Applications of Synchrotron Radiation in Biology and Biotechnology Zehra Sayers Sabanci University, Turkey Chair, SESAME Scientific Committee UPHUK III Bodrum, Turkey Sept. 17-19, 2007
Applications of Synchrotron Radiation in Biology and Biotechnology
Zehra SayersSabanci University, Turkey
Chair, SESAME Scientific Committee
UPHUK IIIBodrum, TurkeySept. 17-19, 2007
SYNCHROTRON RADIATION (SR)
H. Winick
Acceleration of charged particles results in emission of electromagnetic radiation.
Initially thought as nuisance because of energy loss from accelerated particles.Importance recognized by early ’60s.
At low electron velocity (non-relativistic case) radiation is emitted in a non-directional pattern.
When the electron velocity approaches the velocity of light radiation is emitted in the direction of motion and the radiated power goes up dramatically.
SR: Production
High flux and brightnessPulsed time structure Tunability
Polarized (linear, elliptical, circular)
Small source size
Partial coherence
High stability
Flux = # of photons in given /sec, mrad
Brightness = # of photons in given /
sec, mrad , mrad , mm2
(a measure of concentration of the radiation)
SR: Basic properties
Continuous spectrum characterized by c = critical energy
c(keV) = 0.665 B(T)E2(GeV)
eg: for B = 2T E = 3GeV c = 12keV
(bending magnet fields are usually lower ~ 1 – 1.5T)
Quasi-monochromatic spectrum with peaks at lower energy than a wiggler
1 (keV) =
K = where is the angle in each pole
1 = u
(1 + ) ~ (fundamental)K
2
U
+ harmonics at higher energy
0.95 E2 (GeV)K u
(cm) (1 + )2
undulator - coherent interference
wiggler - incoherent superposition
bending magnet - a “sweeping searchlight”
SR:Storage rings, bending magnets and insertion devices
klystrons generate high power radiowaves to sustain electron acceleration, replenishing energy lost to synchrotron radiation
electron gun produces electrons (at e.g. 80 keV)
linear accelerator/booster accelerate e- which are transported to storage ring (at e.g. 7 GeV)
the storage ring circulates electrons and where they are bent - synchrotron radiation is produced
beam lines transport radiation into “hutches” where instrumentation is available for experiments
special “wiggler” insertion devices used to generate x-rays
SR: Practical Production and Delivery to Users
SR: Biological and Biotechnological Applications
“Biologists” are involved in 4 types of experiments at SR sources:
Macromolecular Crystallography.
Spectroscopy.
X-ray Diffraction and Scattering from non-crystalline systems.
Imaging.
WHAT ARE THE ADVANTAGES OF USING SR TECHNIQUES IN BIOLOGY?
MACROMOLECULES OF LIVING SYSTEMS
• Special architecture at molecular structure level;
Nucleic acids (DNA, RNA), Proteins, Lipids, Carbohydrates.
• Examples:
DNA Proteins
• Hierarchical Organizational at larger scale:
Static and dynamic structures.
Cytoskeletal dynamicsChromatin fibre dynamics
FUNCTIONAL ORGANIZATION
• Examples:
SCHEME FOR FUNCTIONAL STUDIES
Structural BiologyExperimental MethodsModelling
BioinformaicsConservation analysisCluster analysis
Molecular biologySite directed mutagenesis
Activity measurements Enzyme kineticsLigand interactinsActivity under perturbation
Test structural modelsMake functional predictions
Test functional predictionsMake structural predictions
STRUCTURE AND FUNCTION RELATIONSHIP
• Experiments: Static and Dynamic
measurements of structural parameters.
• Calculations: Prediction of structure, structural change where and how.
SR offers a wide selection of powerful experimental tools for determination of structural parameters.
Time resolved data for establishment of correlation between structural change and function.
MACROMOLECULAR CRYSTALLOGRAPHY
Determination of structure of macromolecules at atomic resolution.
Applications include:
Therapeutic drug design Enzyme mechanisms Supramolecular structure Molecular recognition Nucleic acids Structural genomics High-throughput crystallography
SR sources; high intensity, small beam size, and collimation.
The MAD (multi-wavelength anomalous ddiffraction) phasing method readily applicable with tunable radiation at SR sources,
MACROMOLECULAR CRYSTALLOGRAPHY
SR offers possibility of usingMicrocrystalsLarge unit cell crystals
Cryo-crystallographyMinimizing radiation damageImprovement of data quality
Automated crystal mounting robot
Crogenic robotic crystal transfer system
FedEx Crystallography!!!
SSRL, SAM
MACROMOLECULAR CRYSTALLOGRAPHY: Highlights
Nobel Prize 2003
Mechanism for the voltage dependent K-ion channel.
Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait, and R. MacKinnon, “X-ray structure of a voltage-dependent K+ channel,” Nature 423, 33 (2003).
Nobel Prize 2007
Mechanism for RNA polymerase II.
Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, P. Cramer, D.A. Bushnell, J. Fu, A.L. Gnatt, B. Maier-Davis, N.E. Thompson, R.R. Burgess, A.M. Edwards, P.R. David, and R.D. Kornberg, “Architecture of RNA polymerase II and implications for the transcription mechanism,” Science 288, 640 (2000).
SPECTROSCOPY
X-ray absorption spectroscopy EXAFS; atomic arrangements, bond distance, coordination no., symmtery,
XANES: valence,
Magnetic circular dichroism: spin-orbit magnetic moments
X-ray fluorescence spectroscopy Quantitative analysis of elemental distribution
Far and near Infra-red, VUV spectroscopy
Vibrational spectroscopy
Hard X-ray spectroscopy:Extended x-ray absorption fine structure (EXAFS) spectroscopy, X-ray absorption spectroscopy (XAS), Near-edge x-ray absorption fine structure (NEXAFS) spectroscopy, X-ray absorption near-edge structure (XANES) spectroscopy, X-ray magnetic circular dichroism (XMCD)
Investigations of geometric and electronic structure.
Sensitive to element, oxidation state and symmetry of the molecules.
Tunability of SR is essential.
Investigation of silent Zn in Metalloenzymes
Zn K-edge EXAFS as a function of time.O. Kleinfeld, A. Frenkel, J.M.L. Martin, and I. Sagi, “Active site electronic structure and dynamics during metalloenzyme catalysis,” Nat. Struct. Biol. 10, 98 (2003).
Investigation of elemetal composition of cancerous lung tissue can be compared with that of healthy tissue by X-ray fluorescence mapping measurements. An optical micrograph of lung tissue is shown together with specific maps showing Fe, Cu and Zn distributions in the boxed area of the tissue.SSRL
Imaging and Spectroscopy
X-RAY SCATTERING AND DIFFRACTION FROM NONCRYSTALLINE SYSTEMSLow resolution data on the size and shape of the molecule can be obtained.
Time-resolved data in response to a perturbation on the system.
Protein solutions, fibers. Biomaterials: membranes, lipid micelles.
Measurements can be made at small (SAXS) and/or wide angles (WAXS) depending on the system.
Complementary data to crystallography, electron microscopy and spectroscopic measurements.
Applications include:Protein (DNA)-ligand interactions.Drug delivery.Material characterization.Time-resolved changes instructure.
X-RAY SCATTERING AND DIFFRACTION FROM NONCRYSTALLINE SYSTEMS
Examples:
Bacterial crystals
Rat tail tendon
IMAGING
Absorption contrast imagingPhase contrast imagingFluorescence ImagingFull field imagingDiffraction enhanced imagingTopographyTomography
X-RAY THERAPY
Targeted and dose-controlled therapy.
CLOSER LOOK SMALL ANGLE X-RAY SCATTERING (SAXS) FROM PROTEIN SOLUTIONS
SMALL ANGLE SOLUTION X-RAY SCATTERING
• Small angle X-ray scattering results from inhomogeneities in the electron density in a solution due to macromolecules dispersed in the uniform electron density of the solvent (0).
A solution of macromolecules
Solute: protein, DNA, polymer (p)
Solvent (0)
• Scattering pattern is determined by the excess electron density of the solute, (r)
(r) = (p-0)c(r) + s(r) = av c (r) + s (r) (1)
Where p = the average electron density of the particle.
av = the average electron density of the particle above the level of the solvent (contrast).
c (r) = dimensionless function describing the volume of the solute (with the value 1 inside the particle and 0 elsewhere).
s (r) = fluctuations of the electron density above and below the mean value (independent of the contrast).
•In an ideal solution all particles are identical and randomly positioned and oriented in the solvent.
•Scattering pattern contains information about the spherically averaged structure of the solute described by a distance probability function p(r)
•p(r) is the spherically averaged autocorrelation function of (r) and r2p(r) is the probability of finding a point inside the particle at a distance between r and r+dr from any other point inside the particle
Dmax
• For a globular particle p(r) has two main regions
a. A region of sharp fluctuations due to neighbouring atom pairs (0.1 nmr 0.5 nm) and of damped oscillations due to structural domains
(i.e -helices in proteins)
b. A smooth region corresponding to intramolecular vectors.
• Beyond Dmax p(r) vanishes
• The scattering curve also contains two regions:
a. Small angle region; information on the long range organization (shape) of the particle
b. Large (wide) angle region; internal structure of the particle (deviations from p)
Large distances only contribute at low angles.
