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Biochimie 95 (2013) 912e921

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Biochimie

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Research paper

Protein L-isoaspartyl-O-methyltransferase of Vibrio cholerae:Interaction with cofactors and effect of osmolytes on unfolding

Tanaya Chatterjee a,*,1, Aritrika Pal a,1, Devlina Chakravarty a, Sucharita Dey b,Rudra P. Saha a, Pinak Chakrabarti a,b,*aDepartment of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, IndiabBioinformatics Centre, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India

a r t i c l e i n f o

Article history:Received 30 July 2012Accepted 13 December 2012Available online 26 December 2012

Keywords:MethyltransferaseFolding intermediateOsmolytesProtein modeling and simulation

Abbreviations: AdoMet, S-adenosyl methionine;cysteine; bis-ANS, 4,40-dianilino-1,10-binaphthyl-5,50-dichroism; DLS, dynamic light scattering; FRET, flutransfer; GdnHCl, guanidium hydrochloride; ITC, isotPIMT, L-isoaspartyl-O-methyltransferase; TMAO, trime* Corresponding authors. Tel.: þ91 33 2569 3253; f

E-mail addresses: tanaya_chatterjee@yahoo.comboseinst.ernet.in (P. Chakrabarti).

1 Joint first authors.

0300-9084/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.biochi.2012.12.013

a b s t r a c t

Protein L-isoaspartyl-O-methyltransferase (PIMT) is an ubiquitous enzyme widely distributed in cells andplays a role in the repair of deamidated and isomerized proteins. In this study, we show that this enzymeis present in cytosolic extract of Vibrio cholerae, an enteric pathogenic Gram-negative bacterium and isenzymatically active. Additionally, we focus on the detailed biophysical characterization of therecombinant PIMT from V. cholerae to gain insight into its structure, stability and the cofactor binding.The equilibrium denaturation of PIMT has been studied using tryptophan fluorescence and CD spec-troscopy. The far- and near-UV CD, as well as fluorescence experiments reveal the presence of a non-native intermediate in the folding pathway. Binding of the hydrophobic fluorescent probe, bis-ANS, tothe intermediate occurs with high affinity because of the exposure of the hydrophobic clusters duringthe unfolding process. The existence of the probable intermediate has also been confirmed from limitedtryptic digestion and DLS experiments. The protein shows higher binding affinity for AdoHcy, incomparison to AdoMet, and the binding increases the midpoint of thermal unfolding by 6 and 5 �C,respectively. Modeling and molecular dynamics simulations also support the higher stability of theprotein in presence of AdoHcy.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Accumulation of mutated, less active and toxic biomolecules,such as DNA, RNA, proteins, lipids, as a result of side reactions ofnormal metabolic processes leads to the loss of cellular function [1].Among these, proteins are subjected to a variety of spontaneousdegradation processes, including oxidation, glycation, deamidation,isomerization and racemization. To rectify these modifications,organisms have developed enzymatic repair pathways, someidentified and some yet to be identified.

It iswell reported in literature that proteins containing L-aspartyland D-aspartyl residues are more susceptible to racemization and

AdoHcy, S-adenosyl homo-disulfonic acid; CD, circularorescence resonance energyhermal titration calorimetry;thylamine N-oxide.ax: þ91 33 2355 3886.

(T. Chatterjee), pinak@

son SAS. All rights reserved.

isomerization reactions and can have altered structures therebycausing the most common types of aging-related protein damage[2,3]. The reaction proceeds through the formation of a transientcyclic succinimide intermediate which spontaneously hydrolyzesto either the L-aspartyl residue or to the abnormal L-isoaspartylresidue [4]. Protein L-isoaspartyl-O-methyltransferase (PIMT) issuch a repair enzyme that recognizes altered aspartyl residues andeventually converts them to normal L-aspartyl residues [5]. Itactually catalyzes the transfer of the methyl group from S-adeno-sylmethionine (AdoMet) to the isoaspartyl residues in the proteins,resulting in the production of an isoaspartyl methyl ester andS-adenosyl homocysteine (AdoHcy) as a by-product [1].

PIMT is important in the repair actions against diseases likehomocystinuria and uremia [1]. In homocystinuria, the level ofAdoHcy in the plasma increases, inhibiting the enzyme function,while in the case of uremia, AdoHcy level in the erythrocyteincreases leading to the inhibition [6,7]. Moreover, in humanreduced expression of PIMT and increased accumulation of thealtered form of tubulin can cause epilepsy [8]. Substrates of PIMTinclude a wide range of proteins starting from tubulin, synapsin I,ovalbumin, calmodulin, lysozyme, as well as amyloid b-protein,which is known to be responsible for Alzheimer’s disease [9,10].

T. Chatterjee et al. / Biochimie 95 (2013) 912e921 913

Literature studies on PIMT have revealed its existence in bothdomains of life, prokaryotes as well as eukaryotes [11e14]. Reducedsurvival in PIMT-knockout mice (beyond 6 weeks) [15] and Escher-ichia coli (in the stationary phase) [16], and the involvement of PIMTin the long-term survival of Caenorhabditis elegans [17] emphasizethe biological importance of this protein. While extracts of someenteric bacteria have been tested for methyltransferase activity,there is no study on PIMT from Vibrio cholerae [14]. There isa sequence similarity of 67% between PIMT from V. cholerae andhuman (Fig. S1), which is reflective of the house keeping role of theenzyme in damaged protein repair. As V. cholerae has an extraor-dinary capability to adapt to different environments and is known tosurvive under stressed conditions that may influence the rates ofmany spontaneous processes, including the modifications of L-aspartyl and L-asparaginyl residues, it would be interesting tounderstand the role of the enzyme in this species.

