Association Properties and Unfolding of a βγ-Crystallin Domain of a Vibrio-Specific Protein

The βγ-crystallin superfamily possesses a large number of versatile members, of which only a few members other than lens βγ-crystallins have been studied. Understanding the non-crystallin functions as well as origin of crystallin-like properties of such proteins is possible by exploring novel members from diverse sources. We describe a novel βγ-crystallin domain with S-type (Spherulin 3a type) Greek key motifs in protein vibrillin from a pathogenic bacterium Vibrio cholerae. This domain is a part of a large Vibrio-specific protein prevalent in Vibrio species (found in at least fourteen different strains sequenced so far). The domain contains two canonical N/D-N/D-X-X-S/T-S Ca2+-binding motifs, and bind Ca2+. Unlike spherulin 3a and other microbial homologues studied so far, βγ-crystallin domain of vibrillin self-associates forming oligomers of various sizes including dimers. The fractionated dimers readily form octamers in concentration-dependent manner, suggesting an association between these two major forms. The domain associates/dissociates forming dimers at the cost of monomeric populations in the presence of Ca2+. No such effect of Ca2+ has been observed in oligomeric species. The equilibrium unfolding of both forms follows a similar pattern, with the formation of an unfolding intermediate at sub-molar concentrations of denaturant. These properties exhibited by this βγ-crystallin domain are not shown by any other domain studied so far, demonstrating the diversity in domain properties.


Introduction
The surge in genome sequencing of diverse species continues to provide a wealth of uncoded information, on the features and functions of homologous proteins or domains. One of such domains is the bc-crystallin domain found in proteins from microorganisms to vertebrates [1]. A bc-crystallin is a globular domain made up of two Greek key motifs, each containing eight b-strands as seen in the classical structure of lens protein, c-crystallin [2,3]. There are differences in the sequence of these domains from various proteins, except the presence of signature sequences of bccrystallins. In more diverse cases, it is even difficult to clearly identify a sequence as of bc-crystallin domain due to high variations even in the signature sequence. Structurally, these domains have similar topology [4][5][6][7][8], and are thought to be designed as a stable domain during evolution [9][10][11]. While individual bc-crystallin domains adopt stable b-sheet folded structure, a few rare cases, wherein a bc-crystallin is intrinsically unstructured which folds upon binding Ca 2+ , have been identified [12,13]. Presence of a bc-domain in several pathogenic species arouses questions about their functions in these species. It has been predicted that Yersinia crystallin might play a role in the low Ca 2+ response in this pathogen, as this domain is largely unstructured in the apo form, which refolds in the presence of Ca 2+ , thus implicating this protein in Ca 2+ -dependent virulency [12].
In our attempts to study more members from pathogenic species, we have selected a protein containing bc-crystallin domain in the genome of Vibrio cholerae, using the sequence of Yersinia crystallin as template sequence. This hypothetical protein is named vibrillin. Due to the absence of some of the key signatures of Greek key motifs in vibrillin domain, the motifs were earlier classified as S-type motifs (on par with Spherulin 3a) [7] and not as A-type and B-type motifs of Protein S or lens c-crystallin [14]. Our analysis suggests that vibrillin is a Vibrio-specific protein presents in many Vibrio species, but not in the genome of any other species till now. We report that this domain possesses diverse properties, not exhibited so far by any other bc-crystallins, including Spherulin 3a. The domain undergoes dynamic self-association between monomeric, dimeric and oligomeric forms in concentrationdependent manner which is partially influenced by Ca 2+ . We provide extensive data on equilibrium unfolding of oligomeric (octameric) form that demonstrates the presence of an intermediate state before protein dissociates as monomer. Quaternary feature of intermediate state of dimeric form is different than that of octameric forms, as intermediate state of dimeric population associates into oligomers, whereas partially unfolded intermediate state of octameric vibrillin forms insoluble aggregates and Ca 2+ assists in dissociation of the oligomeric protein to its dimeric form.

