Polyproline fold—In imparting kinetic stability to an alkaline serine endopeptidase

Biochimica et Biophysica Acta 1834 (2013) 708–716

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Polyproline fold—In imparting kinetic stability to an alkalineserine endopeptidase

Sonali B. Rohamare a, Vaishali Dixit b, Pavan Kumar Nareddy c, D. Sivaramakrishna c,Musti J. Swamy c, Sushama M. Gaikwad a,⁎a Division of Biochemical Sciences, National Chemical Laboratory, Pune-411008, Indiab Department of Biotechnology, Tilak Maharashtra Vidyapeeth, Pune, Indiac School of Chemistry, University of Hyderabad, Hyderabad 500046, India

⁎ Corresponding author at: Division of Biochemical Sciratory, Dr. HomoBhabha Road, Pune, 411008, India. Tel.: +25902648.

E-mail address: [email protected] (S.M. Gaikwa

1570-9639/$ – see front matter © 2013 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.bbapap.2012.12.007

a b s t r a c t

Article history:Received 23 July 2012Received in revised form 11 December 2012Accepted 13 December 2012Available online 24 December 2012

Keywords:Nocardiopsis sp.Serine proteasePolyproline foldConformationKinetic stabilityDifferential scanning calorimetry

Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsissp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme inpresence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functionalactivity and conformational transitions of the enzyme were studied under various denaturing conditions.Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0 M concentration, organic solventsviz. methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymo-trypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with ahigh energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzymewas observed in 1 M GdnHCl upto 8 h, in organic solvents (75% v/v) for 72 h and in presence of proteolyticenzymes. The polyproline fold remained unaltered or became more prominent under the above mentionedconditions. However, it diminished gradually during thermal denaturation above 60 °C. Thermal transitionstudies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibilityof denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPIIhelix being the global conformation of a non structural protein, an alkaline serine protease, from a microbialsource, imparting kinetic stability to the protein.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Nature has bestowed some uniqueness to all the living things inorder to survive in respective environments. Extracellular proteasesproduced by microorganisms help them by breaking complexproteins to simple peptides, a more accessible form of food for themicrobes. As these proteases often encounter harsh environmentsthey have evolved over time and exhibit remarkable stability undervarious denaturing conditions. Some of these proteases have highfree-energy kinetic barrier separating the folded and unfolded statesand hence prefer to remain in the folded state even under relativelyharsh conditions. These proteases and other proteins that are difficultto unfold are referred to as kinetically stable proteins. The stability ofa protein can be of two types: i) thermodynamic stability, which isrelated to the equilibration between a low amount of unfoldedand partially-unfolded states with the native, functional protein,and ii) kinetic stability, where the native protein and the non-

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functional proteins are separated by a high free-energy barrier. Kinet-ically stable proteins are difficult to unfold compared to thermody-namically stable proteins [1]. Kinetic stability also imparts resistanceto denaturation by SDS and proteolytic digestion to such proteins[2]. There is no evidence of any standard structural pattern responsiblefor kinetic stability. The physical basis of kinetic stability differs fromprotein to protein. Characteristics such as presence of hydrophobicresidues on the surface, presence of disulfide bonds, metal bindingsites and electrostatic interactions, etc. have been shown to increasekinetic stability. Manning and Colon have reported high content of βsheet structure in kinetically stable proteins [2]. The α lytic proteaseand Streptomyces griseous protease B are synthesized with apro-region and their folding is dependent on existence of the pro re-gions. The released mature enzymes are kinetically trapped in the na-tive state by large barriers to unfolding [3].

The present work is about a serine protease from the actinomy-cetes, Nocardiopsis sp. NCIM 5124 (NprotI). The proteases fromactinomycetes, especially non streptomycete actinomycetes, havenot received much attention compared to those from bacteria. Thereare some reports on production and preliminary characterization ofproteases from actinomycetes [4–7]. However, very few reports ondetailed structural characterization of the proteases from alkalophilic

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actinomycetes are available, like, acid resistant Protease A fromNocardiopsis alba [8]. Structural studies of the enzymes from actino-mycetes will undoubtedly improve the understanding of the factorsresponsible for their varied structural stability.

