Molecular Vision 2006; 12:581-587 <http://www.molvis.org/molvis/v12/a64/> Received 26 January 2006 | Accepted 22 May 2006 | Published 24 May 2006

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The interaction between αA- and αB-crystallin is sequence-specific

Yellamaraju Sreelakshmi, K. Krishna Sharma
 
 

Departments of Ophthalmology and Biochemistry, University of Missouri, Columbia, MO

Correspondence to: K. Krishna Sharma, Departments of Ophthalmology and Biochemistry, University of Missouri, Columbia, MO, 65212; Phone: (573) 882-8478; FAX: (573) 884-4100; email: sharmak@health.missouri.edu

Abstract

Purpose: We have previously shown that residue 42-57 (TSLSPFYLRPPSFLRA; recognition sequence 1 or RS-1) and residue 60-71 (WFDTGLSEMRLE; recognition sequence 2 or RS-2) in αB-crystallin play a role in oligomerization and subunit interaction with αA-crystallin. When we created multiple mutations in αB-crystallin in RS-1 and RS-2 at S53(T), F54(G), L55(G), W60(R), and F61(N), we found that these mutations destabilized the protein, and the protein precipitated. When the individual mutations were created at F54, W60, and F61 in αB-crystallin, protein stability was not affected, but the mutations had an effect on oligomerization and subunit interaction with αA-crystallin. To find out whether the sequence specificity of these residues is important for the overall function of αB-crystallin, we inverted the 54-60 sequence such that 54FLRAPSW60 became 54WSPARLF60 using site-directed mutagenesis. We studied the effect of inversion on oligomerization and subunit interaction with αA-crystallin.

Methods: Mutations were introduced using site-directed mutagenesis and the mutant protein, expressed in Escherichia coli BL21(DE3)pLysS cells, was purified by ion-exchange and gel filtration chromatography. The mutation was confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone-like activities of wild-type and mutant proteins were compared using alcohol dehydrogenase and citrate synthase. Subunit exchange between αA- and αB-crystallin was monitored by fluorescence resonance energy transfer (FRET). For this purpose, purified αB- and αBinvert-crystallin were labeled with Alexa fluor 350 whereas Alexa fluor 488 was used to label αA-crystallin.

Results: The inversion of residues 54-60 led to homooligomers that were 38% smaller in size than their wild-type counterparts. The inversion also reduced the tryptophan fluorescence intensity by 50%, as compared to that of wild-type αB-crystallin. This suggests that Trp54 is less exposed than Trp60. Inversion of residues did not affect the total hydrophobicity in αB-crystallin. Secondary structural analysis revealed a slight increase in the α-helical content of αBinvert-crystallin protein as compared to wild-type αB-crystallin. Except for an increase in the ellipticity of the αBinvert-crystallin mutant, no change was observed in the tertiary structure, as compared with that of wild-type αB-crystallin. Chaperone-like function was similar in the αBinvert-crystallin mutant and wild-type αB-crystallin. The inversion of residues decreased the subunit exchange rate with αA-crystallin by two fold.

Conclusions: This study establishes for the first time that proper orientation of residues contributing to RS-1 and RS-2 sites in αB-crystallin is important for homooligomerization and optimal subunit interaction with αA-crystallin.

Introduction

α-Crystallin, a member of the small heat shock protein (sHSP) family, constitutes about 50% of the total lens protein [1,2]. The average molecular size of α-crystallin is about 800 kDa, though sizes ranging from 300 kDa to greater than 1,000 kDa have been reported [3]. α-Crystallin is composed of two highly homologous subunits, αA- and αB-crystallins, each with a molecular mass of 20 kDa [3]. These subunits can form both homo- and heterooligomers, and both types of oligomers exhibit chaperone-like function [4]. Heterooligomer with a 3:1 ratio of αA- to αB-crystallin mostly exists in vivo and possesses greater thermal stability than either αA- or αB-crystallin alone [4].

Ample evidence shows that the NH₂-terminal region plays a critical role in oligomerization of sHSPs [5-7]. A sequence between residues 19-71 of αB-crystallin appears to be involved in oligomerization and subunit interaction with αA-crystallin [5-9]. Spin labeling studies [10] and studies employing yeast two-hybrid system [11] and a mammalian two-hybrid system [12] reveal a role for the α-crystallin domain and the entire COOH-terminal domain of αB-crystallin in interaction with αA-crystallin. Also, COOH-terminal truncation studies in αA-crystallin point to a critical role of Arg163 in oligomerization of αA-crystallin [13].