Cloning and over-expression of a bc-crystallin domain from Vibrio cholarae
The genomic DNA of Vibrio cholarae was a kind gift from Dr. Rukhsana Chowdhury, IICB, Kolkata, India. The region of interest (173 to 256 amino acids) from a hypothetical protein (accession number NP_230470) of Vibrio cholerae was cloned in pET21a expression vector (Novagen) at NdeI and BamHI sites. Vibrillin was over-expressed in the bacterial strain E. coli BL21(DE3) (Invitrogen) in LB medium after induction with 1 mM isopropyl thio-b-D-galactopyranoside (IPTG) for 10 h at 37uC.

Purification of recombinant protein
Vibrillin formed inclusion bodies during over-expression. The protein was refolded from insoluble fraction using an on-column refolding procedure. Briefly, inclusion bodies, isolated by sonication and centrifugation of cell pellets, were further washed with 1 M urea and 0.1% CHAPS. Washed inclusion bodies were solubilized in 50 mM Tris buffer, pH 8.5, containing 3.5 M urea, 1 mM DTT (buffer A) and loaded on a pre-equilibrated Q-Sepharose column with buffer A. The protein was refolded by setting a gradient between buffer A and buffer A without urea. After this step, protein was eluted using a linear gradient of 0 M to 1.5 M NaCl. The fractions containing protein were concentrated by ultra-filtration using an Amicon cell with a 5 kDa molecular mass cut off membrane. The concentrated protein was finally purified by Superdex-75 gel filtration column on a Bio-Rad Duo-Flow purification system.

Hydrodynamic volume
Hydrodynamic volumes of vibrillin were determined by gel filtration on a Superdex-75 (Wipro GE) column, calibrated using standard molecular mass markers. The gel filtration was performed in 50 mM Tris-Cl (pH 7.0), 100 mM KCl, containing either 1 mM EDTA or 5 mM Ca 2+ at a flow rate of 0.4 ml/min.

Glutaraldehyde cross-linking
Both forms of vibrillin (oligomeric and dimeric) (1 mg/ml) was incubated with 0.8% glutaraldehyde for 40 min. The reaction was stopped using 1 M Tris buffer, pH 7.5. SDS-gel loading buffer was added to the reaction samples, boiled and analysed on 15% SDS-PAGE calibrated with appropriate molecular mass markers. The protein bands were visualized by Coomassie Brilliant Blue R250 dye.

Dynamic light scattering
Dynamic-light scattering measurements were performed on a SZ-100 Horiba instrument at 20uC. Approximately 0.5 mg/ml of protein was diluted in 50 mM Tris, pH 7.5, 100 mM KCL and centrifuged at 14,000 rpm for 5 minutes before placing into the cuvette.

Analytical ultracentrifugation (AUC)
AUC was carried out on a Beckman Optima XL-I analytical ultracentifuge (Beckman Coulter, Fullerton, CA, USA) operating in sedimentation velocity mode with an aluminium centerpieces. Protein samples (0.5 mg/ml) prepared in 50 mM Tris pH 7.5, 100 mM KCl were centrifuged at a speed of 60,000 rpm at 20uC in a Ti60 rotor and data were collected using 280 nm absorbance optics for a total of 98 scans with the rate of one scan per every 6 minutes. The data were analyzed using the c(s) method in SedFit software to compute the molecular weight, from the AUC data using non-linear regression fitting of the sedimenting boundary profile with Lamm equation: which describes the concentration distribution c(r, t) of a species with sedimentation coefficient s and diffusion coefficient D in a sector-shaped volume in the centrifugal field v 2 r.

Isothermal titration calorimetry (ITC)
Ca 2+ -binding to vibrillin was evaluated by ITC on a Microcal VP-ITC (Microcal Inc., USA). The protein solutions and CaCl 2 (10 mM) were prepared in a Chelex-treated 50 mM Tris, pH 7.0 containing 100 mM KCl buffer. 1.4 ml of protein solution (100 mM) was used for the binding experiment at 30uC. Aliquots of 2 ml of CaCl 2 as ligand solution were injected for each titration. Appropriate blank was obtained by titrating the buffer with identical concentrations of Ca 2+ . The curve fitting of the data after subtraction with appropriate buffer blank was performed using the software Origin (version 6.0) supplied by Microcal.