Protease I from Nocardiopsis sp. (NprotI) is an alkaline serineprotease with a molecular mass of 21 kDa, optimum temperature of60 °C and optimum pH of 10.0 for activity. The protein is rich inglycine (17%) and proline (10%) [9]. NprotI, a monomeric protein, al-though of 21 kDa, can leak through 3 kDa membrane, indicating ananomalous behavior due to a possible unusual shape of the protein.In the present report, the structural and functional transitions ofNprotI have been studied in the presence of chaotropic agents, alco-hols, proteases and at high temperature by biophysical and assaybased techniques. The CD spectrum of the native protein, structuraltransitions of the enzyme in presence of GdnHCl and a distinctisodichroic point in various denaturing conditions indicated convincingpresence of polyproline II helix (PPII) in NprotI.

In general, the polyproline II structure is encountered in proteinsonly locally where sequential proline residues are present, exceptfor collagen and related structures [10]. The PPII structure is presentlocally in RNA polymerase II C-terminal domain, the high molecularweight (HMW) subunits of wheat glutenin, PEVK segment of thegiant elastic protein titin, and in dehydrins which are stress relatedproteins in plants [11–14]. We believe this is the first report of PPIIhelix being the global conformation of a non structural protein froma microbial source. In the present study the role of PPII conformationin imparting kinetic stability to the protein has been reported.

2. Materials and methods

2.1. Materials

Guanidine hydrochloride (GdnHCl) was obtained from SigmaAldrich Ltd., USA. Trypsin, chymotrypsin and proteinase K wereobtained from SRL, India. All other reagents including buffer com-pounds and organic solvents used were of analytical grade. Solutionsprepared for spectroscopic measurements were in MilliQ water.

2.2. Production and purification of NprotI

The organism was isolated from an oil contaminated marine sitenear Mumbai harbor (India). The protocol used for the productionand purification of NprotI was as described earlier [9]. Briefly, theculture broth of Nocardiopsis sp. NCIM 5124 was obtained by fermen-tation in a medium containing 1% starch, 1% casein, 0.1% K2HPO4, 1%Na2 CO3, 0.2% glucose, pH 10.0 after incubation for 108 h at 30°Cand 200 rpm. NprotI was purified from the cell-free supernatant bytwo successive cation exchange chromatographic steps at pH 5.0and at pH 9.0 [9]. The purified enzyme was stored at pH 5.0 whereit exhibits maximum stability at 2–8 °C.

2.3. Enzyme assay

Protease activity was determined essentially by the method ofKunitz [15], as described by Laskowski [16]. It was modified asfollows: Protease activity was determined by incubating 3 μg of theenzyme in 300 μl of 1% casein (substrate) at pH 10 (300 μl, 50 mMcarbonate buffer) at 60 °C for 20 min. The reaction was stopped bythe addition of 900 μl of 5% TCA and the reactionmixture was allowedto stand for 30 min. Any precipitate formed was then removed bycentrifugation and absorbance of the supernatant was read at280 nm. One unit of protease activity is defined as the amount of en-zyme which releases 1 μmol of tyrosine per minute in the assayconditions.

2.4. Treatment of the enzyme with GdnHCl

To study the effect of GdnHCl on the function of the enzyme, sam-ples of NprotI (50 μg each) were incubated with various concentrationsof GdnHCl (0.5–6.0 M), in 20 mM sodium acetate buffer, pH 5 for15 days at 25 °C. Suitable aliquots were removed at regular time inter-vals and assayed for enzyme activity. The readings were corrected forblank samples containing respective concentration of GdnHCl withoutthe enzyme.

2.5. Treatment of the enzyme with solvents

For the assay-based studies NprotI (30 μg) was incubated in 75%(v/v) of methanol, ethanol, propanol, acetonitrile (ACN) and dimethylsulphoxide (DMSO) at pH 5 for 72 h. The enzyme solvent systemswere incubated in static condition and the solvents were miscible inthe buffer. The incubation mixtures were kept in tightly closed vialsand change in the volume was monitored throughout the period,and no changes were detected in the volumes of the samples. Aliquotswere removed at regular time intervals and assayed for the enzymeactivity.