Here we focus on structural aspects of PIMT from V. cholerae(swissprot entry C3LS22), using several biophysical techniques, likefluorescence and circular dichroism (CD) spectroscopy, isothermaltitration calorimetry (ITC), dynamic light scattering (DLS) etc. Thethermodynamics of unfolding of PIMT using guanidium hydro-chloride (GdnHCl) as the chaotropic agentwas studied using steady-state fluorescence and far-UV circular dichroism spectroscopy. Theprotein showed a three-state unfolding pathway. The intermediatestate was characterized using near-UV CD spectroscopy, as well asits binding to 4, 40-dianilino-1,10-binaphthyl-5,50-disulfonic acid(bis-ANS), a hydrophobic fluorescent probe, using fluorescencespectroscopy. The intermediate formed had a tendency to aggregatewith time which was experimentally established using dynamiclight scattering studies. Fluorescence resonance energy transfer(FRET) provides further evidence for the hydrophobic binding site ofPIMT. The secondary structural content of the protein was deter-mined using far-UV CD spectral data. Quenching of the tryptophanresidues (Trp110, Trp132 and Trp135) by acrylamide and KI revealedthe exposure of these residues to the solvent in the native state.

Binding of the substrate and release of the product are twoimportant processes in the enzyme activity pathway. In the presentstudy AdoMet being the substrate and AdoHcy being the by-product of PIMT’s catalytic pathway, their binding to PIMT hasbeen studied using ITC and CD spectroscopy and thermal unfolding.The thermodynamic parameters obtained from the ITC datarevealed that the binding of PIMT to both is enthalpy driven.

Osmolytes are low molecular weight small molecules oftenreferred to as “chemical chaperons”, which are found inside the cellsand protect the cellular proteins from environmental harsh condi-tions such as high temperature, high salt, or high urea [18]. In thedenatured state the peptide backbone is more exposed to theosmolyte solution and an increase in the free energy due to osmo-phobic effect shifts the equilibrium from the denatured to the nativestate [19e21]. There are three classes of osmolytes known, namelyamino acids and their derivatives, polyhydric alcohols, andmethylamines [22]. The first two are known as compatible osmo-lytes and the last one is known as counteracting osmolyte in contextto the perturbations they cause to the protein under physiologicalconditions [23]. In this paper we have studied the unfolding of PIMTin presence of glycerol (compatible) and TMAO (counteracting) tofind out if the osmolytes can reverse the unfolding equilibrium ofPIMT toward folding under stressed condition.

2. Materials and methods

2.1. Materials

V. cholerae strain O395 was received as a gift from Dr. A. C.Ghose. Acrylamide, IPTG, PMSF, GdnHCl, AdoMet, AdoHcy, sorbitol,

TMAO and glycerol were purchased from Sigma Chemicals(St. Louis, MO). Pfu DNA polymerase, restriction enzymes BamHIand XhoI, dNTPs were purchased from Fermentas. T4 DNA Ligasewas obtained from New England Biolabs, and Ni-NTA SuperflowAgarose was purchased from Qiagen. Radioactive [3H]AdoMet waspurchased from MP Biomedicals. All other chemicals, obtainedfrom Merck (India), were of analytical grade.

2.2. Cloning, overexpression and purification of recombinant PIMT

Genomic DNA of V. Cholerae strain O395 was isolated usinggenomic DNA isolation protocol. Cloning was carried out usingthe primers 50-CCGTGAGGATCCATGGCTAACCCAAAAGC-30 and50-GGGCGTTCTCGAGCGGAAAGAGTAAAGCGTC-30 into BamHI andXhoI restriction sites using pET28a vector following the standardprotocol [24]. That the plasmid contains the correct sequence of thepcm gene was confirmed by DNA sequencing which showed theincorporation of 6X His-tag at the N-termini (Fig. S2). Expressionwas carried out into E. coli BL21 competent cells using isopropyl-1-thio-D-galactopyranoside (IPTG) at a final concentration of 1 mMand the culture was grown overnight at 16 �C. After harvesting thecells at 5000 rpm for 10 min at 4 �C, sonication was carried out inlysis buffer containing 50 mM KH2PO4, 300 mM KCl, 10 mM imid-azole and 5% glycerol, pH 7.5. After removal of cell debris bycentrifugation at 12,000 rpm for 30 min, the soluble fraction wasloaded into previously equilibrated Ni-NTA column (QIAGEN). Afterwashing with 20 mM and 50 mM imidazole containing buffers, therecombinant PIMT was purified using a gradient of 50e350 mMimidazole. The purified protein was run into 10% Trisetricine gelfollowed by staining with coomassie blue dye. Protein concentra-tion was determined spectrophotometrically using the extinctioncoefficient of 25,440 M�1cm�1 as provided by the ExPASy Proteo-mics server (http://expasy.org/sprot/). Mass spectral analysis wasperformed which confirmed a mass of 26,420 Da (expected M.W.from ExPASy server is 26,486 Da).

