadrupoles) of the charge dislribution derived from ab in/t/o Hartree-Fock quantum wave functions. In this procedure the enzyme is subdivided in small fragments end the total elec- lrostatic potential is computed as a sum of the potential calculated from these fragments. For the calculation of the total molecular interaction between the inhibiter and active site's mnino acids we used the Kitatwa/Morokuma decomposition scheme within the Hartree-Fock level. Our results show that the residue ASPI58 plays an important role in the active site of papain and in the inhibitory pro~ss by leupeptin. Another important conclusion is that the effect of counter ions is crucial for the formation of a region of a negative potential, close to ASP158, in the active site.

P391 Identification of Side-chain clusters in Protein Structures by a Graph Spectral Method

N.Kannan and S.Vishveshwara Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.

Side-chain clusters in protein structures which are important from protein structure and folding point of view are detected using a Graph Spectral Method. Protein side-chain interactions are represented by a labeled graph in which the nodes of the graph represent the Cp atoms and the edges represent the distance between the C~ atoms. The Laplacian matrix of the constructed graph is diagonalized. Clustering information is obtained from the vector components associated with the second lowest eigenvalue and cluster centers are obtained from the vector components of the top eigen values. The method uses global information for clustering and a single numeric computation is required to detect clusters of interest. The approach has been adopted to detect variety of side-chain clusters which are important from protein structure and folding point of view. Cluster centers show a good correlation with the ~F values obtained from protein engineering methods to probe the transition states of folding. Expanded clusters are detected near the active and binding site of the proteins studied. The method is also used to detect domains in protein structures and conserved side-chain clusters in topologically similar proteins.

P392 A DATABASE ANALYSIS OF GLYCOSYLATION SITES IN PROTEINS

T. Hema Thanka Christlet and K. Veluraja Department of Physics, Manonmaniam Sundaranar University, Tirunelveli - 627"012, Tamilnadu, INDIA

An analysis of N-glycosylation and O-glycosylation sequences was carried out in an attempt to throw more light on the glycosylatiOn process. For N-glycosylation Asn-X-Ser/I'hr is the necessary but not sufficient condition, and for O-glycosylation till dale no consensus sequence is found out. For N-glycosylation analysis, 452 consensus Asn-X-Ser/Thr sequences were selected from PDB database in which Asn is on the surface of the protein. In the case of O-glycosylation 265 experimentally predicted Ser/Thr sites were selected from O-GLYCBASE database for analysis. In N-glycosylation a proline residue at X position prevents glycosylation, whereas in O-glycosylation proline residues occur preferentially at many positions close to the site of glycosylation. Giy and Ash at X position favours N-glycosylation. In O-glycosylation Gly and Asn residues are less preferred around the site of glycosylation. The aromatic amino acid residues, Phe and Tyr are preferred at X-position in N-glycosylati0n while they are less preferred close to the O-glycosylation site. For O-glycosylation there is positional preference for various amino acids and, in particular, Pro at +3 position strongly favours O-glycosylation. On the conformational aspect, in N-glycosylation, the Ramachandran ( d~,~I ' ) angles around Giy at X-position show a clustering in the region which is disallowed for non-glycyl residues. These results can help the Molecular Biologists and Protein Engineers to identify N-glycosylatable Asn and O-glycosylatable Ser/Thr.

171


P393 Clustering of Psoralen Derivatives using Topological Invariants: a strategy for molecular design

Subhash C. Basak, Gregory D. Grunwald, Alexandru T. Balaban and Kanika Basak* Natural Resources Research Institute, University of Minnesota-Duluth, 5013 Miller Trunk Highway,

Duluth, MN 55811, USA, [email protected]; *St. Xavier's Computer Center, 30 Park Street, Calcutta-700016, India

Modem combinatorial chemistry develops and screens large libraries of chemicals for drug discovery. The cost of exhaustive testing of large number of chemicals could be prohibitive. One method of cost reduction is to select an appropriate set of dissimilar structures for testing instead of screening the entire library. In this study, we have used calculated graph invariants to cluster a large and diverse set of psoralen derivatives in an effort to choose a limited and representative sample of compounds. The utility of this approach in drug discovery and molecular design will be discussed.

P394 A Hierarchical QSAR Approach to Predicting Bioactivity

of Chemicals Using Theoretical Molecular Descriptors Subhash C. Basak, Brian D. Gute, Denise Mills,

Gregory D. Grunwald, David Opitz* and Krishnan Balasubramanian** Natural Resources Research Institute, University of Minnesota-Duluth, 5.O13 Miller Trunk Highway,

Duluth, MN 55811, USA, [email protected]; *Department of Computer Science, University of Montana, Missoula, Montana 59812, USA;

**Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA

A recent trend in computational toxicology is to use theoretical molecular descriptors in predicting toxic effects of chemicals from structure. We have developed a hierarchical quantitative structure-activity relationship (QSAR) approach for estimating physicochemical and biological effects of chemicals directly from structure. This utility of topostructural, topochemical, geometrical, and quantum chemical indices in the development of QSAR models for estimating toxicity of chemicals will be discussed.

P395 SYM - A COMPUTER PROGRAM FOR SIMULATION OF REGULATORY PROCESSES IN

BIOSYSTEMS G I Mihalas, Diana Lungeanu, G Macovievici, Raluca Gruia, Monica Neagu

University of Medicine and Pharmacy, Timisoara, Romania

The simplest regulatory system assumes that the output, y = f(x), which is a function of input x(t), can influence, by feedback, the input. Most biological processes can be modelled by differential equations y = f(x(t), t) and x' = g(y(t),t), and often there are several inputs and outputs. Usual methods for numerical solving (f.i. Runge-Kutta) can be applied for obtaining the time evolution of any of the variables. However, the processes are not instantaneous; we should admit a time lag ~ between the moment of the response release, t, and the moment it is able to influence the input variable, t+~'. Hence, we should rather consider x'(t) = g(y(t-~),~). Introduction of this delay ~ can account for a quite complex behaviour, even for simple systems. We built a computer program (Pascal) admitting a customised set of differential equations, with several operational facilities (autoscaling, choice of represented variables, change of parameter values or graphical representation limits, adjustable iteration step). The program computes the time evolution of the variables involved and can also represent the phase diagram - important for oscillatory phenomena. The program has been applied for several processes: humoral immune response, regulation of protein synthesis in systems of interconnected genes (f.i. p53 - mdm2) and apoptosis of thyme cells.

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P396 N E U R A L N E T W O R K M O D E L I N G TO S I M U L A T I O N OF C O N T R O L OF M E D U L L A R R E F L E X PATTERNS. ~ORTEZ-MAGHELLY C ], DALCIN B 1 & PASSOS EP 2 1Dpt Ci~.ncias Fisiol[gicas, Univ. do Estado do Rio de Janeiro (BR). 2Dpt do Eng. El~trica, Pontificia Univ. Cat61ica do Rio de Janeiro (BR).

The analysis o f nonlinear signs, frequently observed in experiments to study medullar and suprasegmentar sensoriomotor phenomena, becomes more direct by use o f neural artificial networks. So, artificial networks are frequently used to simplify the mathematical modelling o f neurophysiologic phenomena. Indeed, computational simulations become possible the study of the behaviour o f the large neuronal population, starting from small or restrict kind number o f neurones. In present work, we adopted a reduced model o f medullar neuronal circuit o f reflex pattern (neurophysiologic model), and we determined its mathematical equations and its conditions. Then, a artificial neural network related to the model were developed. Neurophysiologic data were insured in these problems and several situations were simulated. The responses given to artificial network were observed and comparated to results from the neurphysiologic model. The results shown that the neurophysiologic model as well as the artificial network can be used to simulate and to study responses to situations o f learning process o f medullar reflex control.

P397 Statistical analysis of the interspike interval series of fusimotor neurones from

decerebrate cats Blesic S ], Ljubisavljevic M l, Milosevic S 2, Stratimirovic Dj 3

i Institute for Medical Research, 2 Faculty of'Physics, University of Belgrade, 3 Faculty of Stomatology, University of Belgrade, Belgrade, Yugoslavia

Sequences of action potentials recorded from fusimotor neurons from decerebrated cats show irregular and complex behavior both in spontaneous activity and during periodic electrical or mechanical stimulation of the muscle. Spike discharges of fusimotor neurons to medial gastrocnemius were registered from filaments dissected free from the nerves to these muscles.

The analysis employed on sequences of interspike intervals (ISis) uses methods of statistical physics. A new technique of detrended fluctuation analysis (DFA) has been applied for quantifying correlation properties of this non-stationary time series. This approach involves calculation of a correlation exponent cx(n) at segment size n, and can be compared with the power spectrum exponent [~(n). The results indicate the existence of a crossover phenomenon that marks a change in a short and long-range scaling behavior in fusimotor activity. DFA exponents for short segments (5 < n < 500) are - 0.5, the value for the uncorrelated, white noise-like signals, while they show prominent increase in a long-range area with values approaching that of the l / f noise. This is consistent with the results of the Fourier analysis. The findings can suggest a probable role of fusimotor activity as a source of intrinsic noise in a stochastic resonance-type mechanism that increases the sensitivity of muscle spindles.

P 3 9 8 Identification of Protein Function through Blending of Sequence Threading and Functional

Complementation by Nandita Bachhawat and Shekhar C. Mande, Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036',-INDIA

As DNA sequences of genomes from various organisms become available, one of the challenging tasks is m assign functions to each of the identified open reading frames. In absence of discernible sequence homology to proteins of known function, this task becomes even more formidable. One of the ways suggested to address this problem, has been with the help of three dimensional structures. We present here a test case using three dimensional structure prediction as applied to the open reading frame Rv0046c of Mycobacterium tuberculosis H37Rv, and its validation by functional assays.

The open reading frame Rv0046c shows a very weak sequence homology to inositol-l-phosphate synthases derived from eukaryotes. The sequence identity is.less than 18%. The encoded protein is substantially smaller than the eukayotic anaJogues, and further critical disulfide bridges important for its activity are not conserved in Rv0046c. The sequence also shows absence of a canonical NAD binding signature, which is a strict requirement for its function. Yet, sequence threading in proteins of known three dimensional structures showed that a NAD binding motif may indeed be present. In close proximity of the putative NAD binding motif, two cysteins are also observed which may play a role for the proposed oxidation- reduction reaction. The results of sequence threading were confirmed by complementing inositol-l-phosphate synthase deficient Sachharomyces cerevisae strain by Rv0046c. The functional complementation shows that this is the first ever reported inositol-l-phoshpate synthase from prokaryotes.

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P399 INVESTIGATIONS ON EVOLUTIONARY CHANGES IN BASE DISTRIBUTIONS IN GENE

SEQUENCES

S Ghosh I and A Nandy 2 tGenetic Engineering and 2Computer Division, Indian Institute of Chemical Biology.,

4 Raja S C MullickRoad, Calcutta 700 032, India, e-mail: [email protected]

Investigations of phylogenetic relationships between species through studies of gene sequences has been engagir, g the attention of molecular biologists. Here we report our findings that indicate that distribution and composition of bases in the coding segments tend to increase towards higher complexity with evolution using a graphical technique for DNA seqeuence representation

A close inspection of the exon clusters of genes from heat shock proteins to globins and myosins shows the effects to be progressively tending towards higher complexity in later genes and species. Recent work of Roldan et al has also shown, that complexity of base organisation in intron segments increases with evolution. Here we present our findings on these and related matters and show that there appears to be a systematic trend towards asymptotic complexity in base organisation in coding regions limited by compulsions to preserve biological diversity.

P400 Genome analysis by introduction of oligostickincss

~LgumlLSaiIfl and Koichi Nishigaki Department of Functional Materials Science, Saitama University

225 Shimo-okubo, Urawa, Saitama 338-8570 Japan

Genorne Science, which is recently being established on the movement of Human Genome Projects, is constantly and abundantly generating a volume of genome sequence information. The next stage of Genome Science must be in developing the tools to analyze and utilize thus generated information. In this view, we devised an analysis method of genome sequences, advancing a computer program composed for predicting random PCR products. This method enabled us to characterize genome structures of E.coli, S.cerevisiae and C.elegans. Positional characteristics of such large genomes were evident by shown, indicating organized genome structures and reminiscences of evolutionary events. These findings were distinct types of information forms obtained by G+C content analysis, oligonucleotide sequence distribution or melting map analysis along the genomic sequence.

P401 G e n o m e - b a s e d Iden t i f i ca t ion o f Species

Koichi Nishigaki and Mohammed Naimuddin Dept. of Functional Materials, Saitama University, Saitama, Japan

Species has been identified based on its phenotypic properties. Now, it is possible to do so based on genome. It is, however, too costly and laborious, and thus unrealistic, to determine the whole sequence of genomic DNA for this purpose solely. On the other hand, it is known that sequencing data of a limited amount often falls short of identifying species. We have developed a powerful and yet convenient technology for identifying species, named Genome Profiling (GP), which is composed of random PCR and TGGE/DGGE. This technology enables us to distinguish between extremely close cells such as bacteria belong to the same species.