2.6. Structural and functional studies in proteolytic environment

Trypsin, chymotrypsin, and proteinase K were used for proteolyticdigestion of NprotI at 25 °C and 37 °C in 20 mM Tris–HCl buffer atpH 8.0. NprotI and each protease were incubated at 10:1 molar ratiofor 24 h. Aliquots were removed at regular time intervals andchecked for activity. There was no interference in the assay readingsfrom the activities of the proteolytic mixture because at 60 °C(pH 10.0) where the NprotI activity was assayed, the other proteaseswere inactive, as confirmed by assaying suitable controls for the otherproteolytic enzymes.

2.7. Circular dichroism (CD) measurements

The CD spectra of the enzyme were recorded on a J-715Spectropolarimeter with a PTC343 Peltier unit (Jasco, Tokyo, Japan)at 25 °C in a quartz cuvette. Each spectrum was accumulated fromfive scans at 100 nm/min with a 1 nm slit width and a time constantof 1 s for a nominal resolution of 0.5 nm. Far UV CD spectra of theenzyme (250 μg/ml) were collected in the wavelength range of200–250 nm using a cell of path length 0.1 cm for monitoring the sec-ondary structure. All spectra were corrected for buffer contributionsand observed values were converted to mean residue ellipticity(MRE) in deg cm2dmol−1 defined as

MRE ¼ Mθλ=10dcr

where M is the molecular mass of the protein, θλ is CD in millidegree,d is the path length in cm, c is the protein concentration in mg/ml andr is the average number of amino acid residues in the protein. The rel-ative content of various secondary structure elementswas calculated byusing CD pro software (http://lamar.colostate.edu/~sreeram/CDPro/main.html). Low NRMSD values were observed for analysis withCONTINLL and SELCON.

2.8. Differential scanning calorimetry

DSC measurements were made on a MicroCal VP-DSC differentialscanning calorimeter (MicroCal LLC, Northampton, MA, USA) equippedwith two fixed cells, a reference cell and a sample cell. DSCexperiments were carried out as a function of scan rate. Sample was di-alyzed extensively against 20 mM acetate buffer of pH 5.0 before re-cording the thermograms. Buffer and protein solutions were degassedbefore loading. All the data were analyzed by using the Origin DSC

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software provided by the manufacturer. The data was fitted to anon-two state transition model. The scan-rate dependence was deter-mined using the methods described by Sanchez-Ruiz et al. [17]. Thescan-rate-dependent shift in Tm for denaturation was fitted to theequation:

Scan rate=Tm2 ¼ AR=Ea�e–Ea=RTm

The plot of ln (scan rate/Tm2) against 1/Tm yields a slope −Ea/R,where Ea is the energy of activation for denaturation, R is the gas con-stant and A is the pre-exponential factor in the Arrhenius equation.

3. Results

Structural and functional transitions of a serine protease fromNocardiopsis sp. NCIM 5124 (NprotI) were studied in presence ofchemical denaturants, organic solvents, proteolytic environment,and at different temperature values.

3.1. Evidence of polyproline II fold in NprotI

The native protein showed a classic far UV CD spectrum ofpolyproline II structure with a positive band at 230 nm and a negativeband at 212 nm (Fig. 1). Below the large negative band at 212 nm, theellipticity decreased in magnitude up to 195 nm below which it isagain negative. The cross over between two bands at 212 nm and230 nm is at about 224 nm. The maximum ellipticity of the negativeband is four times greater than the maximum ellipticity of thepositive band. All these characteristics of the CD spectrum matchedvery well with the VCUD (vacuum uv CD) of polyproline II film castfrom water [18]. NprotI contains 10% proline, 8% alanine, 5% arginine,8% glutamic acid/glutamine, 11% aspartic acid/asparagine and 11%threonine residues [9], all of which have high propensities to be in-volved in the polyproline like structure. Interestingly, the proteinalso contains 17% glycine which has no major role in PPII structure.CD pro analysis of the far UV CD spectrum of native NprotI yieldedthe values of the secondary structure elements as: α-helix—4.2%,β-sheet—41.5%, turns—22.4% and unordered—33.0% (NRMSD 0.025).However, the software does not detect the polyproline fold.