Short distances contribute over a large angular range and at high angles their contribution dominates the scattering pattern.
SCATTERING PATTERN AND THE DISTANCE DIFSTRIBUTION FUNCTION p(r)
Scattering intensity and the distance distribution function are related by a Henkel transformation.
APPLICATIONS
• Determination of radius gyration, radius gyration of the cross section, molecular weight.
• Shape determination; at low angle (2-3 nm) the scattering curve is dominated by the shape of the particle.
• Time-resolved measurements for determination of structural changes during interactions or upon a perturbation on the system.
• Modern methods allow domain structure analysis, possibility of modeling loop domains, analysis of non-equilibrium systems (Svergun and Koch 2002, Current Opinion in Structural Biology, 12:654-660).
METALLOTHIONEINS
6-8 kDa proteins that bind metals in a wide range of organisms.
High cysteine (cys) content (up to 30%) in the amino acid sequence and bind metals through the thiol groups of cys residues.
Metal composition depends on the source and previous exposure to metals. Human liver MT contains mainly Zn, that isolated from kidneys contain Cd and Zn or Cu. In higher organisms MTs represent the only protein that is a natural Cd ligand.
Precise physiological functions are not yet identified; MTs are involved in transport and storage of essential metal ions (Cu and Zn) and detoxification (Cd and Hg).
Durum wheat MT is expressed and synthesized at high levels during exposure Cd.
MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEVAAGQSGEGQFEVAAGQSGEGCSCGDNCKCNPCNCN-terminalN-terminal
DomainDomain-domain-domain
C-terminalC-terminalDomainDomain
-domain-domain
HingeHingeregionregion
C-X-C (or C-X-X-C) are recurring motifs in the amino acid sequence. C: cystein
“Cystein motifs” (cys-motifs) are involved in metal binding.Metal-binding domains are connected by a 42 residue hinge region.Prepare recombinant proteins GSTdMT and dMT.
durum WHEAT METALLOTHIONEIN
Balcali wheat can tolerate higher levels of Cd in soil than C-1252.Bacteria expressing recombinant dMT can tolerate high levels Cd in growth medium.
Amio acid sequence:
Cys-motifs are clustered in the N- and C-termini of the protein forming the metal-binding domains (- and -domains).
Bilecen et al., 2005
The predicted 3D structure of dMT. Cadmium (blue spheres)-binding metal centers at each pole of the dumbbell-shaped molecule are depicted in ball and stick representation with the extended hinge region highlighted in ribbon representation.
MODELING THE STRUCTURE of dMT
Size-exclusion chromatography
Charge transfer band between 250 and 260 nm due to Cd-thiol interactions
UV Absorbance MeasurementsDynamic Light Scattering
(DLS)Measurements
GSTdMT elutes as dimer
SDS-PAGE Analysis Native-PAGE Analysis
PREPARATION AND CHARACTERIZATION of GSTdMT
Size-exclusion chromatography
SDS-PAGE Analysis Native-PAGE Analysis
UV Absorbance Measurements Dynamic Light Scattering (DLS)Measurements
PREPARATION AND CHARACTERIZATION of dMT
EXPERIMENTAL SET-UP FOR SAXS MEASUREMENTS
THE PRINCIPLE OF A SMALL ANGLE X-RAY SOLUTION SCATTERING EXPERIMENT
• The optical system selects X-rays with a wavelength of 0.15 nm and a narrow band-width
• The beam is focused on a position sensitive detector with an adequate cross section at the sample position
• The incident beam intensity I0 is monitored.
• IT is the intensity of the beam transmitted through the sample and IT = I0 exp(-µt), where the factor (-µt) represents the absorbance of a solution of thickness t
• I(s) is the scattered intensity which depends on the scattering vector s defined as
s = 2Sin/λ
where 2 is the scattering angle and λ is the wavelength
X33 camera of EMBL Hamburg Outstation on DORIS STORAGE ring of DESY, Hamburg.Data are collected and reduced using standard softwareReference measurements are made on solutions of bovine serum albumin.
BASIC SAXS DATA REDUCTION
Structural models can be calculated ab initio using software such asGASBOR, SASHA etc and rigid body modelling using MASSA, ASSA etc (EMBL-Hamburg)
METALLOTHIONEINS
6-8 kDa proteins that bind metals in a wide range of organisms.
High cysteine (cys) content (up to 30%) in the amino acid sequence and bind metals through the thiol groups of cys residues.
Metal composition depends on the source and previous exposure to metals. Human liver MT contains mainly Zn, that isolated from kidneys contain Cd and Zn or Cu. In higher organisms MTs represent the only protein that is a natural Cd ligand.