We discuss what is a sufficient amount of data to identify species together with demonstrating data for this. Consequently, we also steps into the discussion on the evolutionary relationship of organisms and genome sequence space. We refer to the profound effect of this technology on the various disciplines in life sciences such as Ecology, Epidemiology and Environmental Chemistry.

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P402 T H E O R E T I C A L ANALYSIS O F T O T A L P R O T E O M E S BY

A M E M B R A N E P R O T E I N P R E D I C T I O N S Y S T E M , SOSUI.

Shigeki MITAKU, Takatsugu HIROKAWA, Mitsuo ONO and Hirotomo TAKAESU

Department of Biotechnology, Tokyo University of Agriculture and Technology,

Nakacho, Koganei, Tokyo 184-8588. Japan. The discrimination of membrane proteins and the prediction of transmembrane helices provide the infol'mation

about the characteristics of proteomes of various biological species. We have analyzed all amino acid sequences of sixteen species by a membrane protein prediction system, SOSUI. The species include eleven eubacteria, three archaea and two eukaryea. Single-cellular organism from mycoplasma to S. cerevisia showed almost constant fraction of membrane proteins, while the fraction was much larger in C. elegance. The constitution of various kinds of proteins is compared among different species.

P403 Discrimination between afferent and efferent patterns by Artificial Neural Network.

MT El Gohary, Abdalla S. Ahmed, and.A.M. Eissa * Biophysics lab, Faculty of Science, AI Azhar University, Cairo, Egypt. In 1986 Rumelhart et al., investigated a wide variety of network designs, they announced the discovery of a method of enabling a network to learn to discriminate between classes of patterns that are not linearly separable, they called the method backward propagation of errors. The two types, motor and sensory patterns being distinguished by the network are identified to the learning network by the desired output fed to the network in the learning process. Many data records representing the two types were processed by different mathematical modeling approaches. The models coefficients are considered to be the features which would be utilized for discrimination between motor and sensory responses. Then the results were compared to the desired output for each record and the percentage of correct estimations was calculated for each one. Non-linear model results have proven to be the best single model in application on neural networks, which confirms the non-linear behavior of the nervous system.

P404 Oligonucleotide composition analysis of genomic sequence data

Hiroshi Nakashima' and Ken Nishikawa 2 ~School of Health Sciences, Faculty of Medicine, Kanazawa University, KanaTawa 920-.0942, Japan 2Center for Information Biology, National Institute of Genetics, Yata 11 i I, Mishima, Shizuoka 411-8540, Japan

The compositions of di- through hexa-nucleotides were analyzed for genomic sequences of 18 organisms, containing the complete genomes of 15 micro-organisms as well as partial genomes of human, Arcbidopsis thaliana and Caenorhabditis elegans. As different organisms showed different nucleotide compositions, we examined whether fragmental DNA sequences from ! 8 organisms were correctly identified in terms of nucleotide composition. The accuracy of identification increased not only as the length of oligonucleotide was increased from di- to hexa-nucleotide, but also as the length of fragmental DNA to he tested was increased from 1,000 bp to 10,000 bp. Using 32 components of trinucleotide (by unifying the complementary sequences together), 82% of 43,045 fragmental DNAs of 1,000 base long from 18 organisms was correctly identified. This result indicat~l that the whole genome of an organism is characterized by a unique (tri-)nucleotide composition irrespective of coding/non-coding sequences. When the same methods was applied only to coding sequences (i.e., genes), the accuracy went up to 87% suggesting that the effect of codon usage, characteristic to individual organisms, improved the accur~y by five percent.

175


P405 The influence of internal noise on currents

driven by ionic pumps

Monica Neagu and Adrian Neagu

University of Medicine and Pharmacy Timisoara, Department of Biophysics and Medical Informatics, P-ta Eftimie Murgu Nr. 2, 1900 Timtsoara, Romania

ABSTRACT Currents generated by ionic pumps, in partic-lar the Na§ + ATP-ase, as a result of applied external electric fields, are analysed. Voltage jumps and ~ g u l a r signals are considered. We also take into account the stocha~c fluctuations of the membrane potential, commoniy assigned to random opening/closure of the neighbonring io~c channels. The study relies on the Astumian - Robemon model with two distinct cOnfornmtional states of the enzyme. The ionic current, averaged over intrinsic noise, is calculated and, by fitting available experimental d~ta the amplitude of random fluctuations of the membrane potential is asessed. New experimental conditions are suggested which could reveal stochastic resonance in the system.

P406 PROCLASS : A Computer program for predicting Structural Class of a Protein

G.P.S.Rae.hava Institute of Microbial technology, Sector 39A, Chandigarh, India

A computer program called PROCLASS has been described which is developed for predicting the structural class of a protein from its amino acid sequence. This program is developed based on a statistical algorithm developed by Chou (1995). The unique feature of this statistical algorithm is that it incorporates the coupling of different amino acid components. This coupling approach distinguishes it from the previous algorithms of class ixediction. In PROCLASS the novel approach of Chou (1995) of prediction of protein stnLcttwal class in a (20-I) D amino acid composition space and the matrices were utilized. This program is written in computer language "C' on alpha Workstation under Digital Unix. Program is freely available from author (email: raghava@Jmtech,ernet.in, Web:http://imtech.emet.in/raghava)

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P407 CORRECTION OF LIPID PEROXIDATION IN TISSUES AND BLOOD UNDER IONIZING RADIATION

TREATMENT BY EXOGENOUS ALPHA-KETOGLUTARATE INJECTION N.Kurgahadc O.Gory. n

Ivan Franko Lviv State University, 290005, Lviv, Ukraine

Lipid peroxidation is considered as biological process of adaptive modulation of membrane structures and organism's metabolic regulation. In the extremal conditions of ionizing radiation, stress and hypoxia the generation of free radicals increases. Enhancement of lipid peroxidation (LP) in-tissues is accompanied with induction of antio.'ddant enzymes (AO). such as supero:dddismutase (SOD), katalase (KAT). glutationperoxidase, synthesis and activation. We ha,,~e study the effect of sodium alpha-ketoglutarate (KG) intraperitoneal injection on the regulator' mechanisms of LP in rat's blood (malonic dialde~-de concentration) and AO under the ionizing radiation treatment 0RT). tt have been shown that 30-days IRT (10, 20 and 30 Roentgen in total) results in LP activation in rat's tissues (hepar, miocard, intestinum muco~ tenue) and blood and in the increase of AO activity. Exogenous KG in therapeutic concentration limits the LP activation and normalizes the AO protection of treated organisms. Our previous data show that changes in the ways of mitochondrial energetic exchange economize oxygen utilization, decrease cells oxygen requirements and. on the organism level lead to decrease of oxygen tensity in blood and in the velocity of it consumption by the tissues. Such changes result in increase of organism's resistance to e.'aremal influences adjoining with pathology.

P408 Laser Generation of High Pressures in the Eye; Exploding Melanosomes and Retinal Damage

Bernard S. Gerstman Department of Physics, Florida International University, Miami, FL 33199

(Email: [email protected]; Telephone: 305 348 3115) Detailed calculations will be reported on that show how sub-nanosecond laser pulses can

damage the retina by producing high pressures. Especially interesting is the fact that not only are large compressive pressures expected. The calculations also show that large tensile stresses at the core of the absorbing melanosome will be produced. These large tensile stresses may result in the explosion of the melanosome. Thus, damage to the retina may occur either due to large amplitude acoustic waves, or due to the explosive breakup of the melanosome. In addition, the size of these pressure effects will be shown to increase as the laser pulse duration is decreased. Therefore, for short enough pulse duration, possibly sub-nanosecond, pressure effects may be the underlying physical mechanism responsible for retinal damage at threshold level laser exposure.

P409 DETERMINATION OF PHYSICAL LOAD INFLUENCE ON PEOPLE BY BIOLUMINESCENCE ASSAY

E.V.Gritsenko I, N.N.Remmel I, O.M.Maznyak 2, V.A.Kratasyuk I 1Institute of Biophysics SB RAS, Krasnoyarsk, Russia, 2Krasnoyarsk State University, Krasnoyarsk, Russia

At present there are different assays of detection of physical load influence on people" physiological, biochemical and others. The main disadvantages of these assays are the duration, the complexity and sometimes the existence of painful sensations (for example, the procedure of blood sampling).

The new fast bioluminescent assay for control has been proposed. This test is based on influence of human saliva on light intensity of coupled enzymatic system NADH:FMN-oxidoreductasc-lusiferase. Three groups of people were involved in our studies. The first was the group of he~. thy children of 4-5 years, their saliva has been tested before and after equal physical exercises. The second was the group of free style wrestlers before and after training. And the last group consisted of students before and after lecture.

Using this method we found 3 types of saliva's effects. All of participants have been separated on three subgroups: 1 the increasing of the fight intensity; 2. the deere~ng of the light intensity; 3. light intensity wasn't change. The saliva's effects depended on personaliti.es. There were the correlation between saliva's effects and content of Ca '2, K +, Na § and activities of enzymes (alpba-amylase, ASAT, ALAT, LDH) and their metabolites concentrations (creatinine, albumin. cholesterol), pH values in saliva.

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P4 t 0 THE USE OF BIOLUMINESCENT BIOTESTS FOR STUDY OF NATURAL AND

LABORATORY AQUATIC ECOSYSTEMS E.N. Esimhekova~ V.A. Kr~_~_c~ak

Institute of Biophysics SB RAS, Krasnoyarsk, Russia The set of biohtminescent tests was developed to monitor water quality in natural and laboratory ecosystems. It consisted of 4 bioluminescem systems: luminous bactmia, coupled ettzyme system NADH:FMN-oxidoreductase-luciferase and triplet enzyme systeans with alcohol dehydrogenasr and trypsin. The set ofbiotests was appfied for a small forest pond (Siberia, Russia), laboratory microecosystems polluted with benzoquinone and a batch culture of blue-green algae. Thereby effvots of natural water compared to those of models of heavy pollution and "bloom" of blue-greens on the bioluminescent tests were revealed. The set of biotests was not affected by a natural seasonal variability of water quality in the unpolluted pond, but responded to the heavy pollution and the ~bloom" ofbhae-greeus. The set ofbiotests could he recommended as the alarm test to control the acute toxicity of natural water bodies.

P411 ENVIRONMENTAL BIOPHYSICS IN WATER QUALITY MONITORING OF SALT LAKES

V.A.Kratasytdr Institute of Biophysics SB RAS, Krasnoyarsk, Russia

A set of bioluminescent toxicity bioassays has been applied for ecological monitoring of salty water of Siberian salt lake Shira near health resort. Methods have been modified according to the salt water' characteristics. It has been shown that the quality of salt water depended on their lake's surface locality: the more distance from health resort the higher quality of water. The results of tests depended on the depth: the maximum effects on luminous bacteria intensity have been found at a depth of 5 and 10 metros. The daily and seasonal properties of water have been investigated. Two kinds of Lake Shira's maps based on biolumine'~rent d-_t-__ have been made: the water toxicity map and the map of the heterogeneity of salt water's characteristics. Bioluminescence assays combine the advantages of speed and sensitivity. The problems and perspectives of bacterial biokLminescent toxicity biotests applications to salty water are discussed.

P 4 1 2 EFFECT OF LOW-FREQUENCY MAGNETIC FIELDS ON THE BLOOD LEVEL OF SH- COMPOUNDS DURING ACUTE PHASE REACTION D. TchitchkarA, S. Koulchitsky, A. Tikhonov, A. German, Y. Pesotskaya, S. Pashkevich, S. Pletnev, V. Kulclfitsky Institute of Physiology, National Academy of Sciences, Minsk 220072 Belarus In experiments on rats, effects o f low-frequency magnetic fields (LMF) on the pattern of immune responses during simulated acute phase reaction following intraperitoneal injection o f l 0 0 ~tg/kg E.coli lipopolysaccharide (LPS) or pyrogen-free saline were studied. After LPS, a biphasic rise by 0.6+0.1 ~ C and 1.3+0.I ~ C, respectively, in colonic temperature and a decrease from 28+6 gm/100 ml to 17+lj.tm/100 ml in the blood content of SH-compounds were observed. A twenty-min exposure to LMF during the first phase of fever markedly reduced the rise in colonic temperature during the second phase of the acute phase reaction and led to a small increase, up to 35-6 O~a/100 ml, in the content of SH-compounds. Thus, a short-term exposure to LMF attenuate~ the temperature response to systemic application of LPS and is accompanied by an increase in SH-compounds, which indicates the ability of LMF to enhance the non- specific resistance of the organism during experimentally simulated acute phase reaction.