3.2. Resistance of NprotI towards GdnHCl

NprotI was not only stable but also showed 1.25 times enhancedcaseinolytic activity in presence of 1 M GdnHCl till 8 h of incubationat 25 °C (Fig. 2a). The enzyme retained about 70% activity in the

Fig. 1. Far UV CD spectrum of Nocardiopsis sp. protease I. The pH of the buffer used was5.0 and the samples were scanned at 25 °C.

Fig. 2. Effect of Gdn-HCl onNprotI. Normalized protease activity profile of NprotI incubatedin different concentrations of GdnHCl at pH 5, 25 °C, for different time intervals. Residualactivity upto a) 12 h 0 M; (■―■), 1 M; (●―●), 2 M; (♦―♦), 3 M; (― ), 4 M; (▼―▼),5 M; (▲―▲), 6 M; (►―►) and b) 24 h to 360 h; 0 M; (■―■), 1 M; (●―●), 2 M;(▲―▲), 3 M; (▼―▼), 4 M; (♦―♦), 5 M; (― ), 6 M; (►―►)c) Far UV CD spectra of NprotI(0.25 mg/ml) incubated in different concentrations of GdnHCl for 16 h;molarity of GdnHClused is indicated in each case.

vicinity of 6 M GdnHCl up to 24 h and about 30% activity even afterfifteen days (Fig. 2b). The structural basis of these transitions was in-vestigated further.

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3.2.1. GdnHCl enhances PPII contentFluorescence measurements: When fluorescence spectra of NprotI

were recorded after incubating NprotI for 24 h with various concen-trations of GdnHCl, no shift was observed in the λmax of the protein(353 nm, excitation wavelength 295 nm) (data not shown).

Circular dichroism studies: The MRE value of the positive band at230 nm remained constant and that of the negative band at 212 nmincreased when NprotI was incubated with increasing concentrationsof GdnHCl from 1.0 M to 4.0 M (Fig. 2c), indicating stability of thepolyproline fold. The presence of isoelliptic point near 222 nm inGdnHCl profile and increase in structural content with increase indenaturant concentration confirmed the presence of PPII structurein NprotI. The rearrangement in the structure, apparently seen as in-creased compactness and maintenance of the polyproline fold can bethe cause of 50% residual activity in presence of 4 M GdnHCl evenafter 15 days.

3.3. Organic solvents enhance NprotI activity

The activity of NprotI increased by about two fold; in the presenceof 90% (v/v) methanol and ethanol, when assayed after 48 h. The pro-tein remained active in other solvents as well, e.g. 75% v/v DMSO till24 h and same concentration of ACN till 48 h (Fig. 3a). The activitydecreased with time; still, total loss of activity was not observed

Fig. 3. Effect of organic solvents on NprotI. a) Normalized protease activity profile ofNprotI incubated in different solvents (75%, v/v) at pH 5.0, 25 °C upto 72 h. b) Far UVCD spectra of NprotI incubated for 24 h in different solvents (50%, v/v). −, native pro-tein; —, methanol; ⋅⋅⋅⋅, acetonitrile; −⋅−⋅−, propanol.

even after 72 h. However, at 90% (v/v) concentration both DMSOand ACN caused total loss of NProtI activity.

3.3.1. Stability of PPII fold in organic solventsThe fluorescence profile of NprotI was studied in the presence of

different organic solvents. The λmax of the intrinsic fluorescence ofthe protein experienced a 2–6 nm blue shift in the presence ofdifferent solvents (data not shown) indicating that the protein adoptsa more compact shape, resulting in a shielding of the exposed trypto-phan residues.

Incubation of NprotI with methanol, propanol, and acetonitrile(all 50%(v/v)) for 24 h stabilized the PPII fold and made it more pro-nounced (Fig. 3b), which could be correlated with the enhancedactivity.