Circular dichroism (CD) spectroscopy
Far-and near-UV CD spectra of vibrillin were recorded on a Jasco-815 spectropolarimeter using 0.01 cm and 1 cm path length cuvettes respectively at room temperature. The change in protein conformation upon binding Ca 2+ was studied by titrating the protein solutions with aliquots of standard CaCl 2 solution.

Thermal unfolding
Thermal unfolding experiments were performed by monitoring the change in ellipticity in far-UV range (208, 216 or 218 nm) on a Jasco J-815 spectropolarimeter as a function of temperature at the rate of 1uC/min using a Jasco peltier system attached to the sample holder. Protein solution (0.1 mg/ml) in a 1 mm path length cuvette was used to monitor changes in ellipticity between 25uC to 85uC. Data obtained were plotted and best fitted using Origin 6.0.

Fluorescence spectroscopy
Intrinsic fluorescence emission spectra of protein samples (0.1 mg/ml concentration) were recorded on a F-4500 Fluorescence spectrofluorimeter (Hitachi Inc., Japan) in 50 mM Tris-HCl, pH 7.0, 100 mM KCl. The spectra (300-450 nm) were recorded at an excitation wavelength of 295 nm in correct spectrum mode of the instrument using excitation and emission band passes of 5 nm each. The effect of Ca 2+ was studied by titrating the protein solutions with standard aliquots of CaCl 2 solution from 10 mM to 5 mM concentrations.
Change in surface hydrophobicity of protein samples in different concentrations of denaturant GdmCl (0 to 2 M) was monitored using bis-ANS (10 mM) as probe. The samples were excited at 395 nm, and emission spectra were recorded between 400 nm and 650 nm. The spectra were corrected for equal concentrations of bis-ANS in buffer.

Equilibrium unfolding studies
Equilibrium unfolding of vibrillin was studied using guanidinium chloride (GdmCl) in the range of 0-6 M concentration with an interval of 0.1 M GdmCl (total of 60 data points for each unfolding transition) in the apo (Ca 2+ -free) and holo (Ca 2+ -bound form) states. Protein samples prepared in increasing concentrations of GdmCl (0 to 6 M) in 50 mM Tris buffer, pH 7.0, containing 100 mM KCl either with 1 mM EDTA or with 5 mM CaCl 2 , incubated for different times (2 to 48 hours) at room temperature, and Trp fluorescence emission spectra were recorded by exciting the samples at 295 nm and unfolding was monitored by following the changes in intrinsic Trp emission fluorescence. The ratios of fluorescence intensity (at 360/320 nm) was plotted as a function of GdmCl concentration and the data were fitted to the following equation, which relates fluorescence intensity to the extent of unfolding. The free energy of unfolding (DG D ) determined from the ratio of native to denatured protein at each GdmCl concentration, was plotted against GdmCl concentration [15]. Equation used for two-state model: Where Y N is signal of the folded protein, Y u is signal of the unfolded forms, m is denaturant dependence of the free energy change in units of kcal mol 21 M 21 , R is the universal gas constant (1.987 cal mol 21 K 21 ) and T is the absolute temperature.