2.3. Size exclusion chromatography of PIMT

Analytical gel-filtration experiments were carried out in anHPLC system AKTA prime Plus. Protein at a concentration of 1 mg/mlwas injected at a time. The columnwas pre-equilibrated with 0.1 Mpotassium phosphate buffer, pH 7.2. Bovine serum albumin(66.5 kDa), RNase A (13.7 kDa), chymotrypsin (24.8 kDa) andovalbumin (44.5 kDa) were used as molecular weight markers. Thevoid volumewas calculated by running blue Dextran. The retentionfactor Rf is represented by the following equation

Rf ¼ ðVe � VoÞ=ðVt � VoÞ (1)

where Ve is the elution volume, Vo is the void volume and Vt is thetotal column volume.

2.4. Limited proteolysis

Tryptic digestion of PIMT was carried out using trypsin at 1:50molar ratio (trypsin/protein) and incubating at 37 �C for 30 min.Reaction was quenched by the addition of 5� protein dye, followedby heating at 95 �C for 15min, subsequent towhich the sample wasrun into 10% Trisetricine gel.

2.5. Fluorescence measurements

All fluorescence spectral data were taken from Hitachi F-3010spectrofluorimeter at a scanning speed of 240 nm per min in 1 cmpath length quartz cuvette having slit widths of 2.5 and 5 nm for

T. Chatterjee et al. / Biochimie 95 (2013) 912e921914

excitation and emission, respectively. Fluorescence emissionspectra were recorded from 310 to 420 nm using 0.1 M potassiumphosphate buffer, pH 7.5. An excitation wavelength of 295 nm wasused to follow tryptophan fluorescence and the wavelength atmaximum emission intensity, lmax was determined.

For the denaturation studies, series of freshly prepared solutionof GdnHCl having concentrations ranging from 0.2 to 6 M in 0.1 Mpotassium phosphate buffer, pH 7.5 were prepared and the proteinwas added to a final concentration of 5 mM. Bis-ANS binding to thenative, partially folded and denatured protein was studied afterexciting the samples at 395 nm and recording the fluorescenceintensity at 495 nm. For FRET measurements, samples were excitedat a wavelength of 295 nm and the spectra was recorded in therange of 300e600 nm. Slit width of 5 nm was kept for bothexcitation as well as emission data.

Solvent exposure of the tryptophan residues in the native statewas accessed using fluorescence quenching techniques. Two typesof quenchers were used viz. acrylamide and potassium iodide (KI)in 10 mM increments and quenching constant (Ksv) was calculatedusing the SterneVolmer equation

F0FC

¼ 1þ KSV½Q � (2)

where F0 is the initial fluorescence intensity, FC is the correctedintensity in presence of quencher and KSV is the SterneVolmerconstant.

2.6. Circular dichroism spectroscopy

Circular dichroismmeasurements were done at 25 �C in a JASCOspectropolarimeter (model J-800) using a cuvette of path length of1 mm. Protein concentration used for the Far-UV CD experimentswere 10 mM in 0.1 M potassium phosphate buffer (pH 7.5) andspectra was recorded at a range of 210e260 nm with a step reso-lution of 0.1 nm, a scan speed of 50 nm per min and a bandwidth of1 nm. Each spectrumwas taken as an average of 3 scans to increasethe signal to noise ratio. The spectra were obtained after baselinecorrection for the buffers with the same denaturant concentrationwere taken care of. The spectra were reported in terms of meanresidue molar ellipticity [q] (deg cm2 dmol�1). The formula used forcalculating mean residue ellipticity is

½q222� ¼ 100qMw=clN (3)

where [q], mean residue ellipticity; q, experimental ellipticity inmdeg; Mw, molecular weight of the protein in Dalton; c, proteinconcentration in mg/ml; l, cuvette path length in cm; N, number ofamino acids of the protein.

For thermal unfolding of the protein and cofactor-bound proteinfar-UV CD spectra were recorded as a function of temperaturebetween 20 and 70 �C in steps of 2 �C with an equilibration time of2 min at each temperature. The temperature dependence of thesecondary structure was estimated from the two-state fitted far-UVCD curves. Near-UV CD spectra were recorded to determine thetertiary structure of the protein. Spectra were taken in the range of350e250 nm using a protein concentration of 1 mg/ml.

2.7. Isothermal titration calorimetry

Isothermal calorimetry has been used as one of the mostquantitative means to determine the thermodynamic parameters,like the binding stoichiometry of the interaction (N), the associationconstant (K), the free energy (DG), enthalpy (DH) and entropy (DS)between the protein and the cofactors, AdoMet and AdoHcy [25].

The ITC experiment was carried out on a VP-ITC microcalorimeter(Microcal, Northampton, MA) at 30 �C. The protein was thoroughlydialyzed for 24 h in 0.1 M potassium phosphate buffer (pH 7.5)before loading. Titration experiments consisted of 25 successiveinjections of either of the cofactors viz. AdoMet or AdoHcy (injec-tion volume 10 mL; conc. 300 mM) into the reaction cell (1.6 mL)containing PIMT (conc. 30 mM) in 0.1 M potassium phosphate buffer(pH 7.5). The titration cell was stirred continuously at 310 rpm. Theheat of dilution of the protein solutions when added to the buffersolution in the absence of cofactor was determined using the samenumber of injections and concentration of protein as in the titrationexperiments. The data were analyzed using a simple one-sitebinding model using Microcal Origin 7.0 software (Origin LabCorporation, Northampton, MA) provided with the instrument. Thebinding constant (K), enthalpy change (DH) and binding stoichi-ometry (N) were determined from curve-fitting analyses.