3.4. Resistance of NprotI towards proteases

Investigations on the resistance of NprotI to GdnHCl and organicsolvents had already shown the unusual structural property of theprotein. The enzyme was checked for its susceptibility towards stan-dard proteases. Again, the activity of NprotI was enhanced (upto 2fold) in the presence of trypsin, chymotrypsin and proteinase K,when both were incubated in 10:1 ratio at 25 °C (Fig. 4a). The en-zyme was found to be stable to proteolysis at 25 °C for 24 h. Initialenhancement in the activity was observed when the reaction mixturewas incubated at 37 °C and there was decrease in activity of NprotIafter 8 h in the proteolytic mixture (Fig. 4b).

3.4.1. Interaction of proteases with PPII fold: Structural enhancementCD spectrum of the mixture of NprotI and trypsin (representing

other proteases) in 10:1 ratio showed compactness in the structurei.e. polyproline fold compared to the structure of NprotI alone(Fig. 4c).

3.5. Sensitivity of PPII fold to high temperature

The Far UV CD spectra of NprotI at different temperatures indicatedgradual decrease in the MRE at 230 nm and 200 nm at and above65 °C, indicating loss in the secondary structure (Fig. 5a). Anisodichroic point could be seen near 218 nm for the thermal unfoldingof NprotI. These points are attributed to the conformational equilibri-um between unordered and PPII conformation [19]. The sigmoidal fitsfor the plots of MRE 212 nm and MRE 230 nm versus temperatureyielded Tm values of 66.2 °C and 71.9 °C, respectively, indicatingpossible existence of different structural domains in NprotI (Fig. 5b,c).

3.5.1. Thermal unfolding of NprotI–DSC studiesThermal unfolding of NprotI was investigated by differential

scanning calorimetry at pH 5, where the protein exhibits maximumstability. A representative thermogram recorded at a scan rate of40 K h−1, corrected for buffer base line is shown in Fig. 6a. Fromthe thermogram the Tm of the thermal unfolding of NprotI was deter-mined as 76.4 °C, whereas the area under the endotherm yielded thecorresponding denaturation enthalpy (ΔH) as 90.2 kcal/mol. DSCexperiments performed at different scan rates clearly showed thatthe unfolding temperature increases significantly and linearly withincrease in the scan rate (Fig. 6b). Further, for each sample, no transi-tion was observed in the second scan clearly showing that thethermal unfolding of NprotI is irreversible. These observations clearlyindicated that the thermal unfolding of NprotI is kinetically con-trolled. Kinetic activation energy for irreversible denaturation wasderived from the scan rate dependence of the DSC transition of NprotIusing Arrhenius plot (Fig. 6c). From the slope of this plot the activa-tion energy for the thermal unfolding of NprotI was estimated to be67.8 kcal/mol.

Fig. 4. Effect of proteolytic enzymes on NprotI. Activity profile of NprotI incubated inpresence of proteases (10:1) at a) 25 °C and b) 37 °C at pH 8.0 upto 24 h. PK, protein-ase K; chy, chymotrypsin; try, trypsin. c) Far UV CD spectra of NprotI incubated in theabsence and presence of trypsin (10:1) at pH 8, 25 °C.

Fig. 5. Thermal denaturation of NprotI. a) Far UV CD spectra of NprotI (0.25 mg/ml,pH 5.0) after incubation at different temperatures between 25 and 90 °C at 5° inter-vals. Samples were incubated for 10 min at each temperature, starting with 25 °C.Numbers indicate temperature at which the spectrum was recorded. ; Sigmoidal fitsof MRE at b) 230 nm and c) 212 nm vs temperature are shown.

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4. Discussion

A very interesting feature of PPII structure is the absence of any in-tramolecular and intermolecular hydrogen bonds, because of whichthe PPII structure, as elucidated by 1H NMR spectroscopy, is nearlyidentical to an irregular backbone structure. Water or other solventmolecules might help in stabilizing the PPII structure by hydrogenbonding to its backbone [20]. PPII can be found as a dominant confor-mation of a protein even when no prolines are present in the sequence

[21–23]. The novel polyproline (PPII) fold in the structure of NprotI wasrevealed by the characteristic far UV CD spectrum of the protein. Thereports on PPII fold have been summarized in Table 1. In most of thecases, the fold has been confirmed by CD spectroscopy. Polyproline IIhelix (PPII, poly-Pro II) is formed when sequential residues adopt Φand Ψ backbone dihedral angles of roughly −75°and 150°, respective-ly. The PPII conformation has been reported to be of great significance

Fig. 6. Differential scanning calorimetric studies on NprotI. a) DSC scan of NprotI in20 mM acetate buffer, pH 5.0. b) Scan rate dependence, and c) Activation energy plotfor NprotI.