Vibrillin is a Vibrio species specific bc-crystallin
A hypothetical protein of 1533 amino acid residues (accession number ZP_01675500) in Vibrio cholerae 2740-80 genome was found to contain two putative bc-crystallin domains (annotated at residues 348-422 and 599-673), predicted by the presence of the signature sequence ( Figure 1A). The homologous protein (with varying sizes) is present in the genomes of twelve different strains of Vibrio cholerae and two strains of Vibrio mimicus, which are annotated as hypothetical proteins, except in two cases where they are named as putative inner membrane proteins probably because of the presence of a short transmembrane domain in the region of 572-594 amino acids ( Figure 1). This transmembrane domain sequence is located before the second bc-crystallin domain in all the strains. The full length Vibrio specific-protein is comprised of 1533 residues whereas the deposited sequences of proteins from other Vibrio strains are not of similar sequences length, though the sequences are identical (Figure 1). The second bc-crystallin domain is common in all the sequences reported so far in the database for Vibrio strains. Vibrillin domain (84 amino acids) was cloned spanning the second domain with additional residues at both ends. This protein is not found in the genome sequence of any other organism available in the database. The sequence of this domain does not have a Tyr corner (a signature sequence with GXY) and a Trp corner in B-type motif, though other characteristic signatures distinguishing a bc-domain, such as YXXXXY/FXG and N/D-N/D-X-X-S/T-S sequences are present in both the Greek key motifs ( Figure 1B). The latter sequence is indicative of the presence of two canonical Ca 2+binding motifs in the domain. Based on these atypical features, we selected this domain (84 amino acids, molecular mass 9.6 kDa) for further analysis.
While purifying we noticed that this protein elutes largely as two peaks during gel filtration chromatography (data discussed below), seen as a single band in SDS-PAGE. We, therefore, investigated the nature of association of this domain in solution.

Homo-domain association
(i) Dynamic association/dissociation between octameric and dimeric forms. The purified vibrillin domain, when analysed for its molecular mass by size exclusion chromatography, was found to elute in two major peaks, suggesting that vibrillin exists largely in two populations in solution as re-confirmed by SDS-PAGE ( Figure 1C) and spectral properties (discussed below). The major fraction of protein (1 st peak) was more symmetrical and eluted at .66 kDa equivalent to oligomer (octameric form) while a minor fraction (a broad 2 nd peak), eluted largely as a dimer ( Figure 1C). It has been further noted that the concentration of protein subjected for gel filtration is a determinant for the relative proportions of both the species in a solution.
We next examined if there was any exchange between these two forms. When the oligomeric form (peak 1) was further analysed to size exclusion chromatography on the same column, it eluted largely in the same state (octameric state). On the contrary, the dimeric fraction (being minor) was concentrated before being resubjected to the column, after which it eluted as two peaks, oligomers and dimers (Figure 2A). These results suggest that though the protein has greater propensity to form oligomers, this oligomerization is concentration-dependent. An additional peak corresponding to monomer at low concentrations of protein (,0.5 mg/ml), was noticed in gel filtration profile, suggesting that oligomers dissociate further if the protein concentration is reduced, and vice versa ( Figure 2B).
A minor fraction of monomeric population seen at 1 and 1.5 mg/ml protein concentrations in the absence of CaCl 2 disappears in the presence of Ca 2+ . In the presence of Ca 2+ , monomeric fraction is seen only when protein concentration is further reduced (0.5 mg/ml) ( Figure 2B). On the other hand, the dimeric population of vibrillin appears to associate as well as dissociate to monomer, dimer, and oligomer (at concentrations between 0.5-1.5 mg/ml) in the absence of CaCl 2 . The oligomeric population elutes as a broad peak (octamer) with a small hump (likely hexamers as per molecular size). In the presence of Ca 2+ , vibrillin elutes only as dimers and oligomers. The oligomeric fraction elutes as a broad peak while monomeric fraction was absent in the concentrations used for analysis (0.5-1.5 mg/ml) ( Figure 2C).
(ii) Formation of higher oligomers. Vibrillin associates as oligomers, as demonstrated by glutaraldehyde crosslinking, dynamic light scattering and analytical ultra centrifugation. Both forms (dimeric as well as oligomeric) have a propensity to form dimer, tetramer as well as octamers as seen by glutaraldehyde crosslinking ( Figure 2D). Very large oligomers could not penetrate the SDS-PAGE pores, though higher oligomers were clustered above 94 kDa marker and could not be resolved by SDS-PAGE. The dynamic light scattering of vibrillin (peak 1 and peak 2) also exhibits the polydisperse nature of the protein with more polydispersity of oligomeric fraction (molecular size range 20-100 nm) and a comparatively narrow range of dimeric fraction (80 nm) ( Figure 2E). Oligomers were observed by analytical ultracentrifugation indicating the polydisperse nature of the protein (Figure 3). The major populations of 18.6 and 79 kDa representing the dimer and octamer were observed in sedimentation profile of peak 1 (oligomeric) vibrillin ( Figure 3A, Figure S3). In addition, further higher oligomers were also seen, though they were not resolved by gel filtration as well as by glutaraldehyde crosslinking obviously due to low level and sensitivity of measurement. Based on analytical ultracentrifuge which is more precise technique, we cannot rule out the presence of higher oligomeric populations. The sedimentation velocity data obtained for vibrillin (peak 2 of Figure 1) exhibit the major populations of 10.6 and 53 kDa, which may represent the monomer and hexamer species; however, higher oligomers of 129 and 416 kDa are also seen as minor fraction ( Figure 3B). Ca 2+ helps in dissociating higher oligomeric (both oligomeric and dimeric) fractions to lower molecular mass populations as compared to its apo forms (Figure 3, Figure S3).