178


P413 Ttp of human articular cartilage: an/n vivo probe for molecular dynamics

Umamaheswar Duwuri, Sridhar Charagundla, Rahim Rizi, John S. Leigh and Ravinder Reddy Department of Radiology, Univ. of Pennsylvania, Philadelphia, PA 19104 USA

Introduction: Osteoarthritis (OA) is a degenerative disease of articular cartilage. It has been proposed that, in the early stages cs OA, proteoglycan (PG) molecules may be lost from the tissue. There has been much interest in developing a non-invasive technique to monitor these changes. We have recently reported that Tip imaging and spectroscopic measurements are specific for PG changes in bovine tissue. Here we show, for the first time, Ttp measurements of articular cartilage obtained from a healthy human volunteer. Methods: A standard Tip pulse cluster was appended to a fast spin-echn imaging sequence and implemented on a 1.5T GE scanner. The knee of a healthy male volunteer was then imaged with this sequence and a birdcage volume coil. T~p was measured by varying the spin-locking time from 10 ms to 120 ms. The Ttp dispersion of the tissue was gauged by obtaining T~p values at spin-locking frequencies (vB~) of 187 and 30 Hz. Juvenile bovine cartilage was also studied at 2T with spectroscopic and imaging techniques. Results: Trap in the human knee was found to be substantially longer than literature values for "I"2 of adult cartilage. Juvenile bovine cartilage was also found to have uniformly longer T2 and Ttp values at all B,'s. Furthermore, adult cartilage did not demonstrate substantial dispersion in the 30-187 Hz frequency.range. However, there appears to be significant dispersion in the 0-30 Hz regime. This indicates relaxation processes occuring on the order of~33 msec. Conclusion: Our data show that T~p is sensitive to the macromolecular structure of cartilage. T~p and T2 both decrease in mature cartilage, indicating a slower time scale for the relaxation processes. Therefore,~dies of T,p relaxation might yield useful biophysical information regarding the macromolecular dynamics and structure of cartilage. This information might be useful for the early detection of OA.

P414 l n - v l v o 11t & 31p M R S and i n - v i t r o I l l N M R studies on bureau breas t c a r c i n o m a

hf, l l hmk , l~ l~ - , O. Coshic @, P.K. Julka #, G.K. Rath # and N.R. J~nnatlmn* Departments of N.M.R.*, SurgeaT@, and R a d i o ~

All India Institute of Medical Sciences, Ansafi Nagax, New Delhi- 110029, Indi~ n-vivo 1H and 31p MRS wexe performed on human breast canc~ lmtie~as suffering from int'fltrating ductal carcinoma along with in-vitro 1H NMR studies on tissue extracts and FNAC/FNAB mmapl~. Volume localized proton MRS (n=80) showed that W/F values were higher in tumorous breast tissues compared to normal tissues. We have documented reomtly ~ the W/F values from proton MR spectra can be used as ~ index to monitor the response of breast tumor to neoadjuvant chumotherap3~. The water suppressed in-vivo proton spectra of malignant breast tissue shows a choline peak at 3.2 ppm in about 70% of the patients studied. The presence of choline was confirmed by in-vitro proton NMR spectra obtained from malignant tissue extract as. well as from FNAC/FNAB samples. The spectra showed in addition to choline, lactate, taurine, succinatc and several other metabolltes. We have also carried out in-vivo 31p MRS at 1.5 T using a doubly tuned surface co t in 15 patieats and 10 volunteers. PME and PDE level increases while PCr level ~ e s in breast cancex patients compared to controls. The relative concentration of inorganic phosphate (Pi) and the high energy phosphate, PCr and NTPs provide vahmble information with respect to tissue enefgetics. Our results suggests that in-vivo 1H & 31p MRS in combination with in-vitro 1H NMR gives a better insight about the tissue biochemistry and metabolism of breast tumors. $ : (1) NMR Biomed., 1998, 11,414-422: (2) Curr. Sci., 76, 6, 1999, 777-782).

P415 M I C R O W A V E I N D U C E D L E A K A G E O F H U M A N E R Y T H R O C Y T E S

Karina Roxana Iliescu, Maria Sajin Department of Pathology, "Carol Davila" Medical University

P.O. Box 15-205, Romania

The effects of low level microwaves (2.45 GHz) on the membrane of human erythrocytes were studied measuring the hemoglobin loss and the osmotic resistance of erythrocytes exposed to different power densities (0.025 - 10.000 mW/cm 2) at different irradiation times. A significant increase of the hemoglobin loss by exposed erythrocytes as well as a strong dependence of the rate of the increase of hemoglobin loss on the initial level of spontaneous hemolysis were observed. It was found that at low power densities (0.84 and 1.36 mW/cm2), the hemolysis degree increases qu~isi-linearily with the exposure time while at higher density (5mW/cm 2) this tendency is reversed after first 10 hours of irradiation. It appears like long-term irradiation exerts a protective effect against spontaneous hemolysis caused by blood ageing. The osmotic fragility test performed on samples exposed to 5mW/cm 2 at different i r radiat ion t imes showed that the osmot ic resistance of exposed erythrocytes increases in time, reaching maximum at the end of irradiation (60 hours) while the osmotic resistance of the controls is constant.

179


P 4 1 6 LIPID PEROXIDE FORMATION IN RAT TISSUES AFTER LOW LEVEL CONTAMINATION

WITH TRITIATEI) WATER

Nicoleta Moisoi, lleana Petcu hlstitute of Physics and Nuclear Engineering, Department of Experimental Phv,~cs, P. O. Box MG-6, R-

76900, Magurele - Bucharest, ROALLVL4

The lipid peroxides level was investigated in different tissues (liver, kidney, small intestine, spleen, bone marrow) of rats exposed to low levels of tritiated water in relation to tissue radiosensitivity, irradiation dose and dose rate domain. The radiation exposure, in the 0 - 50 cGy dose domain, with dose rates in the range of 0.01 - 2 cGy/day, was performed by internal contamination of rats with tritiated water. For the lower dose rates (<0.35 cGy/day) the peroxide levels did not increase for doses up to 10 cGy, while a dose rate of 1-1.75 cGy/day induced an increase in peroxide levels starting with 5 cGy. The increases were more significant for the tissues with higher radiosensitivity: spleen, small intestine and bone marrow. For the 4.2-7 cGy dose domain and very low dose rates, up to 0.1 cGy/day, the peroxide level has an inverse dose rate dependency. The results were discussed in relation to the possibility to induce an adaptive response in peroxide formation after a conditioning iradiation by low level tritium contamination and a challenge dose of 1Gy by fast neutrons irradiation.

P 4 1 7 THE CHEMILUMINESCENCE TECHNIC APLICATION FOR FREE-RADICAL LIPID OXIDATION

DETERMINATION IN LOW DENSITY LIPOPROTEIN A.I. Kuzmenko, R.P. Morozova, I.A. Nikolenko, G.V.Donchenko

A.V. Palladin Institute of Biochemistry, Ukrainian National Academy of Sciences, 9 Leontovich Street, 252030, Kiev, Ukraine.

Lipid free-radical oxidation (LFRO) and its regulation are the task both for the basic and applied life science. In the present study, the LFRO in low density lipoproteins was investigated on D-deficiency model in vivo by chemiluminescence (ChL). The ChL kinetic dependances were recorded by chemiluminometer PCL-01. Data were presented as mear~SD. Average values were calculated by the Student's t-test. The processes of LFRO activation in low density lipoproteins at D-deficiency occured. The chemiluminescence kinetic parameters: the maximum intensity of the first flash and inclination angle tangent of an ascending branch of the second flash grew at D-deficiency in comparison with control group (p<0.02 and p<0.05, respectively). In low density lipoproteins of the animals group maintained on the D-deficiency diet the maximum intensity of the second flash did not change, and the period between first and second flashs decreased (p<0.05). At the same time, the vitamin D3 introduction to the experimental animals diet failed result into reduction of maximum intensity of the first flash, change of maximum intensity of the second flash and increase of a period between first and second flashs. But, statistically reliable inclination angle tangent of an ascending branch of the second flash was reduced (p<0.02). Thus, with the ChL method application the intimal part of the LFRO separate stages are possible to determine.

P 4 1 8 Neurotransmitter Mediating Enzyme Dopamine 13-hydroxylase in the Serum of Patients with Arseniocosis

M.K. Rahman and M.M. Ahmed Depamnent of Biochemistry, University ofDhaka, Dhaka-1000, Bangladesh

We measured the dopamine ~-hydroxylase (DBH) activity in the serum of patients with arseniocosis and normal healthy adults of both sexes. DBH activity dicreased significantly in arsenicosis patients as compared to the normal value. In male patients the mean DBH activity was found to be 24.99 nmolesJmin/ml, whereas the activity in normal healthy individual was 33.52 nmol/min/ml. In female patients, the value was found to be 22.68 nmol/min/ml, where as in normal individual it was 31.33 nmol/min/ml.

180


P419 Geuome profiling : a useful tool for quick study of comparative biology without the need of sequencing.

Mohammed Naimuddln, Takehiro Watanabe and Koichi Nishigaki

Department of Functional Materials Science, Faculty of Engineering, Saitama University,

255 Shimo-okubo, Urawa-shi, 338-8570, Japan

Comparative biology has been a very powerful tool in the studies of the evolution of organisms. Genome

profiling (analysis of random PCR products by way of perpendicular TGGE or DGGE) allows detecting

genomic differences among microorganisms. We carried out a systematic study on the verotoxic E.coli strain,

O157 spread in the field, using this technique. The results obtained by computer-assisted processing were

consistent with those obtained by conventional sequencing of the DNAs of.those microorganisms. We will

discuss focusing on the meaning of small difference in genome profiling observed here, relating them to

'microevolution'. The advantages of this technique over various other related techniques including mass

sequencing will also be discussed.

P420 lntracellular Na + accumulation During Prolonged Ischemic Stress: A Double-Quantum

Filtered 23Na NMR Study. *Y. Rub~, **IL Gi lk~, *R. Skatmay, *IL Ammar, *G. Ure/zkF

*Department of Cardiothoracir Surgery, Carmel Medical Center & Technim~ Israel Imtttute of Technology, Bruce Rappaport F~d~y or MedkCme.

**Department of Chemistry, Technie~ Israel Institute of Technology, Haifa Israel We have studied the intracdhdar Na + ehs,]ges during prolonged ischemia and reperfusioa periods, using

the Double-Quantum Filtered 23Na NMR technique. Male Sprague Dawley rat hearts ~r162 randomized into two experimental groups (~--6). The control group was subjected to the same protocol, but without preconditioning The results indicate significantly lower intracellular Na + aectanulation in the precotutitioning group then in the control group during ischemia and repeffusion periods. This reduction was coupled with improved recovery in the preconditioned group.. In conclusion, The study suggests that ischemic preconditioning can attenuate the intracellular Na + influx levels during ischemia and reperfusion. This attenuation may explain the improved recovery, of the preconditioned hearts.

P421 SLEEP - WAKEFULNESS STUDY'IN-RAT BRAIN BY SIMULTANEOUS EEG AND fMRI RECORDINGS

M.Khubchandani*, H.N Mallick+, V.Mohan Kumar+, N.R. Jagannathan* *Departments of N.M.R and + Physiology,, All India Institute of Medical Sciences, New Delhi, India-110 029.

Local hemodynamic alterations due to changes in the neuronal activity in particular brain loci can be studied by functional Magnetic Resonance Imaging (fMRI). The sleep-wakeful state of an animal is conventionally studied by recording electroencephalography (EEG), electromyography (EMG) and electroculography (EOG). Simultaneous recordings of EEG and MRI would then reveal the precise spatiotemporal orchestration of neuronal activity with sleep-wakefulness.

A stereotaxic apparatus was fabricated using teflon and perpex. Non-magnetic electroaes of EEG, EMG, EOG were implanted in male Wistar rats which were connected to an IC socket fixed to the skull with dental cement. Non- magnetic anchoring screws fixed on to the skull to make a receptacle of dental cement.

The preoptic area (POA) is one of the neural sites which is involved in sleep -wakefulness. We have simultaneously recorded EEG and fMRI (multislice) in awake rats with one of the slices of the images passing through the preoptic area. Preliminary, results obtained indicate that such a study would help us to understand the functional state of brain areas involved during sleep -wakefulness.