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in the unfolded states of proteins [23,24]. It has also been reported tobe involved in molecular recognition [25]. Many proteins such astitin, elastin, abductin are known to contain PPII folds involved inimparting elasticity to them. As many denatured proteins have thebias for PPII, this structure is being studied with a view of modeling

the denatured state or to understand the protein stability determinants[21].

4.1. Resistance of NprotI towards GdnHCl

Initially, we speculated that the high positive charge density (pI ofNprotI, 8.3) at pH 5 on the surface of the enzyme could repel the pos-itively charged guanidinium ions, which in turn might reduce thestructural flexibility of the enzyme leading to a compact structureand retention of the activity. However, the detection of PPII fold inNprotI led us to correlate the stability of the protein with the unusualstructure as also reported in a few other cases [14,26]. The observa-tion of unusual stability of NprotI also led us to suspect the proteinto be a kinetically stable one. The content of the PPII fold of the pro-tein or peptide increases in the presence of chaotropic agents likeurea and GdnHCl. NprotI was found to be more stable than anotherkinetically stable protein milin, which retained 70% activity in 3 MGdnHCl while NprotI retained same amount of activity in the vicinityof 6 M GdnHCl [27].

4.1.1. GdnHCl enhances PPII contentThe activity profile of NprotI in the presence of GdnHCl correlated

well with changes in its secondary structure as revealed by far UV CDspectra. The activation observed in the vicinity of 1 M GdnHCl couldbe due to the pronounced PPII fold as indicated by increased negativeellipticity. The activity of the enzyme decreased to some extent withincreasing concentrations of GdnHCl and remained constant thereaf-ter. The more compact, stable structure formed at higher concentra-tion could have had less access to the substrate. There are reports ofstabilization of proteins due to charge screening effect of Gdn+ orCl− ions at subdenaturing concentrations [28,29]. In case offerrocytochrome c, protein stiffening was the possible cause of stabi-lization of the protein through lowering of conformational entropy[30]. NprotI is unique in its ability to maintain the activity even inthe presence of 6 M GdnHCl, which denatures most proteins.

Presence of an isoelliptic point at 222 nm in the presence of differ-ent concentrations of GdnHCl suggested an equilibrium betweenunordered and PPII conformation. Similarly, an isoelliptic point wasobserved at 218 nm in PEVK peptide in the presence of urea [28].The isoelliptic point near 222 nm in the CD spectra of GdnHCl treatedNprotI and increase in the structural content with progressive in-crease in denaturant concentration strongly support the presence ofPPII structure in NprotI.

Denaturants such as urea or GdnHCl are known to increase thePPII content by hydrogen bonding, predominantly to the peptidebackbone, as seen in polyproline peptides, in PEVK segment of titin,TP1 and C-terminal domain of RNA polymerase [13,28,31]. But, innone of the cases the increase in PPII content has been correlatedwith activity of the protein. The retention of activity and stability ofthe enzyme in the presence of GdnHCl could be a combined effectof decrease in entropy of protein due to repulsion of Gdn+ ionsfrom the surface and the stability of PPII content of the protein.

4.2. Stability of PPII fold in organic solvents

The protein structure and its activity are very much reliant on thenature of solvent used as ultimately it affects the hydrogen bondingpattern (non-covalent interactions) in the protein. Also, water is anecessary component in many hydrolytic reactions, for examplewater molecule acts as a nucleophile in serine protease drivencatalysis.