Ca 2+ -binding properties and conformation
As vibrillin domain has two canonical Ca 2+ -binding motifs, i.e., NDTLSS and NDVISS, in each Greek key motif ( Figure 1B), Ca 2+ -binding to: (i) octameric, and (ii) dimeric fraction was undertaken by ITC. Ca 2+ -binding to vibrillin followed exothermic heat changes as seen in thermograms; (Figure 4A, B). However, due to polydisperse nature of the protein, it is not appropriate to calculate the exact binding stoichiometry. Mg 2+ does not bind to vibrillin, suggesting specificity for Ca 2+ ( Figure S4).
Both forms of vibrillin domain exist predominantly in b-sheet conformation with a minimum at 215 nm in far-UV CD. We observed insignificant change in far-UV CD spectra upon Ca 2+binding ( Figure 5A, B). Both fractions have well defined, tertiary fold as seen in near-UV CD as ( Figure 5C, D). Upon Ca 2+binding, significant conformational changes were seen in Trp fluorescence. The octameric form showed l max at 338 nm and fluorescence intensity quenched upon Ca 2+ -binding with a blue shift in l max to 336 nm ( Figure 5E). The l max of the dimeric form of apo vibrillin protein was 342 nm while it was shifted to 338 nm (4 nm blue shift) in the Ca 2+ -bound form ( Figure 5F). It is inferred from the fluorescence data that the local micro-environment surrounding Trp residues is perturbed with either exclusion of

Oligomerization increases thermal stability
Apo oligomeric vibrillin domain is thermally more stable (T m 46.9uC) than the apo dimeric form (T m 36.5uC) ( Figure 6). The thermal unfolding profiles in both the cases are different, apodimeric form follows a more cooperative transition than the apo oligomeric form. Ca 2+ -binding increases the thermal stability in both the cases significantly (DT m ,10-14uC), but oligomeric form of vibrillin is more stable than its dynamic dimeric form ( Figure 6A, B). In the presence of Ca 2+ , a significant change in ellipticity in 25-45uC prior to transition zone was also noticed during thermal unfolding of oligomeric vibrillin (peak 1), which was less pronounced in case of dimeric mixture (peak 2).