Precise physiological functions are not yet identified; MTs are involved in transport and storage of essential metal ions (Cu and Zn) and detoxification (Cd and Hg).
Durum wheat MT is expressed and synthesized at high levels during exposure Cd.
MSCNCGSGCSCGSDCKCGKMYPDLTEQGSAAAQVAAVVVLGVAPENKAKMYPDLTEQGSAAAQVAAVVVLGVAPENKAGQFEVAAGQSGEGQFEVAAGQSGEGCSCGDNCKCNPCNCN-terminalN-terminal
DomainDomain-domain-domain
C-terminalC-terminalDomainDomain
-domain-domain
HingeHingeregionregion
C-X-C (or C-X-X-C) are recurring motifs in the amino acid sequence. C: cystein
“Cystein motifs” (cys-motifs) are involved in metal binding.Metal-binding domains are connected by a 42 residue hinge region.Prepare recombinant proteins GSTdMT and dMT.
durum WHEAT METALLOTHIONEIN
Balcali wheat can tolerate higher levels of Cd in soil than C-1252.Bacteria expressing recombinant dMT can tolerate high levels Cd in growth medium.
Amio acid sequence:
Cys-motifs are clustered in the N- and C-termini of the protein forming the metal-binding domains (- and -domains).
Bilecen et al., 2005
The predicted 3D structure of dMT. Cadmium (blue spheres)-binding metal centers at each pole of the dumbbell-shaped molecule are depicted in ball and stick representation with the extended hinge region highlighted in ribbon representation.
MODELING THE STRUCTURE of dMT
Size-exclusion chromatography
Charge transfer band between 250 and 260 nm due to Cd-thiol interactions
UV Absorbance MeasurementsDynamic Light Scattering
(DLS)Measurements
GSTdMT elutes as dimer
SDS-PAGE Analysis Native-PAGE Analysis
PREPARATION AND CHARACTERIZATION of GSTdMT
Size-exclusion chromatography
SDS-PAGE Analysis Native-PAGE Analysis
UV Absorbance Measurements Dynamic Light Scattering (DLS)Measurements
PREPARATION AND CHARACTERIZATION of dMT
s (nm-1)
0.5 1.0 1.5 2.0 2.5 3.0
log
(I)
-3
-2
-1
0
1
2
3
GSTdMT
s2 (nm-2)
0.06 0.08 0.10 0.12
ln (
I)
5.1
5.2
5.3
5.4
5.5
5.6
Guinier plot of GSTdMTLinear Fit
s2 (nm-2)
0.06 0.08 0.10 0.12-0.03
-0.02
-0.01
0.00
0.01
0.02
0.03
GSTdMT exists as a dimer in solution.
The monomer has an extended structure.
SAXS DATA from GSTdMT
Data collected from a1.5 mg/ml GSTdMT solution at X33 camera on DORIS storage ring. EMBL Hamburg Outstation.
GST molecules are located in the center of the dimer and dMT molecules extend from the center.
Low-resolution GSTdMT structural model (GASBOR)
ab initio SHAPE DETERMINATION of GSTdMT
s (nm-1)
0.5 1.0 1.5 2.0 2.5 3.0
Log
(I)
-0.5
0.0
0.5
1.0
1.5
2.0
dMT
X Data
ln (
I)
3.2
3.4
3.6
3.8
4.0
4.2
4.4Guinier plot of dMTlinear fit
s2 (nm-2)
0.20 0.25 0.30 0.35 0.40
-0.3
-0.2
-0.1
0.0
0.1
0.2
SAXS DATA from dMT
1.0 mg/ml dMT solution.
Experiments are possible only on SR source.
dMT exists as a dimer in solution with an extended structure.
Asymmetry in the structure of dMT?Implications for Cd-binding?Domain folding?Functional implications.
ab initio SHAPE DETERMINATION of dMT
FUTURE OUTLOOKMacromolecular crystallography
High throughput crystal structure determination.Automated remote screening and data collection.Time-resolved crystallography.Crystallography and SAXS.
X-ray Scattering
Cryo-SAXS.Time-resolved SAXS.High-resolution micro-beam SAXS.Combination with SRCD.
SpectroscopyInfrared microspectroscopy.EXAFS and imaging.
ImagingImaging and spectroscopy.3D tomography.Imaging single particles…..
Useful information can be found at:
1. SSRL website: www-ssrl.slac.stanford.edu
2. www.lightsources.org
Sabanci UniversityF.DedeG. DinlerF. KisaayakU. SezermanH. BudakO.GokceI. Cakmak
EMBL HamburgM.H.J. KochD. SvergunM. RoessleA. RoundM. V. Petoukhov
SESAMEZ. HussainS. HasnainG. VignolaH. Winick
ACKNOWLEDEGEMENTS