NprotI was found to be more stable than another kinetically stableserine protease milin to solvent induced denaturation. Milin retained100% activity in 40%methanol compared to enhanced activity observedin case of NprotI in 90% methanol. Also, milin retained total activity inthe presence of 60% ACN while the activity of NprotI increased in the

Table 1List of proteins with polyproline II fold.

Name Source Function Tech. Used Ref.

1 High molecular weight (HMW) subunits Wheat glutenin Cereal storage protein CD [14]2 Antigenic peptide analog Foot-and-mouth disease virus (FMDV)

serotype 0CD, X-ray

3 Amyloidogenic prefibrillar intermediateof human lysozyme

Human lysozyme ROA

4 Collagen Stiff connective fibre CD, X-ray5 PEVK segment of titin Human Imparts elasticity to the muscle sarcomere CD [26]6 Abductin Bivalve shellfish “Pecten jacobaeus” Elastic function CD7 Elastin Vertebrates Elastic function CD8 PXXP sequence peptide of the p85

subunit of P13-kinaseVertebrates Interaction with Fyn SH3 domain of nebulin,

a giant modular muscle proteinCD [48]

9 Systemin Plants Oligopeptide hormone-like molecule CD [52]10 Cysteine proteinase Trypanosoma brucei Interaction with other proteins, divides two domains From sequence [55]

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presence of 75% ACN [27]. Activity enhancement was observed in fishgut trypsin in the presence of 1-propanol (6.25% v/v) and the activationeffect was amplifiedwith increasing hydrophobicity of the alcohol [32].However, for NprotI, the activation effect decreased with increasinghydrophobicity of alcohols. Rate enhancement in the presence of anorganic solvent octane was extensively studied by Klibanov and co-workers in case of α-chymotrypsin and subtilisin activity. The abilityof enzymes to remain active in organic solvents has been attributedto structural compactness attained which might result in high kineticbarrier for the protein to unfold [33].

Alcohols are known to stabilize the helical secondary structure ofproteins and disturb the tertiary structure [34–36]. Addition ofalcohols to cytochrome C, myoglobin and lysozyme were reportedto increase their helical content [37–39]. Except for Ervatamin C andVlsE, the alcohol induced β sheet structures have not been studiedin detail [34,40]. Several proteins like VlsE from Borrelia burgdorferiand cpn10 from human mitochondria have been shown to attain al-tered secondary structure in the presence of alcohols. Addition ofmethanol to VlsE, an α helical protein, initially induces superhelicalstructure followed by induction of β structure, whereas, in cpn10, aβ sheet containing protein, addition of methanol causes initialunfolding followed by induction of non-native β structure [34]. How-ever, the interpretations of conversion of one element of secondarystructure into another are based on the visual difference in the spec-tra and not on the quantitative estimation based on the softwaresavailable for calculations.

The isodichroic point observed in the far UV CD spectra at 222 nmindicated equilibration between unordered and PPII conformations.Generally, the PPII helix converts to PPI helix in the presence ofaliphatic alcohols such as methanol, propanol, etc. [41–43]. The PPIstructure is characterized by CD with a negative band at 198–200 nm, a strong positive band at 214–215 nm, and a weak negativeband at 231–232 nm [44]. Presence of such bands was not observedin NprotI in presence methanol or propanol. This shows that thePPII conformation of the enzyme is highly stable. A peptide withPPII structure, capped at both C and N termini, has been reported tobe more prone to switch to PPI structure in alcohols than theuncapped peptide [45].

4.3. Interaction of proteases with PPII fold

The enhancement in the activity of NprotI observed in the proteo-lytic mixture is unusual. The enzyme, being extracellular, must haveadapted the structure for survival in harsh conditions. Proteins fromextremophilic organisms have evolved to remain stable in harsh condi-tions like hot, acidic and proteolytic environments. Increased structuralcompactness has been adapted by many thermophilic enzymes forsurvival [46,47]. Our present results suggest that NprotI has becomestructurally more compact in order to survive in conditions that favorproteolysis.