2.8. Dynamic light scattering

DLS, also known as quasi elastic light scattering (Quels) andPhoton Correlation spectroscopy (PCS), is an important tool inprotein characterization and is based onmeasuring the fluctuationsin the laser light that is scattered from the particles in a solutionwithout perturbing the system [26]. It is a measure of the hydro-dynamic size, polydispersity and the extent of aggregation ina protein sample. The fact that large molecules move slower thansmall molecules gives us a correlation function. From this correla-tion function the diffusion coefficient (D) of the molecules can becalculated, which in turn gives the hydrodynamic radius (rH) of theparticles and molecules.

D ¼ kT6phrH

(4)

k being the Boltzmann constant, T temperature, h solvent viscosity.The sample preparation is crucial for the measurements of light

scattering from protein solutions. All chemicals used were ofreagent grade. Deionized and filtered sterile water was used as thesolvent. Protein samples and buffers were also filtered for theexperiment. For each sample, the measure time was 2 min ata temperature of 27 �C. Eachmeasurement was repeated four timesand the result was an average of four measurements. The concen-tration of protein sample used was 0.3 mg/ml.

2.9. Effect of osmolytes on PIMT

To study the effect of osmolyte, viz. glycerol and TMAO on PIMT,tryptophan fluorescence and CD measurements were carried out.Glycerol and TMAO were added to a final concentration of 1 M toPIMT. Thermal unfolding of PIMT in the presence and absence ofosmolytes were followed using far-UV CD. Moreover the effect ofosmolytes on denatured proteinwas studied using the hydrophobicdye bis-ANS.

2.10. Enzyme assay

V. cholerae O395 cells were grown at 37 �C until OD reached 1.0and then harvested by centrifugation and washed twice with bufferA (5 mM sodium phosphate, 5 mM EDTA, 15 mM b-mercaptoe-thanol, 25 mM phenylmethylsulfonyl fluoride, 10% glycerol, pH 7.0).Cells were resuspended in 1.5 ml of buffer A per g of wet cell pelletand were broken by French press treatment. The lysate wascentrifuged at 20,000 � g for 15 min to sediment the cell debris,and the supernatant was recentrifuged at 100,000� g for 90min toobtain a cytosolic fraction which was stored at �20 �C for analysis.

Fig. 1. Protein L-isoaspartyl-O-methyltransferase activity of V. cholerae cell extract withpeptide substrate as a function of time (blank A, peptide substrate :). The blankcorresponds to PIMT, [3H]AdoMet and the buffer (but no substrate).

Fig. 2. Fluorescence spectra of native PIMT (lex 295 nm; D) and in the presence of 1 M(-) and 6 M GdnHCl (B).

T. Chatterjee et al. / Biochimie 95 (2013) 912e921 915

Total cytosolic protein concentration was determined by Bradfordassay. The activity of protein L-isoaspartyl-O-methyltransferase wasdetermined in a 25 ml reaction mixture containing about 500 mg ofthe cytosolic protein as the enzyme source, a methyl acceptor(100 mM, VYP(isoD)HA), the methyl donor S-adenosyl-L-[meth-yl-3H]methionine (6 mM, 1.0 Ci/mmol), and 0.1 M sodium citrate(pH 6.0). After incubation at 37 �C for 30e150 min, the reactionwasquenched using 0.6 M sodium borate, 1% sodium dodecyl sulfate(pH 10.2) and methyl esters were hydrolyzed using 0.1 N NaOH.Peptide [3H]methyl esters were quantified by vapour diffusionassay [27].

2.11. Homology modeling and molecular dynamics simulation

The primary sequence of PIMT of V. choleraewas extracted fromSWISSPROT (http://expasy.org/sprot/). To find the best template formodeling, the sequence was aligned using ClustalW with multiplesequences of PIMT from different sources, human, E. coli andDrosophila melanogaster [28]. The protein from E. coli has thehighest sequence identity (64%) and with known atomic co-ordinates (PDB code, 3LBF) (Protein Data Bank) and this was usedas the template. The structure was built by homology modelingusing MODELLER version 6v0 [29,30]. Secondary structures of theprotein were calculated with the program DSSP [31]. The boundAdoHcy in the E. coli structure was mapped into the modeledV. cholerae PIMT by superimposing the two protein structures [32].Superimposition was done using the module SUPERPOSE in CCP4[33]. A 10 ns simulationwith Generalized Born Surface Area (GBSA)implicit solvent model and parm99SB force field parameters wasthen performed on the PIMT complex using the sander module ofAMBER 10.0 package [34,35]. The molecule was first minimizedfollowed by equilibration at constant pressure for 40 ps. Langevindynamics was performed for 10 ns at 300 K.

Hydrogen bonding between the protein and the ligand waschecked using HBPLUS [36]. Solvent accessibility was calculatedusing NACCESS [37]. A similar simulation and analysis was done forPIMT bound to AdoMet after generating the position of the ligandbased on its known complex structure in Pyrococcus furious (PDBcode, 1JG4) [38]. A third simulation was also performed for the apoprotein structure.