Equilibrium unfolding of vibrillin: different association form, different unfolding transitions
Equilibrium unfolding of oligomeric form was monitored by Trp fluorescence in the absence or presence of Ca 2+ . Fluorescence intensity increased in samples up to 0.7 M GdmCl followed by a gradual decrease with no change in emission maxima. Beyond 0.7 M GdmCl, there was a sharp red shift in emission maximum with complete exposure of Trp residues (l max 352 nm) at 1.5 M GdmCl ( Figure 6C, Table 1). Only a gradual increase in fluorescence intensity was seen in samples containing 0.7 to 6 M GdmCl concentration.
The wavelength emission maxima (l max ) of dimeric vibrillin was 338 nm in the absence of Ca 2+ while a 6 nm blue shift (l max 332 nm) was observed in the presence of CaCl 2 . Dimeric vibrillin follows red shift in wavelength maximum gradually up to 0.8 M GdmCl with complete exposure of Trp residues (l max 352 nm) at 1.5 M GdmCl in the absence of Ca 2+ . The unfolding data of apo dimeric vibrillin could not fit properly, however, in the presence of  Ca 2+ , it follows a two-state transition ( Figure 6, Table 1). A significant gain in stability was observed in the presence of Ca 2+ for both fractions during urea-induced unfolding ( Figure 6E, F).
While GdmCl-induced unfolding data of oligomeric form could not fit to any model and Ca 2+ apparently does not influence stability, Ca 2+ increases the stability of the dimeric fractions (c 1/2 apo and holo proteins are 0.9 and 1.4 M, DG 1.4 and 3.1 kcal mol 21 respectively) ( Figure 7A, B Table 1). Urea-induced unfolding of both fractions indicated a two-state unfolding in the absence or presence of Ca 2+ , plotted by the ratio of fluorescence intensity at 360/320 nm ( Figure 7C-F). As seen, Ca 2+ increases the stability of the dimer significantly (c 1/2 apo and holo proteins are 1.3 and 3.9 M). A similar gain in stability was observed for oligomeric fraction of vibrillin (c 1/2 apo and holo proteins are  (Table 1).
While there was no apparent change in unfolding transitions of holo protein with time (measured as ratio of fluorescence intensity at 360/320 nm at 2, 14, 24 and 48 hours), apo protein unfolding transitions were not linear (scattered data) up to 1.8 M GdmCl concentration ( Figure 8). The non-linearity in fluorescence intensity of apo vibrillin domain after 4 h of incubation may be due to invisible aggregation of partially unfolded species (up to 1.8 M GdmCl), which is inhibited in the presence of Ca 2+ .     vibrillin both in the absence or presence of Ca 2+ was observed in protein samples in 10 mM to 500 mM GdmCl, which decreased at higher denaturant concentrations (Figure 9). These results suggest the presence of an on-pathway unfolding intermediate (UI) state at submolar concentrations of denaturant [16]. This is not similar to that seen in lens c-crystallin, where only partially refolded but not unfolded species form insoluble aggregates [17].  Figure S1).
(ii) Unfolding intermediates of oligomeric and dimeric vibrillin have different quaternary association. We next compared the quaternary association of UI states using gel filtration. UI state of apo-oligomeric vibrillin (at 0.1 M GdmCl) has octameric (a peak hump as hexamer) association with only a minor fraction as dimer (Figure 10 A). At 0.5 M GdmCl in the presence of Ca 2+ , UI state of holo-oligomeric vibrillin has almost similar gel filtration chromatogram (Figure 10 B). The comparative less peak height indicates for protein precipitation, which we observed during unfolding due to aggregation, akin to that seen in the case of cataract associated mutants of cD-crystallin [18]. At 0.2 M GdmCl, Ca 2+ influences the formation of dimer without any additional oligomer formation ( Figure S2). Even at further GdmCl concentrations (0. 5 M), similar dissociation is seen, thus suggesting that while protein is partially unfolded, its oligomeric form is not dissociated completely. Interestingly, UI state of dimeric vibrillin at 0.5 M GdmCl was observed in both apo and holo forms, as noticed by increased intensity of bis-ANS fluorescence (Figure 10 C, D). Furthermore, gel filtration profile also suggests that UI states of dimeric forms had propensity to form polydisperse species (octamer and hexamer) eluted as a peak with a hump. Ca 2+ -binding had no effects on UI state of dimeric form.