181


P 4 2 2 In Vivo MR Mapping of Proteoglycans in Cartilage

Ravinder Reddv. Arijitt Borthakur, *Erik M. Shapiro, Umamabeswar Duvvuri Department of Radiology and MMRRCC, *Department of Chemistry, University of Pennsylvania, Philadelphia, PA, USA

Rapid developments in the chondroprotective therapies have generated tremendous demand for noninvasive methods for detecting degenerative changes in cartilage associated with early osteoarthritis. Changes in proteoglycan (PG) macromolecules present in extracellular matrix of cartilage, are initiating events in early cartilage degeneration. We have been developing methods based on sodium magnetic resonance imaging (MRI) that target these measurements. Here, we demonstrate that sodium MRI is capable of providing quantitative measurement of PG content and for the first time their distribution across cartilage in vivo. MRI experiments are performed on 4T GE scanner using home built radio frequency coils. Preliminary results obtained on bovine models of osteoarthritic cartilage demonstrate that sodium content directly tracks the PG in cartilage. The quadrupolar interaction of sodium in cartilage is sensitive to the macromolecular changes in cartilage matrix. In vivo experiments on human subjects demonstrate that a 3D data set can be obtained in about 10 minutes with a resolution of--600 p,m across cartilage. Comparison of proton and sodium MRI showed that sodium content directly correlates to PG content and its T1 and 1"2, are increased with depletion of PG. Whereas, no consistent changes are observed in proton content and relaxation times as a function of PG changes. In vitro and in vivo sodium MRI'also revealed that there is a large variation of PG content across cartilage. The transitional zone has the highest PG content and superficial and radial zones have the lowest. Potential of these properties in the development of noninvasive diagnostic tools for the early detection of osteoarthrtis will be outlined. Acknowledgements: This work was supported by NIH grants RR02305 and R01-AR45242

P 4 2 3 Detection Of Pathological Features Of Experimental Gastrointestinal Traet(GIT)

Carcinogenesis By Magnetic Resonance I m a g i n g M Gulnaz Begum, Mahaveer N Degaonkar*, S Govindasamy and N R Jagannathan*

Dept. of Biochemistry and Molecular Biology,University pf Madras, Chennai-600025, India. *Dept. of NMR, All India Institute of Medical Sciences, New Delhi-110029, India.

Diet plays an important role in the etiology of GIT cancers. Chronic exposfire to various dietary factors cause an insult to the mucosal architecture and threat its homenstasis. Recent studies related to the diagnosis and prognosis of gastrintestinal cancer, focus on whether screening for an earl.}, stage of cancer can be made effective. In the present study, progressive pathology of GIT in mice exposed to different dietary regimens such as the test agent (mixture of carbohydrates and organic acids and their salts; n=3), test agent+carcinogen (N-nitrosomethyi urea; n--3) and high fat diet+test agent (n---3), was monitored using magnetic resonance imaging. Following initial pilot images in three orthogonal planes, multislice spin-echo TI - and T2-weighted images were acquired in the coronal orientation to characterise the morphological changes. The control animals (n--3) showed an intact GIT, whereas distinct changes in the image characteris6cs were observed for the above different groups of animals. Regenerating cellular environment and disruption as well as diffusion of mucosal layer were dearly visible in the MR images. In addition to these general charactefistics,.mdmals induced with the test agent+carcinogen showed narrowing of the luminal wall (n=2). The animals fed with high fat diet+test agent showed keratinization of gastric wall in the distal body of the stomach and large bowel. The study thus emphasizes the potential of MRI in distinguishing the morphological changes during gastrointestinal carcinogenesis.

P 4 2 4 Dynamic Tip-Dispersion Imaging of H2170: Towards Mapping of Oxygen Consumption

Ravinder Reddy, Sridhar R. Charagundla, Umamaheswar Duvvuri, Rahim R. Rizi, Arijitt Borthakur, Ivan Dimitrov Department of Radiology and MMRRCC, University of Pennsylvania, Philadelphia, PA, USA

Non-invasive monitoring of oxidative metabolism is of utmost important for the evaluation of tissue viability in pathologies such as cerebral and myocardial ischemia, and tumors. Currently available non magnetic resonance imaging (MPd)-methods for measuring oxygen consumption have inadequacies such as low spatial resolution and use of ionizing radiation. A promising MRI

1 7 1 7 based method of monitoring metabolism uses 02 gas. O is a spin 5/2 nucleus and is non-radioactive. Inhaled/consumed 9 9 1 7 9 molecular 170 gas metabohzes into H2 O, which can be detected by 1-70 MRI. However, direct 170 MRI suffers from poor signal

to noise and as a consequence inadequate spatia! and temporal resolution. To overcome this problem, indirect detection methods have been developed 9 We have developed T~p-dispersion imaging sequence to quantitate H2170 using proton imaging. Since this method involves proton imaging, it comes with all the advantages such as high spatial and temporal resolution associated with proton imaging. As oxygen consumption measurements involve changes in H2170 of few mM, the indirect detection methods should be sensitive to these small changes. In this presentation we will describe experiments performed on animal models to demonstrate the feasibility of measuring H21~O distribution in rat brain with a precision of few mM H21~O. Preliminary results obtained on brain following inhalation of 1702 gas and its use in measuring oxidative metabolism will be discussed. Furthermore, strategies involving dynamic polarization transfer to further enhance the sensitivity of the indirect detection methods will be outlined. Acknowledgements: This work was supported by NIH grants R29-NS35625, RR02305 and a grant from Whitaker Foundation.

182


P 4 2 5 The Effect o f Environmenta l Electromagnet ic Field on N a + , K + - ATPase

F r o m the Gills of Oreochromis mossambicus

T.A. K U M O S A N I

Biochemistry Depar tment , Facul ty of Science, P.O. Box 9028, King Abdulaziz University, Jeddah - 21413, Saudi Arab ia

Abst rac t

The Na+,K+ - ATPase (adenosine triphosphalase), the ion pump enzyme located in cell membrane, is a well- defined protein whose properties can be used tb study the mechanism of electromagnetic Field (EMF) interaction with biological system. Electromagnetic Field of 60Hz, and Field strenght of 10 kV/m was applied for 2 weeks on Oreochromis mossambicus to measure its effect on the Na+, K+ -ATPase activity.

The field strength applied showed a decrease m the activity of the enzymes. A decrease which was non- reversible (no return to normal values}, to Na+, K+ - ATPase activity, when the Pre-exposed Fish were left outside the field for a period up to 4 weeks.

P 4 2 6 TREATMENT WITH DEUTERIUM DEPLETED WATER HAS A STRONG ILADIOPROTECTIVE AND

IMMUNOSTIMULANT EFFECT IN MICE W. BILD, I. STEFANESCU, G.TITESCU, 1LILIESCU, C.LUPUSORU, V. NASTASA, I. HAULICA

UNIVERSITY OF MEDICINE AND PHARMACY IASI, ROMANIA Mice fed dining 15 days with Deuterium-Depicted Water (30 ppm deuterium) had a statistically sit, nificant increased survival compared with control groups fed with normal distilled water (150 ppm deuterium) after 8.5 Cry irradiation (61% survival in test group towards 25% in control group). Hematological picture showed maintaining of the normal WBC, RBC and platelct count in test groups. Immunological paranaeters (sermn opsonic and bactericidal capacity, bactericidal capacity of the peritoneal macrophages) showed a marked increase in test groups compared to a severe decrease in the control groups. Auxiliary tests using chemical radiomimeties (hydrochloric ombihine) and immunosupressors (cyclophosphamide) showed a strong protective effect o f deuterium-depicted water against the decrease o f the leukocyte counts and other immunologic parameters. In conditions o f experimental inflammation with subcutaneous-implanted pellets, deuterium-depleted water feeding statistically significant increased inflammatory response, obviated by increased percentages of PM2q and lymphocytes in the peripheral blood and increased phagocytic capacity of the peripheral blood PMN. Experimeutal infectious with K. pneumoniae 506 and S. pneumoniae 558 in mice irradiated or treated with eyelophospham~dr showed increased non-specific immunity parameters. All results show a marked intensification of the immune defenses and increased proliferation of the peripheral blood cells, probably accounting for the radioprotoetive effects.

P 4 2 7 HUMAN MEGAKARYOCYTES AND PLATELETS CONTAIN THE ESTROGEN RECEPTOR 13 AND

ANDROGEN RECEPTOR (AR): TESTOSTERONE REGULATES AR EXPRESSION ~ g i l ] ~ g . W . g , N. Faraday, .M. Nealen, S. Noga, P. F. Bray Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA Coronary Artery Disease (CAD) is the major cause of death of women in Worldwide. Gender differences in vascular thromboses are well known and there is evidence that platelets may be involved in these differences (7hromb Haemost 77:748, 1997). We characterized the expression of the estrogen receptor a (ER r estrogen receptor 15 (ER 1~), progesterone receptor (PR) and androgen receptor (AR) in the megakaryocyte (MK) lineage. Megakaryocytes generated ez rive from normal human CD34+ stem cells, contained RNA for ER 15 and AR which increased with cell differentiation. Immunofluorescence microscopy showed ER 13 protein was present in GPIIb+ megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb+ and GPIIb- cells derived from CD34+ ceils and in HEL cells. Testosterone reduced megakaryocyte and HEL AR expression. Platelets and HEL cells contained ER 13 and AR transcripts and the corresponding platelet proteins were detected by immunofluorescence and immunoblotting. No E R a or PR mRNA or protein were detected in the megakaryocyte lineage. Our immunoblot data for both AR and ER 13 showed the presence of different size proteins in human platelets. In summary, 1) the AR is present in CD34+ and GPIIb+ cells (MKs and platelets), 2) testosterone down regulates AR expression, and 3) ER 13 expression correlates with MK differentiation and is also present in platelets. These findings indicate a regulated ability ofmegakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and tlirombotic diseases.

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H1 : BiophysicalTechnology: Medical and Environmental Biophysics

P 4 2 8 ENZYMATIC KINETICS OF B-,L-CATHEPSIN ACTIVITIES IN HUMAN BLOOD SERUM UNDER THE LUNG

DISEASES T.V.Ananicva, E.A.Lycholat

Ukrainian State Research Institute Medical and Social Problem of Physical Disability, DnicImmctrovsk 320027. ~ e

Kinetic parameters of B- and L-cathepsins were studied in blood serum of four patient groups with all lung diseases: bronchial asthma, chronic bronchitis, acute and chronic pneumonia. Donors serum was used as a control. It have been shown that generally under lung pathologies Km mean wasn't changed, and V ~ was increased by. 2 times for the B- cathepsin; in the case of L-cathepsin both values of IOn and Vmax were increased by 1.5 and 1.4 times, respectively. Investigation on pH-<lependence of the optimal cathepsin activities resulted in, as followed: i) the B-cathepsin maximal activity ~ s found out at pH 6.0 in donor's serum, as well as in patient's one; ii) pH-optimnm of L-cathepsin activity was 5.8 in normal blood; under bronchial asthma two peaks of enzymatic activity were pointed at pH 4.0 and pH 8.0; under chromc bronchitis the pH-optimum was moved into zone of acid means (pH 5.0), some samples showed two peaks at pH 5.0 and pH 7.0; under chronic pneumonia the enzymatic activity peak was observed at pH 5.0; and under acute pneumonia it was at pH 8.0. Thus, chronizalion of lung pathologies moved pH-optimum of the serum L-cathepsin activity into slightly-acid value zone,, and accumulation of them - into ,l~lim~ value zone. Obtained data would be sionifi~nt in diagnostic and treatment of lung disease pmc~____~e~_, because cathepdn B provoked m~lcing ~ from zymogen and cathepsin L was able to bind its.

P 4 2 9 Study of Stmcture-Letivity Correladoq for Bisquateraary Ammouium Antimicrobial Agent by Means

Liquid SIMS and AM1 Quantum-Chemical Claculatioa V.A.Pasbin.qkaya, M.V.Kosevich, S.G.Stepanyan

ILTP of the National Academy of Science of the Ukraine, 47, Lenin ave., Kharkov 310164, Ukraine Cationic surface active compounds, including bisquaternary ammonium salts, are widely used as antimic-

robial agents. Some experimental data evidence the existence of strong correlation between the molecular structure of bisquaternary ammonium compounds and the extent of their activity as antimicrobial drugs. At the same time there is no comprehensive theory concerning molecular mechanism of biological action of these compounds. The aim of the present work was to obtain information on structural and electronic parameters of selected antimicrobial agents decamethoxinum, aethonium, thionium, and a number of model dications and to look for structure-activity correlations. Experimental liquid secondary ion mass spectrometry investigation provided data on stability of the drugs, pathways of their thermal degradation and fragmentation in the gas phase. As a result of quantum-chemical calculations optimised geometry of the dications was obtained and general rules of charge distribution over four alkyl substituents at quaternary nitrogens were established. These rules of charge distribution together with structural parameters could be helpful-in evaluation of adjuslment of the dications to their molecular receptors in microbi-al cells or incorporation into cell membrane. Theoretical data on energy of molecular orbitals, ionisation potential and electron affinity were used in consideration of.another possible mechanism of antimicrobial activity, which involves intervention of the drugs into metabolic reactions, in particular, those in breathing chains of bacteria.