3. Results and discussion

Literature reports methyl transferase activity from cytosolicextracts of various bacteria, viz. S. typhimurium, E. coli, Pseudomonasaeruginosa, Rhodobacter sphaeroides, enteric species Klebsiellapneumoniae, Enterobacter aerogenes, Proteus vulgaris, and Serratiamarcescens [14]. Fig. 1 shows the transfer of methyl group into theadded substrate, an isoaspartyl peptide (VYP(isoD)HA) as a functionof incubation time. With the peptide substrate, V. cholerae PIMTshowed methyl transferase activity above what is seen in the blankdue to the presence of any endogenous isoaspartyl substrates in thecytosolic extract. The manifestation of this activity prompted us tostudy the recombinant protein, which was also found to be enzy-matically active (to be published).

3.1. Oligomeric status of V. cholerae PIMT

In most of the species PIMT is known to exist as monomer[4,5,32], although a hexameric form has also been reported inSulfolobus tokodaii [39]. The oligomeric status of V. cholerae PIMTwas examined by gel-filtration chromatography. Fig. S3 showsa plot of the Rf value against the logarithm of molecular weight. Theelution profile of PIMT exhibited a single peak corresponding to

a molecular weight of 26.6 kDa, confirming the monomeric statusof the protein.

3.2. Equilibrium unfolding of PIMT in presence of GdnHCl

Tryptophan residues serve as intrinsic fluorescent probe sincefluorescence intensity and emission maximum provides informa-tion about the local and overall conformations of the protein. PIMThas three tryptophan residues (Trp111, Trp133 and Trp136) and thefluorescence emission spectra of PIMT in the presence and theabsence of GdnHCl are shown in Fig. 2. Native PIMT shows anemission maximum at 345 nm, indicating that the tryptophanresidues are quite exposed to the polar environment. Uponincreasing concentrations of the GdnHCl there is a significant dropin fluorescence intensity, combined with a red shift in the peakposition, indicating complete exposure of the tryptophan residues.

3.3. Analysis of the unfolding data

The CD and fluorescence monitored observables obtained fromGdnHCl-induced unfolding were fitted into a two-state model toobtain the thermodynamic parameters for the transition betweenthe folded (N) and the unfolded (U) states (Table 1). At eachdenaturant concentration the observed signal S, representing theshift of the fluorescence emission maxima or CD ellipticity at222 nm were fitted to a two-state equation as shown below

Table 1The unfolding of PIMT using GdnHCl: Two-state analysis.

Parameters lmax monitored data CD monitored data

SN 345.54 �18.51SU 350.24 �0.32DGNU kcal mol�1 2.98 � 0.48 4.52 � 1.33mNU kcal mol�1 M�1 0.57 � 0.098 1.50 � 0.37[dNU]1/2 M 5.22 3.00

T. Chatterjee et al. / Biochimie 95 (2013) 912e921916

SNe

NU

RT þ SU

S ¼

�DG

�

e

�DGNU

RT

�þ 1

(5)

The plots of DGNU (unfolding free energy) against denaturantconcentration were analyzed by linear least-squares analysis, byusing the equation

DGNU ¼ DGH2ONU �mNU½dNU�1=2 (6)

where mNU is the dependency of the DGNU on denaturant concen-

tration, which is a measure of cooperativity of unfolding and DGH2ONU

is the free energy change in the absence of denaturant, which isequivalent to the conformational stability of the protein. DividingDGNU by the slope gives the value for the midpoint of transition,[dNU]1/2.

It is well reported in literature that classical unfolding analysessometimes fail to detect partially folded intermediates because oftheir very short life and sparse population [40]. Moreover, thetransitions which occur with the formation of intermediates areassociated with the non-superimpossibility of the curves usingdifferent spectroscopic tools [41]. In the present study the non-superimposition of the observables from CD and fluorescencespectroscopy (Fig. 3) indicates the probable existence of anunfolding intermediate, although the fitting has been done usinga two-state model.

3.4. Identification and characterization of folding intermediate

Different techniques have been utilized for the identificationand characterization of folding intermediate [42e44]. At lowerconcentrations and pH below 11, GdnHCl exists as Gdnþ which

Fig. 3. Unfolding of PIMT using GdnHCl. Fluorescence lmax(B) and ellipticity (C) at222 nm overlaid along with the best fit curves.

electrostatically interacts with the negatively charged residues ofthe protein, thereby causing protein stabilization [45]. However, athigher concentrations it behaves as a classical denaturant causingprotein unfolding. At lower concentration (0.8e1.2 M GdnHCl)a decrease in the fluorescence intensity with the concomitant blueshifted spectrum (lmax 344 nm at 1 M GdnHCl) (Fig. 2) is observed,indicating the existence of a probable intermediate.

The nature of the intermediate was further examined bycomparing its far- and near-UV CD spectra with that in the nativestate. The secondary structural content of PIMTasmonitored by far-UV CD was deconvoluted using CDNN [46]. Far-UV CD of PIMT inthe presence of 1 M of GdnHCl shows increase in the value ofellipticity (Fig. 4a), which corresponds to an increase in a-helicalcontent (53%) as compared to the 35% for the protein alone(Table 2). In the near-UV CD spectrum (Fig. 4b) native PIMT hasa negative band around 284 nm, which may arise due to thecontribution of six Tyr and three Trp residues. The presence of 1 MGdnHCl led to a loss of this band, indicating the absence of tertiarystructure. Thus the unfolding of PIMT by GdnHCl leads to theformation of a non-native intermediate species, with most of itssecondary structure intact (or enhanced) but substantial loss oftertiary structure. Such intermediate species with similar spectralchanges have been observed for a number of proteins [47].