Discussion
The Vibrio species specific protein containing bc-crystallin domains has several atypical sequence features, some of them are noted in Greek key motifs of other members, such as in Yersinia crystallins [12]. Vibrillin domain shows properties not reported by any other bc-crystallin domains. It forms concentration-dependent oligomers and exists in solution in several forms ranging from high oligomers to dimer (also monomer as a minor fraction if protein concentration is ,0.5 mg/ml). It appears to be the only known bcdomain which forms such oligomers, though many individual bc domains form homodimer via domain pairing [19]. Individual domains of lens cB-crystallin do not associate and remain monomer in solution [20]. Ca 2+ is not known to influence the domain association of other bc-crystallins, including those with canonical Ca 2+ -binding motifs. On the other hand, Ca 2+ shifts the association towards dimeric form at the loss of monomers at low protein concentrations (in the apo form) of vibrillin.
Vibrillin has two canonical Ca 2+ -binding motifs, i.e., NDTLSS and NDVISS, and binds Ca 2+ as seen by ITC. The difference in binding affinities of bc-crystallin domains is due to the variations in the amino acids in the N/D-/N/D-X1-X2-T/S-S motif [21]. Ca 2+ -binding affinities of various bc domains range from 4 mM to 100 mM [22,23]. The affinity for Ca 2+ could not be calculated owing to polydisperse nature of vibrillin domain. This protein does not undergo any notable conformational changes, which is a common feature of most bc-crystallins except those which are intrinsically unstructured in apo form [12,13,19,24].
Since vibrillin sequence is not similar to lens bc-crystallins, it serves as an interesting domain for comparing such distantly related proteins. It has been shown that stability of bc-crystallin domains is tuned by N/D-/N/D-X1-X2-T/S-S motif [25]. Unfolding of vibrillin depicts several unusual facets, despite having two Ca 2+ -binding canonical motifs. This domain is not highly stable as seen by unfolding studies. While Ca 2+ increases the stability of vibrillin protein to some extent when unfolded by GdmCl, the Ca 2+ -induced gain in stability is much more significant when unfolded by urea. An unfolded intermediate with more exposed hydrophobicity was also observed. Our data demonstrate novel facets of unfolding of an oligomeric protein.
The intermediate state retains the original association (octamer) and does not dissociate to monomer, though Ca 2+ favours more dissociation towards dimerization. On the other hand, the intermediate state of dimeric protein aggregates rather than dissociates. Partially unfolded intermediate species of oligomeric vibrillin tend to form insoluble aggregates, which is not similar to that seen in case of human cD-crystallin (which forms only during refolding) [18]. Several mutations in lens c-crystallins are known to alter the properties making them more prone to aggregate in partially unfolded form [18,26,27]. Though molecular basis of such aggregation propensity is not known, it has been recently suggested that aggregation of c-crystallin during refolding is due to C-terminal domain swapping [28]. While lens c-crystallin may be considered as an extensively modified form of vibrillin domain (sequence similarities of 43% to N-terminal domain, and 36% to C-terminal domain), such studies could provide further clues for the non-crystallin like properties attained by these members with extensive sequence modifications.
A few microbial bc-crystallins, such as Protein S from Myxococcus xanthus and Spherulin 3a are associated with stress in Ca 2+dependent form. As seen from our data on the role of Ca 2+mediated stabilization, vibrillin may not be involved in such stressrelated function in Vibrio. It would be important to investigate its role in biofilm formation as its formation is known to play a role in Vibrio pathophysiology and survival strategies [29]. Incidentally, Ca 2+ has been implicated in decreasing the proteins related biofilm formation in Vibrio species [30,31]. In summary, we have characterized a bc domain of a Vibrio-specific protein. This domain associates in solution forming oligomers with a fraction of the population being in dimeric state. Ca 2+ -binding influences the self-association to some extent. Both oligomeric as well as dimeric populations form intermediates during unfolding which follow the similar pathways and dissimilar aggregation. It would be worth to explore the role of this protein in virulency or in Vibrio-specific functions. Supporting Information Figure S1 Change in the surface hydrophobicity (demonstrating the resonance peak) of (A, B) octameric and (C, D) dimeric vibrillin in the presence of GdmCl monitored by bis-ANS fluorescence. Vibrillin samples were prepared in the absence or presence of CaCl 2 (5 mM) at different concentrations of denaturant, GdmCl. bis-ANS (10 mM) was added to the protein solution and incubated for 30 min and samples were excited at 395 nm, emission spectra were recorded between 380 nm and 650 nm. The increase in scattering at 395 nm is seen at 0.