P 4 3 0 LOW-DOSE IRRADIATION INFLUENCE ON FREE-RADICAL PROCESSES IN LUNG TISSUE

E.A.Lvcholat, T.V.Anamcva. S.V.Antonyuk Ukraim'an State Research Institute Medical and Social Problem of Physical Disability,

Dnieprol~trovsk 320027, Ukraine The resptrato~, ways and lung ts known to be one of the main tissues - targets resi~ustble tar rammion pathology

development. Ionizing radiation action results in non-stability of membrane processes in mucous membrane of respiratory ways and alveolarepithelium. Ray violation is shown to be radiate endotoxisis with free-radical process intensification and radiotoxin accumulation. Oxidative-antioxidative system imbalance is one of the mnin mechanisms of pneumopathia. Purpose of the present research - investigation of free-radical processes in lung under chronic low-dose irradiation. Expenments were carried out using Wistar rat-males alter chronic (for 25 days) whole-body. X-irradiation with low dose (0.01 Gy per day at dose rate of 0,04 mC-5'/c). Homogenatcs of lung were examined by indices evaluating levels of lipid pcroxidation (LPO) and antioxidative activity. Obtained experimental data showed essential accumulation of LPO products on 1 't day after complete irradiation: malonic dialdehyde (MDA) level was i n ~ by 7(P~. On 5 th day MDA concentration was lower slightly, 10ut it exceed the control level by 29%. At the same time obvious superoxiddismutase (SOD) activation was measured after radiation exposure. However integrative antioxidant index was decre_~___u:d accordingly by. 40% and 25%. Thus, in spite of considerable tension of antioxidative system, LPO processes had been prevailing and its phenomenon underlie pneumopathia development caused by chronic low-dose irradiation exposition.

184

H1 : BiophysicalTechnology: Medical and Environmental Biophysics

P 4 3 1 Laser Therapy in the Complex Treatment of Rheumatoid and Infections Non-Specific Arthrites

R., Khachatryan, H. Arakclian, A. Kumar, S. Ayrapctyan, V. Mkhcyan, S. Agadjanyan, A. Khachatryan Medical Center of WU of Armenia 3. Kasian Sir. 375033 Yerevan/Armenia

Clinical uses low energy HcNc, Rubi and GaAs lasers in both sepalate and complimentary method of basic medicaments treatment at medium and high (2-rd and 3-rd degree) of active rheumatoid arthritis.

Laser therapy has been used for 143 patients, at age of 15-60 years, out of them 58% patients feels better, 30.5% improvement and 11.5% cases no changes observed. Further study continues in the direction of the effect of laser beam in biological tissue.

Special methodology of laser therapy is proposed of receiving a steady therapeutic effect.

P 4 3 2 ~ v i t y correlatiom of bioinsccticidcs fiom neem

~xh0anl,V.Katmleesw~-=r~ l and l~Mals~1,Geeths Gopalskrishnan 2 and T.R.OovindacAari 3. University of Madras,600 025 India and

2 SPIC Science Foumi~on, 600 032,1ndia.

Amdirachtins belong to the family of telranortritzrpenoids and show maximum sntifee&nt end ecdysisactivity while a few other neem components end,bit low but different levels of activity. These compounds have two bulky groups connected by s single bon& Oysta] structures of three s z s d / n ~ snd. a few bioective componmts like azsd/md/ones, nimbin, salmmin andtheir derivatives have been solved to correlate the sm~cture and function. The

features ncccs__,myfor the minimum activity and for enlmnc, cment have been identifioct The studies rcvml that the modified decalin moiety is necessary for the minimum activity and the relative orientation of the bulky groups determines the degree of activity.

185

H2 : BiophysicalTechnology : Biomaterials and Biosensors

P433 BIOMATERIALS FOR BONE SUBSTITUTES

Meera ~ Department of Physics, gani Dutgavati University, Jabalpw (India)

Treatment of fractures cr severe damage or disease of bone involves surgical intervention by mteoconductive fixation. The ultimate clinical success of biomaterials intended for use as bone mbetimte dep~ds largely upon its mechanical strength, structure, composition and biccompatibility.

In case of critical size defects in bones, bone grafting is done or demineralised bone powder in combination with bioactive ceramics or certain polymers mixed with bone morphogmetic wotein are introduced at the defect sites. The compositions m'e completely replaced by newly induced bone within a few weeks. In certain cases of major damage artificial implants are used for replacemmt. Considering the required strmgth md rigidity, r metals end alloys are commonly used. A coating of osteoccoductive ceramics like hydroxyapatite, enhances the bonding of implants with the bone Imd facilitat~ new bone formation.

There is a mismatch of mechanical properties of cortical bone and metallic implants. Due to mess protection, the bone structure formation is mechanically infln-ior. Several workers have tested rmcdmble polymers filled with osteoconductive ceramics, whi& provide sufficient support during healing period, permits woper healing by primary callus formation and resorbs in time. Polyhydroxyacids, poly(L-lectide), poly-mlfone, triA~n resin, polyaminoacids etc. are the main polymer matrices and hydroxyapatite, carbon fiber etc. are main fillers trader investigation. The medmnicsl and clinical testing demonstrate a good conformation to meet the requirements.

P434 Effects of Drying and Naaotopography on Biomolecule Distribution at Surfaces

Ph/lliv Lowe', Andrew Badiey b and David C. Cullen" "Cmnfieid Bioteclmology Centre, Institute of BioScience and Technology, Cranfield University,

Cmnfield, Bedfordshire MK43 0AL (Great Britain)

bUnilever Research, Colworth Laboratory, Shambmok, Bedfordshire MK44 ILQ (Great Britain)

The physical adsorption and ~ e q u e n t &'yins of biomolecules on surface is common throushout the clinical diagnostics indus . . The atomic force microscope has been used to study the molecular consequences of these procedures. It has been found that a range of biomoleculcs (monoclonal antibodies, DNA and other Wotein molecules) adsmbed as monolayers on polystyrene microtitre plates am/HOPG ma'fac~ dewet upon diyin8 rcsuhinv~ in an array of network - like distributions of biomolecules at the micrometer and sub - micrometer scales.. To further explore the possible effects of nano-topography on the molecular re-distribution of biomolecules during r highly oriented pyrolytic graphite, with a surface topography consisting of hydrophobic basal plane areas disrupted by edge plane defects, has been studied. For this mrface, de-wetting during drying of adsorbed biomolecule layem appears to be significantly influenced by the edge plane defects.including specific parameters inch as the edge Diane defect deusitv.

P435 Cyanine dye as proteia~6-sheet sensor for fluorochroming in histopathology

H. Hermel ~), W. Schmahl 2), H. M0hwald ~) l)Max-Planck-Institute of Colloids and Interfaces, D-14476 Campus Golm, Germany 2)Institute of Animal Pathology, Neuropathology, Ludwig-Maximilians-University, 1)-80539 Munich, Germany

We have found recently that in the spectra of some cyanine dyes the dye monomer band shifts bathochromically in the presence of protein-13-sheet structure, but never in the presence of ct-helices. This is a surface-effect which has opened the possibility to detect protein-[~-sheets in tissue sections. The composition of the tissue staining bath and of the following washing dips determines the colouring of the various tissue compartments. Additionally, more intensity on stain can be provoked in connection with xylene as washing and coverglass mounting medium. Especially at the f'mal low dye concentration adsorbed at the stained and washed tissue the coefficient of the shifted M-emission is close to 10t2LM'lcm'l and the quantum yield is about 0.3. These high values are the reason .for an outstanding bright appearance of the fluorescence of the J3-sheet containing tissue compartments.We have stained neural tissues of different animals and found a selective labelling of amyloid, including small deposits not observable by conventional amyloid staining in histopathology. Furthermore, it was possible to detect a very distinct affinity pattern to nuclear proteins and nuclear membranes and to the myelin wrapping around unstained axons in the central and peripheral nervous system.

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H2 : BiophysicalTechnology : Biomaterials and Biosensors

P436 B A C T E R I O R H O D O P S I N A N A L O G U E S BASED ON A Z O B E N Z E N E C O M P O U N D S

Anii K. Singh*, Nirmalya Majumdar, Joydip Das and Kartha S. Madhusudnan Department of Chemistry, Indian Institute of Technology, Bombay, Powai, Mumbai - 400 076, India

Using chromophore substitution technique, bR-analogues based on azobenzene-enal chromophores have been prepared and characterized. The azo chromophores covalently bind into the retinal pocket as indicated by competitive binding and fluorescence studies. These bR analogues exhibit altered and pH sensitive uv-vis absorption characteristics and an altered photocycle kinetics. The decay of M intermediate is slowed down with varying degree of light-induced proton pump activities.

P437 C h a n g e s o f the refract ive index o f b a c t e r i o r h o d o p s i n d u r i n g the p h o t o c y c l e

AndrOs D~r t, LorAnd Kelemen t, L~szl60roszl t, Andr/ts H~mori:, Jeremy J. Ramsden ~ and PAl Ormos t t:Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, P.O.Box 521,

H-6701 Szeged, Hungary ::Research Institute of Solid State Physics and Optics, Hung. Acad. Sci., P.O.Box 49, 1525 Budapest, Hungary

~:Dept. of Biophysical Chemistry, Biozentrum, Ch-4056 Basel, Switzerland

Non linear optical (NLO) materials change their optical properties upon external effects in general, most prominently the index of refraction upon illumination. They offer great possibilities in optically active devices, applications in optoelectronics. Bacteriorhodopsin, primarily due to the immense absorption shifts during the photocycle (> 200 nm between the K ~nd M forms), is expected to exhibit a large change in the refractive index as can be estimated from the Kramers-Kronig relation. We explored the possibility to apply bacteriorhodopsin as an active nonlinear material in combination with optical waveguides, the fundamental component of integrated optoelectronic devices. Using waveguides into which the light is coupled by grating, we determined both the static optical properties of bacteriorhodopsin as well as the changes of the refractive index during the photocycle. The waveguide with grating coupler in combination with bacteriorhodopsin provides a proof-of-concept optically controlled optical switch - the basic unit for promising complex optoelectronic applications.

P438 Replacement of porphyrin moiety in horseradish pen)xidase and activity studies of the

reconstituted enzyme D Savitri and Chanchal K Mitra, School of Life Sciences University of Hyderabad,

Hyderabad 500 046, INDIA. e-mail: [email protected]

The heine group in horseradish peroxidase (HRP) enzyme has been replaced with a manganese porphyrin and the activity of the reconstituted immobilised enzyme has been electrochemically studied. Manganese porphyrin with an amino side chain was covalently immobilised to the surface of a activated glassy carbon matrix using a long spacer arm. The characteristics of the porphyrin paste electrode were studied with substrates like hydroquinone and riboflavin. The enzyme horseradish peroxidase immobilised onto the matrix in a similar fashion was also characterised electrochemically. The berne group of HRP was removed by acid treatment and the apoenzyme was isolated. A modified enzyme was reconstituted using the manganese porphyrin immobilised on the glassy carbon matrix. The reconstituted HRP retains its activity. There is a small shift seen in the cyclic voltaramograms in the peak positions for the reconstituted HRP from that of the native enzyme. The effects of directly linking the porphyrin moiety to the electrode surface and the replacement of porphyrin in the HRP were studied to investigate the possible influence on the electron transfer characteristics of the enzyme. The detailed experimental procedure and results will be presented. Keywords: modified electrode, manganese porphyrin, enzyme reconstitution, horseradish peroxidase

187

H3 : BiophysicalTechnology : Innovative BiophysicalTechniques

P439 Single molecule mechanics and models of the myosin motor.

Toshio Yanagida123,Seiji EsakP, Yuji Kimura z, Tomoyuki Nishida 2, Yosiyuki Sowa I 1 Dept. Physiology and Biosignaling, Graduate School of Medicine, Osaka Univ., Suita Osaka Japan 2 Dept. Systems and Human Science, Graduate School of Engineering Science, Osaka Univ.Toyonaka Osaka Japan

3 Single Molecule Process Proj. ICORP, JST, Mino Osaka Japan

Recently, we have found that a single S1 molecule processively moves along an actin filament with regular steps of -5.5 nm (Nature, 397, '99). As myosin heads are known to bind two adjacent actin subunits, a myosin head may walk along actin by using these two binding sites, without detaching from the actin filament as an inchworm. The actin-myosin interface may be important for the generation of force rather than the lever arm. To examine this idea, we measured displacements caused by chimera of dictyostelium myosin HMM with and without LC binding domain and skeletal myosin LMM, which were incorporated into long rod filaments and correctly oriented The average displacement of the chimera without LC binding domain was -5.8 nm (-12 nm after correction for the stiffness) was similar to that with it. The recent results that single one-headed kinesin 0noue et al., US '99BS meeting) and KIF1 (Nakata et al., Science, '99) can processively move along a microtubule are consistent with this idea. I will discuss how a single one-headed motor can processively move during one biochemical cycle of ATP hydrolysis.