Bis-ANS, a fluorescent probe that binds to hydrophobic regionsof protein, is used for the characterization of intermediates whichhave large clusters of solvent- exposed hydrophobic patches[48,49]. Here we find that biseANS binds strongly to the interme-diate state (1 M GdnHCl) with a resultant increase in its fluores-cence intensity (excitation 395 nm and emission 495 nm) incomparison to the native and the denatured state (6 M GdnHCl)(Fig. 5). These results, therefore, complement those from the otheranalyses described above, and strongly support the existence ofa non-native intermediate in the unfolding pathway of PIMT.

Due to the formation of the hydrophobic surface patches in theintermediate, non-specific hydrophobic interactions can occur,leading to the aggregation of the protein [50]. DLS experimentswere performed as a function of time to note the time dependenceof the aggregation process for PIMT (Fig. S4). It was found that thediameter in case of native PIMT is 4.7 nm, which increased to7.27 nm in the presence of 1 M GdnHCl. There was further aggre-gation and the size continued to increase with time.

3.5. Partial trypsinolysis of the native and the folding intermediateof PIMT

Proteins in the intermediate state are known to have consider-able secondary structural content and greater flexibility of sidechains, because of the loss of some of the tertiary structural inter-actions [51]. In our study, the conformational flexibility of foldingintermediate of PIMT has been investigated using limited proteo-lytic digestion. Since proteolysis is governed by the stereochemistryand accessibility of the protein substrate, minor conformationalchanges can also be detected by this technique.

Undigested PIMT yields a band on 10% Trisetricine SDS PAGE,which corresponds to a molecular mass of 26 kDa that has beenused as a control to monitor the proteolytic activity of trypsin onthe native PIMT and the folding intermediate. Upon tryptic diges-tion the 26 kDa band disappears with the appearance of a lowermolecular weight band (Fig. 6). In presence of 0.5 M GdnHCl, theextent of proteolysis has increased as can be seen from theconcomitant decrease in the intensity of the 26 kDa band alongwith the appearance of other low molecular weight bands.However, in presence of 1 M GdnHCl we observe the reappearanceof the intense 26 kDa band along with few other bands. The aboveresults suggest that at the concentration of 1 M GdnHCl, PIMT

Fig. 4. (a) Far-UV spectra of native PIMT (D) and PIMT þ 1 M GdnHCl (A) and (b) Near-UV CD of native PIMT (6) and PIMT þ 1 M GdnHCl (A).

T. Chatterjee et al. / Biochimie 95 (2013) 912e921 917

assumes a more native-like conformation (relative to that in pres-ence of 0.5 M GdnHCl), its increased susceptibility to proteolysis isindicative of the enhanced conformational flexibility in the inter-mediate state.

3.6. Solvent accessibility of tryptophan residues in the native stateof PIMT

For studying the solvent accessibility of the tryptophan residuesof PIMT, flourescence quenching experiments were followed usingtwo types of quenchers viz. acrylamide and potassium iodide (KI)(Fig. 7). Iodide, an ionic species usually quenches tryptophan(s)near the surface, while acrylamide being non-ionic and small insize can quench tryptophan(s) near the surface, as well as in thehydrophobic interior of the protein through diffusion [52]. Evalu-ation of the acrylamide and iodide quenching constants (Ksv) givenby SterneVolmer plot tells us about the solvent accessibility of thetryptophan residues. As KI and acrylamide give similar quenchingeffect (Ksv of 15.0 M�1 and 12.9 M�1, respectively) for the nativeprotein, it is likely that all the three Trp residues (Trp111, Trp133and Trp136) are exposed to the solvent. This result has also beenverified theoretically using the model developed for PIMT, dis-cussed later.

3.7. Fluorescence resonance energy transfer involving the foldingintermediate

FRET is a useful experimental approach to determine thebinding affinity of a particular probe to protein [53,54]. Overlap ofthe Trp emission spectrum of PIMT with the excitation spectrum ofbis-ANSwasmanifest in energy transfer from the donor (Trp) to theacceptor (bis-ANS) fluorophore (Fig. S5). In the absence of bis-ANSTrp residues of PIMT (in presence of 1 M GdnHCl) showedmaximum emission at 345 nmwhich diminished upon addition ofincreasing concentration of bis-ANS. Since bis-ANS does not exhibitfluorescence upon excitation at 295 nm, the observed emission at

Table 2Derived secondary structure content (%) of PIMT alone and in presence of 1 M(the intermediate) and 3 M GdnHCl, and osmolytes.

NativePIMT

PIMT þ 1 MGdnHCl

PIMT þ 3 MGdnHCl

PIMT þ 3 MGdnHCl þ 1 Mglycerol

PIMT þ 3 MGdnHCl þ 1 MTMAO

a- helix 34.7 52.7 12.9 15.3 37.3b- sheet 16.2 9.8 20.1 18.2 15.2Random coil 48.8 38.2 67.2 64.9 46.8

At the concentration of 3 M GdnHCl the protein showed significant denaturation, asevident from the substantial loss of a-helical content (in the far-UV CD spectra), aswell as bathochromic shift of Trp residues (in the fluorescence spectra). Use ofa higher concentration of GdnHCl led to an increase in the signal to noise ratio,which debarred the proper interpretation of the CD spectra.

495 nm is exclusively due to the binding of the probe to thehydrophobic patches of PIMT.