NprotI might resist interaction with other proteases by enhancingstructural compactness as well as by maintenance of the polyprolinefold. There are many reports on the involvement of PPII fold inprotein–protein interactions [9]. The classical SH3 domain is usuallyfound in proteins that interact with other proteins and lead to forma-tion of specific protein complexes. They bind proline-rich peptides inthe other protein. The PXXP sequence peptide of the p85 subunit ofP13-kinase interacts with Fyn SH3 domain and it goes from anunstructured form to PPII-type helix upon binding. Also the PEVKmodule, with polyproline fold, of titin interacts with SH3 domain ofnebulin during IZI assembly in muscles [48,49]. M. P. Williamson inhis review on structure and function of proline rich regions in pro-teins has emphasized that the binding of the polyproline rich regionto ligands is not specific. This allows binding to a wide range ofligands e.g., salivary PRPs binds to a variety of polyphenols and othersubstrates [25]. The polyproline structure thus appears to be capableof undergoing minor conformational changes in order to interactwith a variety of ligands. Lack of intra-chain hydrogen bonds possiblymakes such conformational changes easier. As the fold does not possessinternal hydrogen bonds it tries to get stabilized by interactingwith theligands appearing in the close environment. Thus, the polyprolinestructure is capable of sensing the surrounding environment andadapts its structure accordingly.

NprotI was more prone to proteolysis at 37 °C, which could be dueto some structural changes and/or exposure of proteolytic cleavagesites. Milin was found to be more stable to proteolysis than NprotI.It was stable in presence of other proteases even at 37 °C for 24 h,which could be due to the presence of extensive glycosylation inmilin, which probably rendered the proteolytic cleaveage sites lessaccessible [50].

4.4. Sensitivity of PPII fold to temperature

Gradual loss of ordered structure at and above 60 °C indicatedmelting of PPII fold (isodichroic point 218 nm), a transition not ob-served in chemical denaturation of NprotI. Such isodichroic pointshave been seen in temperature dependent spectra of many PPIIcontaining peptides, e. g., in the consensus sequence CTD eight repeat(R8), such point was observed at 213 nm in water as the temperatureincreased from 2 °C to 60 °C [11], in aqueous solutions for poly (Lys)at 203 nm [21], poly (Glu) at 209 nm [51], and systemin, a naturaloctadecapeptide, at 209 nm [52].

CD studies indicated presence of two structural domains in NprotIwhich is also reported in other proteases, e.g., a serine protease fromN. alba contains a domain bridge and a large β hairpin structure [8].The alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43 alsoconsists of 2 domains [53]. The scan rate dependence as observed inNprotI has also been studied in kinetically stable proteins such as the li-pase and its variant from Thermomyces lanuginose [54]. The lower

715S.B. Rohamare et al. / Biochimica et Biophysica Acta 1834 (2013) 708–716

transition temperature observed in CD analysis of NprotI could be dueto the lower scan rate.

5. Conclusions

The unusual stability of NprotI towards high concentrations of de-naturing agent, organic solvents and proteolytic enzymes makes thisenzyme an interesting candidate for structural investigations. Thestability was attributed to the presence of PPII conformation observedfor the first time as a global conformation in a nonstructural proteinof microbial origin. The structure-function relationship of NprotI isinfluenced by PPII fold. The PPII fold of the enzyme is more stabletowards chemical denaturants and proteolytic enzymes than to thephysical denaturant i.e., heat, which could be attributed to the factthat PPII helix does not have any internal hydrogen bonds for stabiliza-tion. The chemical reagents might be fulfilling the need of PPII helix forhydrogen bonding while heat could be disrupting the other stabilizingnon-covalent interactions. Since the enzyme is kinetically stable, it isimportant to investigate the folding and unfolding rate constants andhalf life of the enzyme. The exact implication of presence of PPII helixin NprotI, apart from providing kinetic stability, is yet to be understoodand requires further investigations.

Acknowledgements

The authors are thankful to Dr. M. Fernandes, Organic ChemistryDivision, NCL, for allowing the use of CD spectropolarimeter. Authorsare also thankful to Ms. Ashwini Chaudhary, TMV, for her timely help.SR, PKN and DS were supported by Senior Research Fellowships fromthe CSIR, New Delhi, India. The DSC equipment used in this study wassupported by a UPE grant from the UGC (India) to the University ofHyderabad.

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