P440 The "Pore Model" Can Explain the Dynamics of Some Experimentally Recorded Parameters of Spherical

Membrane During the Electroporation Process M. Radu

Institute of Physics and Nuclear Engineering "Horia Hulubei", Department of Experimental Physics, PO Box MG-6, Bucharest-Magurele, R76900, Romania

The "pore model" describes in a satisfactory manner the electrical breakdown of the planar lipid membrane (Waver et al., Bioelectrochem. Bioenerg., 41 (1996) 135-160). We adapt this model in order to describe the electroporation of spherical membrane introducing some changes imposed by the spherical shape (the distribution of the induced potential on the membrane surface, the specific pore formation energy and the dependence of the potential, as a first approximation, only on the local value of the membrane conductivity). With these changes, we used the model to simulate the dynamics of the potential and of the local membrane conductivity distributions duringand after the electric pulse and to calculate the number of pores per cell and total aria of pores per cell. We Used in our simulations appropriate values for the model's parameters in order to compare the theoretical predictions with some experimental evidences. Our results are in a good agreement with the experimental records reported in ele~i~oporation experiments done on the sea urchin eggs (Hibino et al., Biophys. J., 64 (1993) 1789- 1800) and on the evythrocyte ghosts (Sowers et ai., FEBS Lett., 205 (1986) 179-184).

P441 Endohedral Metalofullerenes: New Molecular Paramagnetic Probes for EPR oxymetry and NMR

Tomography V.K. Koltover, Ya. I Estrin, L.A. Kasumova, V.P. Bubnov, E.E. Laukhina

Institute for Chemical Physics Research, RAS, Chernogolovka, Moscow Region, Russia

Concentration and diffusion of oxygen are critical matters in b'io-medical experiments. EPR techniques have already been suggested to measure oxygen. For example, lithium phtalocyanine crystals and fusinite demonstrate EPR signals that are highly sensitive to pO2. However, to measure intracellular oxygen concentrations, one needs a molecular probe. The encapsulating metal inside the fifllereue cages M@Cs2 0VI= La, Y, etc.) may provide for such molecular EPR rrobes. Under careful deoxygenation, the solutions of La@Crz in o-dichlorbenzene demonstrate octet EPR signals with the individual line-width value 0.14 G at room temperature. This value grows with increase in the air-oxygen pressure. Similarly, the solutions of Y@Cs2 under careful deoxygenation demonstrate doublet signals with the line-width value 0.12 G that increases with oxygen concentration. The effects of line-width broadening by oxygen are of reversible linear character that makes it possible to use La@Cs2 and Y@C82 for measurement of oxygen in microheterogeneous systems of biomedical importance. Furthermore, these new compounds hold much promise for as paramagnetic agents for in vivo imaging. [Research sponsored by the Russian Foundation for Basic Research, grant #98-03-33243a and the State Program "Current Directions in Condensed Matter Physics", subprogram "Fullerenes and Atomic Clusters", grant 98078-99010].

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P442 ELECTRICALLY STIMULATED S ~ Y METABOLITES ~ I O N THRO[K~ PLANT TISSUE

(XJLTt~E : A NOVEL APPROACH

P.A]IV DOTTA BIOLOGICAL SCIENCE GROOP, BITS, PILANI 333031 INDIA

The application of electric field through suspension cultures containing plant tissue stimulates the secondary metabolites secretion in medium without effecting tissue viability. The metabolites were subsequently isolated from the culture medium. Kallstroemia pubescens callus induced from stem was shake cultured in a liquid medium and pulsed 20 times with an electric field (max. voltage 3000V; time constant 5 MS attenuated pulse) and the amount of diosgenin and yamogenin secreted into medium were measured. They were 2o5- 6.8 mg/l and 0.8-1.6 mg/l respectively, as compared to ~ 0 secretion for that without the application of the electric field (control). The respiration activity was also analysed in electrically treated and control plant tissues.

P443 Comparative study of Gadodiamide (Gd-DTPA-BMA) and Gadopentitate dimeglumine (Gd

DTPA) contrast agents in MRI of exper imenta l a l l erg ic encepha lomye l i t i s (EAE) lesion of central nervous system.

~ , P. Ragbunathan, Rama Jayasundar and N. R. Jagannathan Department of NMR, All India Institute of Medical Sciences, New Delhi-110029, India.

Paramagnetic contrast media have been shown to be of great value in magnetic resonance imaoing 0V[R[) of central nervous system (CNS). Gd-DTPA which is a negatively charged ionic contrast agent (osmolality 1940 mosmol/kg) and Gd-DTPA-BMA, a non-ionic contrast agent (osmolality 789 mosmol/kg), are the most preferred MR contrast agents in use because these are well-tolerated, safe and gives better image contrast enhancement.

The usefulness of intravenous gadolinium containing contrast media is evaluated by the increased confidence level with which the lesion border is dearly differentiated, better discrimination between edema and lesion, etc. It has been observed that the contrast enhaned MRI in multiple sclerosis (MS) shows an increase in the number of lesion detected. However, a comparative study of the efficacy of these contrast agents in demyelinafng disease such as the EAE (model of MS) has not been studied.

We report a longitudinal comparative study of both non-ionic and ionic contrast media to evaluate the diagnostic efficacy in rat EAE model. EAE was induced using lysophosphotidyl choline (LPC) in white wistar rat brain (n=12). Various parameters such as signal enhancement, signal-to-noise ratio and contrast-to-noise ratio were calculated and compared to assess the relative efficacy of these contrast agents.

P444 A new approach to the measurement of I/near electrical parameters

of isolated cardiac myocytes using a square voltage stimulus

Pavel Nordic Milan Marko and Ivan Zahradnlk Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic

Linear electrical behaviour of cardiac myocytes can be described by an equivalent circuit, consisting of membrane capacitance C., membrane resistance R~, and series resistance R s. While there is a lot of techniques for measurement of passive cell parameters, none of them is suitable for simple, reliable and fast simultaneous recording of all three parameters during an electrophysiological experiment. We have developed a new method based on the integration of specific segments of the cell current response to a voltage step. In contrast to other methods based on a voltage step stimulus, this technique doesn't require an exponential analysis or nonlinear regression and iteration. All three parameters are calculated using three variables obtained from the cell current response: the peak current I~ the integral of the current transient I,, and the integral of the steady-state current I,. Based on this method, an analyzer was consVucted using the 8-bit microcontmller DSS000 (Dallas Semiconductor, USA), a generator of the voltage stimulus, and an analog unit for recording of I~ I, and I,. The analyzer can be used with any patch-clamp amplifier and, with the use of a built-in calibration routine, it can correct the errors arising from limited frequency bandwidth of the signal path. The values of the parameters can be read from a LCD display (DV-40400, Dema Electronic, FRG) and are also available on analog output channels.

189


P 4 4 5 Evanescent excitation fluorescence microscopy revealed that the topology of the ventral membranes of Swiss 3T3 cells adhered to glass substrate is affected by the actin cy toske le ton .

Hiroaki Hirata, Hidetake Miyata, Kazuo Ohki Physics Department, Graduate School of Science, Tohoku University, Sendal 980-8578, Japan.

Reflection interference microscopy has demonstrated that the ventral membranes of cultured cells are not flat but "bumpy", and separated from the substrate by 50-100nm except the area called focal adhesions. Since the membranes tension exists which tends to round up the cells, some mechanism should operate to maintain the separation, and hence, the bumpy topology. We observed by evanescent excitation fluorescence microscopy the ventral membranes of the Swiss 3T3 fibroblasts cultured on a glass substrate. By labeling the cell menbranes with DilCI6(3), we could observe the focal adhesions as sporadic fluorescence patterns of the ventral menbranes: 55% of the cells exhibited sporadic pattern of the contact indicating bumpy topology of the ventral membrnaes; 34% of the cells exhibited uniform contact. In both cases one end of the stress fiber seemed to coincide with the focal adhesion, the other end terminating in the cell interior. In the presence of 20mM 2,3-butanedinne monoxime which inhibits stress fiber contraction, cells could still adhered to and spread on the substrate, but the populatioin of the cells possessing the sporadic contact decreased to 24%, while population of the cells with the uniform contact increased to 59%. Thus, the contraction of the stress fibers during the establishment of cell adhesion is necessary for the formation of the bumpy topology of the ventral membranes. (Supported by a Grant from Takeda Science Foundation.)

P 4 4 6 T h r e e - d i m e n s i o n a l l y L o c a l i z e d P h o t o b l e a c h i n g

J. Balafl, P. Sengupta and S Maiti Department of Chemical Sciences, Tara Institute of Fundamental Research,

Homi Bhabha Road, Colaba, Mumbai 400 005, India.

Photobleaching with multiphoton excitation is localized in all three dimensions. Existing theories for fluorescence photobleaching recovery (FPR) are inadequate for describing this general case: they are limited to two dimensions, and diffusion during photobleaching usually is assumed to be negligible. We develop a theory for three-dimensionally localized photobleaching, where the illumination is considered to be uniform in a small spherical volume (expected to be an adequate approximation for many experimental situations). Analytical solutions are obtained for two cases: a) weak photobleaching (concentration omside the excitation region relymin~ to,staiR) and b) steady state (tiug -~ co). The steady state solution, possible only in three dimensions, suggests a straightforward experimental method that yields quantitative information on the two-photon absorption cross section 0-2, quantum efficiency of photobleaching 0b and the diffusion constant D. Relative change in fluorescence between the initial and the steady state is shown to be proportional to the quantity 0-2~b/D. Measurement of these quantities for various dyes of biological significance is currently underway.

P 4 4 7 Scanning FJectrochemical Microscopy: A Novd Technique for Imaging Transport and PermetbHity in Membranes

and Tissues M. Gonsalves, A.L. Barker, J.V. Macpherson, D. O'Hare, C.P. Winlove and P.R. Unwin

Department of Chemistry, Univers/~ of Warwick, Coventry CV4 7AL, U~ The transport of ions and molecules through membranes and tissues is a general phenomenon underpinning a number of major processes in biological systems. There are several key questions to addrem wbea investigating permeability and ,li~u~rt processes in these systems. Is the rate oftrans~n through or across the membrane or tisme uniform or spatiaily iocalised. If the transport is iocalLsed, wlam~ are the aotive sites and can the local transport rates be ~ ~ ekctrochemical m/croscopy (SEC.M) is a technique ideally suited to addreui~ t ~ . . q ~ . . I n . SE .CM, a .tip.electrode with micron or sub-micron dimensions can be positioned with mummetre 1 .eyel ~ m .three " .dh~mions ~ to the surface of a sample. The wnple, which may be a biological membrane or tmue, ts ~ m a solution containing a target species that cam be detected electrochemically, and the magnitude of the ele~ocJaemi~ response depends on the rate at which the .tarF~et molecule or ion is transported to the electrode. In particular, as the electrode is scanned over the tissue surface in the imaging mode, areas ofldgh ~ are revealed by mhanccmentain the dcctmde response. We illustrate the ~ of ~ SE.CM as a ~ ~ ..r~o .h~. ,t~mport ~ . .tlw~. _ ~ studi.'es of ~ a specialis~ tissue found in diarthrodial joints which is rugmy suSCelmble to delalitatmg aiseases men as osteoarthritis, using SECM we have found for the first time that there are variations in the penneabi~es of anion& cations and neutral molecules (e.g. oxygen) with location witlfm the tissue on a micrometre length scale. The application of SECM to other biological ph~om~m (l~niculwrly membrane transport) will also be considered.

190


P448 Absolute two-photon absorption cross-sections o f biologically relevant chromophorea

m e a s u r e d by far-f ield Z-scan P. SemlutTta* J. Balaji* R. Phillip H, S~ Banerjee #, G. Ravindra Kuma/ and S. Maia*

*Departmem of Chemical Sciences & ~Del~ment of Nuclenr and Atomic Physics Tam Instittae of Fundamemal Research, Homi Bhabha Road, Colaba, M , mhai 400 005, India

Multilloton excitation (MPE) has added new capabilities tO biological microsco~, fluorescence correlation spectroscopy and fluorescence photobleaching recovery techniques. Optimal exploitation of MPE in biology would require quantitative knowledge of the absolute two photon absorption cross section (a2) of the relevant chromophores. We obtain an analytical expression for the non-linear Uansmittance of a pulsed Gaussian beam through a specingn of arbitrary thickness, valid both in the near and in the far riel& This suggests a simple but sensitive technique for measurin~g absolnte cr2 Only the transmitted energy needs to be measured as the sample is translated along the beam (i.e. the ~ o n ) in macroscopic steps far from the focus Our set-up uses amplified pulsed lasers at 805 and 1064 nm and a pulse-amplitude controlled triggering scheme that avoids averaging errors. The set up is far less sensitive to beam focusing distortion and positioning inaccuracies compared to the prevalent near field Z-scan technique, and the ability to use long path 'length cells makes measurement of much lower values of o2 possible. The a2 of Rhodamine 6G is measured to be about 9.3 GM r measurement of other fluorescent and non-fluorescent chromophores of biological ~gnificance is underway

P449 Complex observation in electron microscopy - toward single molecular DNA sequencing.