3.8. Effect of the binding of the cofactor AdoMet and AdoHcy to theconformation of PIMT and its thermal stability

During the course of repair mechanism involving PIMT, AdoMettransfers a methyl group thereby producing AdoHcy, both of whichare involved in various metabolic pathways crucial for importantphysiological functions [55]. Since PIMT binds to both AdoMet andAdoHcy, the conformational changes of the protein upon binding toboth the cofactors are important in studying the protein function.

The cofactor induced conformational changes of PIMT afterbinding to AdoMet and AdoHcy were studied by far-UV CD. Thedeconvoluted data showed an increase in a-helix for both thecofactors (AdoMet 37.4% and AdoHcy, 40.3%) at the expense ofrandom coil, as compared to that for the free PIMT (a-helix 34.7%)(Fig. 8 and Table 3).

Temperature-dependent CD profiles of PIMT and PIMT bound toAdoMet and AdoHcy were recorded at a wavelength of 222 nm tostudy the thermal unfolding of the secondary structures of theprotein (Fig. 9). To fit the change of CD at a single wavelength asa function of temperature T, Gibbs-Helmholtz equation was used.

DG ¼ DHð1� T=TMÞ � DCpTMð1� ðT=TMÞ þ ðT=TMÞlnðT=TMÞÞ(7)

TM is the melting temperature, DH is the change in enthalpy andDCp is the change is specific heat capacity from the folded to theunfolded state. The midpoint of the unfolding transition TM,determined from the sigmoidal fits to the plot of q222 withtemperature, was found to be 48, 53 and 54 �C respectively, for

Fig. 5. Bis-ANS binding to PIMT alone (6) and in presence of 1 M (A) and 6 M (B)GdnHCl, based on fluorescence intensity at 495 nm.

Fig. 6. Limited proteolysis of PIMT. Lane 1: Native PIMT, Lane 2: PIMT þ trypsin, Lane3: PIMT þ 0.5 M GdnHCl, Lane 4: PIMT þ 0.5 M GdnHCl þ trypsin, Lane 5: Molecularweight marker (Fermentas), Lane 6: PIMT þ 1.0 M GdnHCl, Lane 7: PIMT þ 1.0 MGdnHCl þ trypsin.

Fig. 8. Far-UV CD of native PIMT (6) and AdoHcy (B) and AdoMet (:) bound PIMT.

Table 3Secondary structure (%) of native PIMT alone and in presence of AdoMet andAdoHcy.a

Native PIMT PIMT þ AdoMet PIMT þ AdoHcy

q222 �24.78 �28.84 �31.19a- helix 34.7 37.4 40.3b- sheet 16.2 14.9 13.9Random coil 48.8 46.6 46.0

a Using the CD data provided in the first row.

T. Chatterjee et al. / Biochimie 95 (2013) 912e921918

PIMT and PIMT bound to AdoMet and AdoHcy. The increasedmelting temperature indicates that the binding of the cofactorsmake the protein more stable.

The thermodynamics of binding of both the cofactors AdoMetand AdoHcy to PIMT were measured using ITC (Fig. S6), and theparameters are summarized in Table 4. The interaction hasa favorable enthalpy change (DH < 0) that is offset partially byunfavorable entropy (DS < 0). Negative values of enthalpy andentropy are consistent with the characteristics of weak van der

Fig. 7. SterneVolmer plots for quenching of tryptophan fluorescence of native PIMT inpresence of the quencher KI (D) and acylamide (:).

Waals interactions in the complex formation. The polarizability ofthe ligand in binding to a protein during complex formation largelycontributes to the negative values for the parameters [56]. Overall,the binding constant for AdoHcy is one order of magnitude higherthan that for AdoMet. A similar observation has also been reportedfor the binding of the two cofactors to a methyl transferase fromB. thetaiotaomicron [57].

3.9. Effect of osmolytes on the native and denatured state of PIMT

Osmolytes induce folding of protein as a result of solvophobiceffects on the peptide backbone [58]. At physiological concentra-tions of osmolytes the folding equilibrium shifts toward the nativestate [59]. The wavelength at maximum fluorescence of nativePIMT does not show any significant change in presence of either ofthe osmolytes (glycerol and TMAO). However, the denaturedprotein (3M GdnHCl) showed blue shift (347 nm vs. 350 nm) in the

Fig. 9. Temperature induced unfolding of native PIMT (open circle), PIMT with AdoMet(shaded circle) and PIMT with AdoHcy (filled circle).

Table 4Thermodynamic parameters of cofactor binding to PIMT.

Parameter AdoMet AdoHcy

N (protein:cofactorstoichiometry)

0.56 � 0.03 0.21 � 0.01

K (binding constant, M�1) (1.42 � 0.16) � 105 (2.72 � 0.31) � 106

DH (binding enthalpy, cal/mol) (�7.63 � 0.46) � 103 (�4.03 � 0 0.91) � 104

DS (entropy change, cal/mol$K) �1.57 �103.40DG (free energy change,

kcal/mol)�7.12 �8.89

Fig. 10. Bis-ANS binding to denatured PIMT (B), denatured PIMT in presence of 1 Mglycerol (:) and in presence of 1 M TMAO (-).

T. Chatterjee et al. / Biochimie 95 (2013) 912e921 919

presence of TMAO; in the presence of glycerol there was, however,no appreciable change in lmax.

Far-UV CD spectra were monitored to study the secondarystructural content of PIMT in the presence of osmolytes. Theaddition of TMAO led to the enhancement of the ellipticities at222 nm in the CD spectrum. The deconvoluted data revealeda residual a-helical content of 12.9% for PIMT in 3 M GdnHCl, whichincreased to 37.3% in presence of 1M TMAO (Table 2). The changewas nominal (15.3%) in presence of glycerol.

Fig. 10 shows the effect on fluorescence intensity due to bis-ANSbinding to the denatured protein and the changes brought about bythe presence of 1 M TMAO and 1 M glycerol. The denatured statebinding features are essentially unaltered in the presence of glyc-erol. However, in the presence of TMAO, a fairly high degree of bis-ANS binding is observed to the denatured protein. Since more

Fig. 11. PIMT-AdoHcy (after 10 ns of simulation), in cartoon representation, along with a detof AdoHcy. Peptide segments containing the residues in contact (Table S1) are: Ile52eIle58, Gthese residues are located in loop regions, except 88e91 and 107e108 in helix, and 57e58

backbone is exposed in the unfolded state, TMAO preferentiallydestabilizes the unfolded state shifting the equilibrium moretoward the native state. Glycerol does not have a similar kind ofeffect. Likewise, we did not find any significant effect of sorbitol onthe equilibrium denaturation profile of PIMT. It may also be addedthat the aforementioned osmolytes did not exhibit any stabilizationeffect upon urea-induced unfolding of PIMT.

3.10. Structural models, dynamics and energetics

To get an insight into the binding of the cofactors by PIMT, wemodeled the apo and the complex structures and performedmolecular dynamics studies. Structures at the end of 10 ns simu-lations do not show much change in the protein backbone or theposition of the ligand (Fig. S7); PIMT-AdoMet shows some fluctu-ations before convergence. The ligand AdoHcy (and likewise,AdoMet) is located in a cavity which is almost buried within theprotein core (Fig. 11), as was observed in the E. coli and Pyrococcusfuriosus structures [32,38]. Due to ligand binding 227 �A2 surfacearea of the protein gets buried, contributed by 29 residues,with Arg105 and Thr148 providing the major change. The ligandAdoHcy forms 11 hydrogen bonds with the protein. The residuesthat are within 4.5 �A of the ligand are provided in Table S1. Thechange in energy (averaged over the simulation period) betweenthe PIMT-AdoHcy complex and the unbound protein and freeligand is �74.5 kcal/mol, whereas for the PIMT-AdoMet complex itis �12.5 kcal/mol, which indicates that (under the limitations ofthe simulation which cannot accurately replicate solvent effects)the PIMT-AdoHcy complex is more stable than PIMT-AdoMet. Theresults are in conformity with the thermal unfolding data, whichhad indicated a higher stability of protein on binding cofactors, andthe ITC data that suggested a somewhat stronger binding withAdoHcy. Our results indicate a higher affinity of V. cholerae PIMTtoward AdoHcy as compared to AdoMet as has been seen inB. thetaiotaomicron methyl transferase [57]. The higher affinity forAdoHcy may be the reason why it is assumed to be an inhibitor ofPIMT [32].

During the course of the simulation, movement was foundmostly at the C-terminal flexible region of the protein (and also atsome other positions, such as Arg105, Thr148 mentioned above),owing to the closing of the openings of the cavity where the ligandis bound. On the part of the ligand, there was very little movement,possibly be due to scarcity of space in the apparently closed cavity;only the hydrocarbon tail region was found to have moved whencompared to that in the crystal structure.

ailed view of the binding site showing stick representation of the residues within 4.5 �Alu81eAla91, Thr102eLeu109, Gly130eTrp133, Thr148eLys153, and Phe199eVal203. All, 81e82 and 102e104 in strands.

T. Chatterjee et al. / Biochimie 95 (2013) 912e921920

4. Conclusions

The cell extract from V. cholerae is capable of repairing alteredL-isoaspartyl residues. The biophysical characterization of recombi-nant PIMT from V. cholerae indicates the existence of a non-nativeintermediate in its unfolding pathway induced by GdnHCl. Theintermediate contains pronounced secondary structure but lacksa defined tertiary structure, which is one of the main characteristicsof the “molten globule” state. The retention of the native-likestructure in the PIMT intermediate is indicated by limited proteo-lytic experiment. The effects of osmolytes, viz. glycerol and TMAOonthe refolding of PIMT showed that TMAO is more effective onrefolding of PIMT. The binding of the cofactors, AdoHcy in particular,increases the helical content of the protein and stabilizes the proteinagainst thermal unfolding. The features of cofactor bindingmayhaveimplications for the activity of the enzyme and its correlation withthe structure, the solution of which is underway.

Authors contributions

Conceived, designed the experiments and wrote the paper: TC,AP and PC. Performed the experiments: TC, AP and RPS. Dockingand simulation studies: SD and DC. Analyzed and interpreted thedata, prepared figures and tables: TC, AP and PC.

Acknowledgments

TC has been supported by grants from the Department ofScience and Technology; PC is a recipient of JC Bose NationalFellowship. Research fellowships from the Council of Scientific andIndustrial Research (AP and RPS), Indo-French Centre for thePromotion of Advanced Research (DC), and the Department ofBiotechnology (SD) are acknowledged.

Appendix A. Supplementary material

Supplementary material related to this article can be found athttp://dx.doi.org/10.1016/j.biochi.2012.12.013.

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