Nagayaka K., Darter R., Sugitani S. and Murata K. National Inst. Physiol. Sci. Myndaiji-cho, (~-'~_ aki 444-8585, (JPN).

A novel observation scheme, termed the complex observation, which is able to reconsm~ wave functions of scattering waves in their intrinsic complex form, has recently been developed and applied to electron microscopy. This observation is based completely on the coherent optics and comprised of a double or triple experiment consisted of twin experiments restoring two linear terms corresponding to the real and i n ~ n a r y part of complex images and if necessary, an additional experiment to cancel the square term which is in company with either linear terr~ Manipulation of the phase of primary waves penetrating through objects without scattering is the key in this observation. The basic scheme is applied to settle the long-standing issue in electron microscopy that the point resolution and the contrast is impaired by the optical ~ s of modulated contrast transfer, which originates from lens aberrations and defocusing A computer simulation performed under an ideal experimental ~ondition illustrates a modulation-free image with a drastic improvement in the contrast as weIl as the point resolution compared with the originals utilized for the image reconstruction. Low resolution (1 nm) complex electron micrographs obtained for gold films, organic materials, membrane lm)teins, TMV have shown enormous imixovement in image quality. With the high resolution version, which is now under construction, this may open a way to determine the sequence of DNA in its single molecular level.

P450 Detection and Selection of Single Biomolecules with

Hydrodynamic Flow in Microstructure Michael GOsch', H. Blom", P. Thyberg*, Z. F61des-Papp', G. BjOrk", J .Holm", T. Heino" and RudolfRigler*

Dept. of Medical Biophysics, Karolinska Institutet', Royal Institute of Technology"& I M C " , Stockholm, Sweden

Our set-up enables us to detectand differentiate between two different types of single dye molecules (TMR-4-dU'IT and Rh-Green-5-dUTP). To select a sins molecule, the detection volmne element must be large, so that all molecules flowing in the channel are detected and also be sufficiently small to reduce the background. Therefore we used a detection volume element, defraction limited in one and outspread in the other direction (Craussian curtain).

A fast hydrodynamic flow (up to 50 mm/s), a Gaussian curtain in the microstructure and a flow switch in the microsecond-range makes it possible to select sins compounds from biological and chemical libraries of high diversity (10 l~ - l015 elements). The selected compounds can interact specifically with target molecules (receptors, enzymes) and act as agouists/antagonists to invent new drugs. Such a selection procedure is also required for a fast DNA-sequencing set-op. For sequencing, a selected bead with an attached DNA strand can be caught and held by a laser tweezer in the laminar flow. The DNA is enzymatically cleaved, the fluorescent nucleotides are released and transported to the detection volume element base by base.

191


P451 Double Frequency NMR Measurements of Amide Proton Exchange Rates with Solvent Water

Protons Using "C/'SN Labeled Proteins.

Masashi Yokochi § Fuyuhiko Inagaki*, and Masami Kusunoki* *Graduate School of Pharmaceutical Science, Hokkaido University, Sapporo, 060-0812, Japan;

*Department of Physics, School of Science and Technology, Meiji University, Kawasaki, 214-8571, Japan.

In order to reveal the dynamical property of a signal transduction protein, we have developed a new NMR method (alternative to MEXICO method) to determine a set of amide proton exchange rates with solvent water protons and the associated auto- and cross- relaxation rates, which, in principle, cancover the dynamic range of 1 kHz-0.05 Hz at any physiological pH. This method requires two sets of HSQC or HMQC data on the amide proton magnetization I(t) as a function of mixing time (t) that are taken at two separated frequencies (e.g. 500 and 600 MHz), with use of at least three kinds of ~3C/~N isotope filters and both ra~ative and nonradiative decays of inverted water magnetization, W(t). The spin dynamics between each I(t), W(t) and a spin group (an amide N,'an alpha-proton and an effective proton) was solved in the simplest &coupling scheme by adopting the model free analysis. This technique'was tested for ~3C/~N labeled human ubiquitin at 25~ and pH 7. Both the determined exchange rates and order parameters showed a good correspondence to the X-ray structure, revealing a reasonable dynamical property of the ubiquitin.

P452 FLUORESCENCE CORRELATION SPECTROSCOPY: EXTRACELLULAR AND INTRACELLULAR MOLECULAR DYNAMICS, by E.K. Matthews* & J.Pines ! , Department of Pharmacology* and Wellcome/CRC Institute i , University of Cambridge, Tennis Court Road, Cambridge CB2 1Q J, UK. Expression of fusion proteins with green fluorescent protein (GFP) is now used widely for the localisation of target proteins in living cells, including cyclins which control mitotic activity. With fluorescence correlation spectroscopy (FCS), which combines the detection of GFP-labelled molecules by high resolution confocal optics with autocorrclation analysis of the fluorescence signal, we have measured the molecular dynamics of events in an optically defined target volume of only lfL.The autocorrelation function G(t) --- 7~(l+t/x,) q) and since ~ = <N> "l at t--0, the number of fluorescent molecules <aN> in the target volume can be defined, together with the corresponding translational diffusion coefficient (Dr) obtained from the autocorrelation decay constant, Tr. In these experiments COS-7 cells were transfected with the cDNA for a fusion protein of human Bl-cyclin and MmGFP, or with MmGFP alone. Using an Ar § laser for FCS, the molecular number and diffusional properties (Dr) of GFP were first determined in free solution. Subsequently, a spatial profile of intracellular dynamics was established, monitoring the GFP or GFP-B1 cyclin fluorescence intensity signal (kHz) at discrete spatial foci defined in the z plane within the intracellular compartment. In pre-mitotic cells, Bl-cyclin accumulates p~gressively and is known to be associated with microtubules. It was therefore of importance that nocodazole (51~g/ml), which disrupts microtubules, reversibly increased the mobility (Dr) of GFP-BI cyelin within a defined intracellular target volume. Thus FCS provides an important new method for analysing the spatial and temporal dynamics of mitosis and other intracellular processes.

192

K : Biophysics in 21st Century

P453 ELECTROMAGNATIC BIOPHYSICS IN XX1 CENTURY

Yu. P. Chukova Krasnopresnenskiy Ecological Fund, Malaya Gruzinskaya St. 6 - 42 ,123242 , Moscow, Russia

Bioeffects under the electromagnetic radiation have been studied more than 100 years. Characteristic feature is disconnection of different parts of photobiology. It is so strong, that the attempts of consideration the common character of different phenomena are practicaly absent.

This situation will be changed in XX1 century, so far as the thermodynamic theory of interaction electromagnetic radiation with bioobjects was worked out. The most important and interesting result of this theory is the existance two region of the interaction, which have different laws of conversion the electromagnetic energy into Helmholz free energy. These regions are well known from the equilibrium radiation theory: W. Wien region (visible light, ultraviolet, X-rays, 3, - radiation) and Rayleigh-Jeans region (radiofreqencies). Infrared radiation is the boundary. The efficiency dependence of bioeffects on the absorbed power is quite different in these regions. In Wien region the efficiency and absorbed power have the logarithmic relationship. In Rayleigh-Jeans region this dependence is very strong. This theory has the explanation for reasons of Purkinje effect existance, logarithmic dependence of photomotion of organisms, lethal ultraviolet effect on the bacteria und Chishevskiy cycles in heliobiology. Reference. Yu. P. Chukova - Bioelectrochemistry and Bioenergetics, 1999, 5453

P454 Reliability Concept as a Trend in Biophysics

Vitaly K. Koltover Institute for Chemical Physics Research, RAS, Chemogolovka, Moscow Regiola, Russia

The new field of biophysics, in dealing with the problem of reliability, incorporates the studies on systematization and classification of failures in biosystems, the investigations of quantitative characteristics and the mechanisms of failures and renewal processes, the elucidation of possible ways of evaluating molecular failures in functional breaks. It also includes tile elaboration of methods for testing the reliability in biological systems and predicting the failures. In engineering, reliability is defined as the ability of a device to perform its function for a given time under given conditions. The same intuitive definition and idea of the theory of reliability have been used in attempted explanation of the problems of creating reliable biological systems from unreliable components *. The intention of this mini review is to present some examples of the reliability analysis of biosystems at different organizational levels - from enzymes to cells, organisms, and species and to show that this reliability-theory approach to the problems of biophysics is heuristic. In part, there are many examples in engineering when a growth of noise predicts wear-out failures of physical devices. Inasmuch as the phenomenon of growth in variability for transient processes is common for biological systems, a simple method of testing the reliability at all functional levels can be set on this basis. * V.K. Koltover. Reliability concept as a trend in biophysics of aging. J. Theor. Biol., 1997, 184, 157-163.

193

Miscellaneous

P455 Influence of 7~-hydroxycholesterol on glutathione status and nitric oxide production in macrophages: In

v/tro studies Gcctanjali Bansal, Urea Singh and NIP Bansal, Department of Biophysics, Panjab University, Chandigarh-

160014, India Glutathione slams of macrophages in the arterial wall has been suggested to play a critical role in

controlling foam cell formation during atherosclerosis. Previously it has been shown that e ~ to oxidised low density lipoproteius (Ox-LDL) leads to increased gintathione levels and decreased nitric oxide (NO) production by macropbages. Oxysterols, particularly those oxidised at C7, are presumed to be mediators of atherogenic effects of Ox-LDL. The protective action of selenium, an integral structural part of redox active selenoenzymes like glutathione peroxidases, against atherosclerosis is well documented. The present study examined the effect of 7[3-hydroxycholesterol and selenium supplementation on macropbage glutathione slams and NO production. Incubation of murine peritoneal macrophages with increasing concentrations of 7[3- hydroxycholesterol for 24 hours led to dose-dependent reduction in cellular glutathione content as well as nitrite levels in the medium. Treatment with sodiu?n selenite elevated cellular glutathione levels and nitrite levels. Our results suggest that 7[~-hydroxycholcsterol may exert its proatherogenic effect by inhibiting glutathione synthesis and nitric oxide production by macrophagcs present in the arterial wall and thus, impairing the cellular antioxidant defense system. Our results further emphasise the need of means to enhance ghtathione slams of macrophages which may attenuate the ~therosclerotic process. Further studies in this regard are in progress.

P 4 5 6 Hormone Receptors in the Receptor Database (RDB)

~ , Tastuya NAlcAno and Tsuguchika Kaminuma

Division of Chem-Bio Informatics, National Institute of Health Sciences

1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, JAPAN

We have developed the Receptor Database (RDB), using an object-oriented database

management system ACEDB (A Caenorhabditis elegans). RDB represent various aspects of

r ecep to r s effectively, for i n s t ance , p ro t e in s t r u c t u r e , func t iona l s i tes , D N A sequences , etc. The

sources i nc lude s t a n d a r d i n t e r n a t i o n a l biological d a t a b a s e such a s t h e up - to -da te d a t a b a s e of

PIR, S w i s s Pro t , PDB, G e n B a n k a n d GDB. Now we focus a t t e n t i o n to h o r m o n e recep tors a n d

the u s a g e o f RDB, espec ia l ly t h e s te ro id h o r m o n e receptors w i t h t h e app l i ca t ion to the

e n d o c r i n e d i s r u p t o r problem.

P457 LDL-receptor expression and deiodinase activity in experimental atherogenesis during selenium

supplementation in rats BPS Kang, U Smgh and MP Bansal,Department of Biophysics, Panjab Umversity, Chandigarh-160014,

India Hypercholesterolemia is associated with hypothyroidism resulting from decreased clearance of

low density lipoprotems (LDL) because of a defect m receptor mediated pathway of LDL catabolism. T3 is knogla to merease LDL-R level which has long been proved to be protective against cholesterol induced atherogenesis. This process being oxygen free radical mediated, antioxidants are being used expermentally for their preventive action against the disease process. Selemum, a potent antioxidant has recently found to be associated with type I-5'-monodeiodinase (DI), which convert 1"4 to 1"3 m peripheral tissues.

Present study was designed to see the expression of LDL-R and DI activity in three treatment groups of rats viz. Control. high fat diet fed and HFD+Sc supplemented. Expression of LDL-R was done in rivo by evaluating the 125I labelled LDL clearance rejected to rats. DI activity was measured in liver and aortae. Results of the present study indicated that LDL-R expression decreased m HFD fed group but Se supplementation led to no~malization of the expression of LDL-R. Although DI activity did not show any significant change m aortae but hepatic DI was decreased sis on HFD feeding. Further, Se supplementation led to increase m DI activity. Thus, the study depicted the alteration of LDL-R expression as well as DI activity on HFD feeding and selenium act as the regulating element for the said effect.

194

Miscellaneous

P458 Determining the orientation of coordinate system for epicardial strains report considering the direction of

the axes of torsion of the left ventricle Bo~ut Kiru, Neja Potocnik and Vito Starc

Institute of Physiology, School of Medicine, University of Ljubljana INTRODUCTION: To determine mechanical properties of passive cardiac muscle from surface kinematics, appropriate coordinate system for reporting epicardial swains is necessary. We defined left ventrienlar (LV) self coordinate system coinciding main axes of deformations: radial symmetrical extension, torsion and elipticalization, using two dimensional epicardial kinematic data. METHODS: Six isolated guinea pig hearts were arrested in diastole and a catheter was inserted through the aorta into the LV chamber for the controlled injection and withdrawal of saline in up to 10 steps of 100 B! each followed by 5 s pause. On the epicard of LV free wall four optical markers were fixed around the central one in the form of crux, which long axes was visually oriented in base to apex direction. The planar projection was used for markers tracing using specific computer program and one CCD camera. Motion of the LV as rigid body was eliminated. The simulation of epicardial kinematics was constructed using four parameters: c~, angle between self coordinate system (which origin we assumed to coincide with central marker) and visually defined base to apex direction; 1%~, 1%~,~ elongation coefficient; t coefficient of torsion. For all volume steps of LV loading or unloading all four parameters were calculated by least square method. Parameters l%~t, k ~ and t were assumed to change linearly with the LV volume, o~ was assumed volume independent. RESULTS: The orientation of LV self coordinate system varied in the range of +_200 from visually oriented base to apex direction.

P 4 5 9 In- V'D,o EPR studies on irradiated seeds: Identification of hydrated electrons and nitrogen centred radicals.

Lata I. Shukla 1, V. Natarajan, 2 T.P.A. Devasagayam, 3 M. D. Sastry, 2 P.C. Kesavan 4. ~.School of life sciences, Jawaharlal Nehru University, New Delhi, 2Radio Chemistry Division, 3Biosciences Group, BARC, Mumbai, 4M.S. Swaminathan Trust, Chenai.

The enhancement of radiobiological damage by oxygen, called ' Oxygen- Effect', is central dogma of radiobiology. This oxygen effect seems to be related to the radiation derived free radicals. Barley seeds forms on ideal test system to examine the mechanistic aspects of oxygen- effect. The barley, seeds (IB 65), (Hordeum Vulgare) ~2.4% moisture were exposed to deuteration, in- vapour form before 60 Co gamma-irradiation, (cumulative dose 750 Gy at 770 K). The EPR spectra were recorded using Bruker ESP- 300, EPR spectrometer at X- Band frequency. DPPH was used field marker. Variable temperature assembly was employed to vary. the temperature of sample in EPR cavity. TL measurements were made in 770- 3200 K.

The deuteration of moisture in seeds improved the resolution of EPR spectra of barley seeds exposed to gamma- irradiation. It led to identification of nitrogen centered radicals and hydrated electrons. The EPR signals were detected mainly from the endosperm part of seeds. A quintet with I = 1, might be arising from damaged arginine / histidine residue in protein part and/or DNA present in endosperm part of the seeds. The results suggests that at least two distinct radical species in gamma-irradiated barley seeds are possibly responsible for radiation damage.

P 4 6 0 METHOD OF ELECTRON SPIN RESONANCE IN INTERNAL MEDICINE

R. Sayfutdinov Institut of Surgery, Medical University.

Russia, Irkutslc

Objectives. The change of the oxidation-reduction processes playes significant part in pathogenesis of diseases. The mdex of intensity of them are the paramagnetic centres. They are represented by metals of variable valency and Free Radicals. Metod$. We studied with method of'Electron Spin Resonance (ESR/EPR) plasma, erythrocytes, saliva, nose secret, gastric and duodenum contents, bile, faeces, siuovial liquid, contents of the cyst received from volunteers and the patients with ischemic heart disease, iron deficiency anemia, ulcer, cholecystitis, rheumatoid arhritis and osteoarthrosis. l~esults. At investagated biological liquids are elucidated the structure of the paramagnetic centres and the role of them were detertrdned at pathogenesis of some diseases. Conclusions. The method of Electron Spin Resonance (ESR/EPR) is allowing to define more exactly pathogenesis of diseases, to differentiate them and to study methabolism of some medicine.

195

Miscellaneous

P461 A METHOD OF SEARCHING DENSITY-DEPENDENT GROWTII CONTROLLING FACTORS IN MICROBIAL POPULATIONS Adamovich V. V., Rogozin D. Yu., Degermendzhy A. G. Institute of Biophysics (Russian Academy of Sciences, Siberian Branch) The method is to evaluate the sensitivity coefficient (SC) of steady-state concentration of the factor to its variation at the chemostat input. SC determines regulation of microorganism growth by the factor under study. SC value is in the ranges from 0 to 1 corresponding to maximum and minimum regulation level. Our earlier published theorem about SC "quantization" proves that the sum of SC of all factors is equal to the difference between the number of these factors and number of species in the community under study and makes possible to define completeness of the list of factors under study. The method was first applied for continuous culture of Candida utilis yeast. Glucose concentration and pH of the medium in the acid range served as factors to measure SC coefficients in different ranges. The SC "quantization theorem" was shown to be valid for certain steady-states attesting to the completeness of the list of factors under study.

P462 Revitalization of NMR of oriented systems

C.L. Khetrapal "'~. K.V. Ramanathan s and G.A. Nagana Gowda s University of Allhabad, Allahabad-211 002, INDIA

~Sanjay Gandhi Post-Graduate Institute of Medical Sciences, Lucknow-226 014. INDIA $Indian Institute of Science, Bangalore-560 012, INDIA

NMR spectroscopy of oriented systems provides the only available method for the precise determination o! molecular geometries in the liquid state. Due to rapid increase of the spectral complexity with the increase in the number of interacting nuclei, the use of the technique has till recently been restricted to the study of rather small molecules. However, recent advances in the field such as the discovery of liquid crystals with low order parameters, the use of high magnetic fields for orienting the molecules and the possibility, of obtaining the deuterium spectra in the natural abundance of deuterium have revitalized the field. Such developments promise a bright future for this method to study more complex systems such as bio-molecules and will be discussed and the utility demonstrated with specific examples.

P463 A N O V E L A P P R O A C H IN C H R O M A T O G R A P H I C S E P A R A T I O N

Ghimire, Kedar Nath, Central Department of Chemistry, T.U. and Ishida Masaru, Tokyo Institute of Technology, Tokyo, Japan.

A new recycling technique named as "Sandwiching Recycle" is proposed based on chromafographic separation. This technique has been found to be quantitatively effective with relevance to preparative scale separation of multi-component mixture. Various interesting features behind sandwiching recycle technique disclosed its superiority over conventional methods.

196

Miscellaneous

P464 MODULATION OF pH-TENSION RELATION BY TROPOMYOSIN: INVESTIGATION USING THIN

FILAMENT-RECONSTITUTED CARDIAC MUSCLE FIBERS

H. Fujita & S. Ishiwata Department of Physics, School of Science & Engineering, Waseda University. 3-4-1

Okubo, Shinjuku-ku, Tokyo 169-8555, Japan

Effect of tropomyosin (Tin) on pH-isomelric tension relation was investigated using thin filament-reconstituted

cardiac muscle glycerinated fiber~ in the pH range of 6.0 - 7.4. Thin filaments were reconstituted from purified G-

aclin with either bovine cardiac Tm or rabbit skeletal Tm in conjunction with cardiac troponin (Tn) or skeletal Tn in

accordance with the method previously described (Fujita et al., Biophys. d., 71, 2308-18) and the pH-isometrie

tension relation was examined. Results showed that isometric tension decreased linearly with a decrease in pH and

the slope of pH-lension relation was dclcrmincd by the type of Tm isoforms regardless of the type of"In isoforms.

Furthermore, closely similar results were obtained in fibers reconstituted with aclin and either cardiac or skeletal Tm

without Tn. These rcsults demonstrate that Tm but not Tn modulates the pH dependence of active tension. P465

PARADIGM-DEPENDENT ALTERATION OF NEURAL SUBSTRATES FOR CLASSICAL EYEBLINK CONDITIONING IN CEREBELLAR LTD-DEFICIENT MICE

Y. KISHIMOTO, l S. KAWAHARA, I M. SUZUKI, z H. MORI, ~ M. MISHINA, ~ and 'School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

2School of Medicine, The Univcrsity of Tokyo, Tokyo, and CREST, JST Corporation, Saitama, Japa~ To examine correlation between synaptic plasticity (cerebellar long-term depression, LTD) with behavior (the classical eyeblink conditioning), we used cerebellar LTD-deficient mice which lack the glutamate receptor subumt 52 (GIuR82). In contrast to the molecules gene-targeted in the previous studies, the expression of GluR~2 is limited to the dendritic spines of cerebellar Pufldnje cells. TMtefore, GluR32-knockout mice could be "an animal specifically deficient m cerebeUar LTD"

The cerebellar LTD-deficient mutant mice received classical eyeblink conditioning. In the delay paradigm in which a tone (CS) overlapped temporally with a periorbital shock (US), the mutant mice exhibited a severe impairment in learning. However, in the trace paradigm in which a stimulus-free interval intervened between the CS and US, the mutant mice learned as succossfiflly as the wild-type mice. These findings indicate that cerebellar LTD is essential in the delay paradigm. but not in the trace paradigm, suggesting a paradigm-dependent alteration of neural substrates for classical conditioning.

P466 Dynamic Electrophoresis of Colloidal Particles in a Concentrated Dispersion

H. Ohshima t, A.S. Dukhin 2, V..N. Shilov 3, and P.J. Goetz 2

~Faculty of Pharmaceutical Sciences and Institute of Colloid and Interface Science, Science University of Tokyo, 12 lchigaya Funagawara-machi, Shinjuku-ku, Tokyo 162-0826, Japan 2Disperion Technology Inc., 3 Hillside Avenue, Mount Kisco, NY 10549, USA 31nstitute of Biocolloid Chemistry, Academy of Sciences of the Ukraine, Kiev, Ukraine

Electmphoretic mobility measurements of biological cells and their model colloidal particles in an dectrolyte solution are a powerful method for determining their electrical suffice properties. In the present paper we propose a general theory of dynamic electrophoresis of charged colloidal particles in concentrated dispersions in an external electric oscillating field on the basis of a cell model. Expressions are obtained for dynamic electrophoretic mobility and colloid vibration potential (or colloid vibration current) of the dispersion, the latter of which is an experimentally observed quantity, as a function of the zr potential, size, and volume fraction of the particles, the frequency of the applied field, and the electrolyte concentration. An Onsager relation between electrophoretic mobility and colloid vibration potential is also discussed.

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Miscellaneous

P 4 6 7 Luminescence Behaviour of Prodan in Reverse Micellar Envirnonments

B. SENGUPTA, J. GUHARAY and P. K. SENGUPTA Biophysics D/vision, Saha Institute of Nuclear Physics, Calcutta 700 037, India.

The hydrophobic fluorescence probe Prodan has been examined in membrane mimetic model systems

consisting of representative reverse micelles formed by Triton X-100 (neutral), AOT (anionic) and

CrAB (cationic). The steady state fluorescence emission characteristics as well as decay kinetics are

found to exhibit remarkable dependence on the nature of the surfactant. Relevent emission parameters

have been utilised to infer the microenvironmets of the fluorophore in the reverse micelles at different

water / surfactant molar ratio (w 0 ).

P 4 6 8 Physical State Necessary for Biological Self Organisation

ILK. Mishra Dept. of Biophysics, All India Institute of Medical Sciences, New Delhi-110 029, India

Biological systems when alive are best recognised as conglomorates of process-structures i.e. processes as communicating strucUa'es. Prigogine by his epochal contribution laid down the laws for structure formation in a far- from-equilibrium regime. These are applicable to much of organisation in living systems also. However as regards the physical state necessary to permit required responses to stimulus in internal millieu, as in well as external-internal context requires the following : (i) 'Life' ~ c atoms and bonds, (ii) 'Loose' stmcaa'e [i.e. rather close intramolecular and intermolecular association energies with low deformational barriers] (iii) thermodynamically open system away from equilibrium and (iv) energy ptunps. All effective pumps appear to depend on bosonic elements (infrared to visible) level photons, phonons and excitions, even plasmons have been demonsWated in ribosomes. Using Okhuma's Hamiltonian used in plasma physics the first three boson fields are shown to generate, solitary and even solitonic waves which constitute then an energy communicating network. Amongst other consequences are emission of RF and microwaves and coherent hiophotons (Popp). These constitute the necessary foundation for interacting process ensemble characterising 'Life'.

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Contributory presentations/posters, XIII International Biophysics Congress in New Delhi in 1999

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