By using peptide scans, we previously identified two regions in αB-crystallin, recognition sequence-1 or RS-1 (residues 42-57) and recognition sequence-2 or RS-2 (residues 60-71), involved in interaction with αA-crystallin [8]. By creating site-directed mutations, we also confirmed that RS-1 and RS-2 regions play a critical role in subunit interaction with αA-crystallin [8,9]. During the course of our study to understand the significance of RS-1 and RS-2 regions in αB-crystallin, we made a multiple mutant of αB-crystallin, with mutations at S53(T), F54(G), L55(G), W60(R), and F61(N) and overexpressed the protein in Escherichia coli. When we made an attempt to purify the protein, we found that the protein precipitates as it gets purified (unpublished results). We also found that partially purified protein failed to protect thermal aggregation of citrate synthase (unpublished data). However, when individual mutations were made in αB-crystallin at F54(G; unpublished results), W60(R), and F61(N) [9], we found that the proteins were stable and all of them exhibited chaperone-function like wild-type αB-crystallin. In addition, F54(G; unpublished data) and W60(R) mutations affected the oligomeric size of αB-crystallin [9], whereas, the F61(N) mutation affected the subunit interaction with αA-crystallin [9]. Our results suggest that S53, F54, L55, W60, and F61 may be involved in specific interactions with other residues in the core of αB-crystallin and thereby, play a role in its quaternary organization. If the multiple mutations disrupt those specific interactions, quaternary organization of αB-crystallin may be lost, affecting its function. In order to delineate between a residue-specific and a sequence-specific role of these residues in the oligomeric assembly and subunit interaction of αB-crystallin with αA-crystallin, we inverted residues 54-60, constituting part of the RS-1 region and the intervening sequence between RS-1 and RS-2 regions. Site-directed mutagenesis was used to invert residues such that 54FLRAPSW60 became 54WSPARLF60. After overexpression and purification of this αBinvert-crystallin mutant, we studied the interaction between this αBinvert-crystallin protein and αA-crystallin and compared it with wild-type αB- and αA-crystallin interaction. To the best of our knowledge, this is the first report of inversion of a stretch of amino acids in α-crystallin subunits. There are reports of the deletion of residues [14], domain swapping [15,16], the addition of residues [17,18], etc., in αA- and αB-crystallin. In two in vitro studies from our laboratory, we demonstrated that the reverse peptides of mini-αA-crystallin [19] and mini-αB-crystallin [20] failed to show chaperone function, unlike their normal sequence counterparts. Also, we found that reverse RS-1 peptide does not bind to αA-crystallin in vitro, unlike the normal RS-1 peptide [8].

The results described in this paper clearly demonstrate the sequence-specificity of αA- and αB-crystallin interaction.

Methods

Construction, expression, and purification of wild-type αA-, wild-type αB-, and mutant αB-crystallins

Human αA- and αB-crystallin cDNA (obtained from Dr. J. M. Petrash, Washington University, St. Louis, MO) were cloned into pET23d vector (Novagen, San Diego, CA) at the NcoI/HindIII site. The αBinvert-crystallin mutant was constructed using a Quik-Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Mutation was confirmed by automated DNA sequencing. The protein was expressed in Escherichia coli BL21(DE3)pLysS cells (Invitrogen, Carlsbad, CA), as described by Horwitz et al. [21] and was purified according to the method previously described in the literature [22]. On the basis of SDS-PAGE profile, the recombinant protein used in this study was more than 95% pure and its mass, confirmed by nanospray QTOF mass spectrometry, matched the expected theoretical mass of the wild-type protein.

Molecular size determination by dynamic light scattering

To obtain native molecular mass of recombinant αB-crystallin, protein samples in 0.05 M PO₄ buffer containing 0.15 M NaCl, pH 7.4, were passed through the TSK4000 gel filtration column connected to a HPLC system, which is online with a multi-angle laser light scattering detector Dawn-EOS (Wyatt Technology, Santa Barbara, CA) and a refractive index detector (Shimadzu, Columbia, MD). Molar mass and polydispersity were determined as described previously [9].

Tryptophan fluorescence

The intrinsic fluorescence spectra of wild-type and mutant αB-crystallin were recorded using a Jasco spectrofluorimeter FP-750. Protein samples of 0.2 mg/ml in 0.05 M PO₄ buffer containing 0.15 M NaCl (pH 7.4) were excited at 295 nm and emission spectra were recorded between 300-400 nm.

1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS) fluorescence

To 0.2 mg/ml of wild-type and mutant proteins taken in 0.05 M PO₄ buffer containing 0.15 M NaCl (pH 7.4), 10 μM 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS) was added. The samples were excited at 385 nm and the emission spectra were recorded from 400-600 nm using a Jasco spectrofluorimeter.

Circular dichroism studies

Changes in protein secondary structure were measured by far- and near-UV CD spectra in an AVIV circular dichroism spectrometer. Protein concentration was 0.2 mg/ml for far-UV and 3 mg/ml for near-UV measurements. The reported CD spectra are the average of eight scans. Secondary structural elements were determined according to a self-consistent method of Sreerama and Woody [23].

Chaperone-like activity

The ability of the wild-type and mutant proteins to prevent protein aggregation was determined using citrate synthase (CS) and alcohol dehydrogenase (ADH) as the substrates.

Citrate synthase aggregation assay

Citrate synthase (CS), 75 μg (Roche Molecular Biochemicals, Indianapolis, IN) in 1 ml of 40 mM HEPES-KOH buffer (pH 7.4), was heated at 43 °C for 1 h in the presence of 10, 25, and 50 μg of mutant and 50 μg of wild-type proteins. The extent of aggregation was measured by monitoring the light scattering at 360 nm in a Shimadzu spectrophotometer.

Alcohol dehydrogenase aggregation assay

Aggregation of alcohol dehydrogenase (ADH), 400 μg (Sigma, St. Louis, MO), was carried out at 37 °C in 0.05 M PO₄ buffer containing 60 mM NaCl and 100 mM EDTA (pH 7.0) in the presence of 10, 25, and 50 μg of mutant and 50 μg of wild-type αB-crystallin. Light scattering was measured up to 100 min.

Determination of protein stability

Stability of wild-type αB- and the αBinvert-crystallin mutant was determined at 37 °C and 43 °C, the temperatures at which the chaperone assays were performed. Next, 100 μg each of αB-crystallin wild-type and αBinvert-crystallin mutant were separately incubated in 0.05 M PO₄ buffer containing 0.15 M NaCl, pH 7.4, in the absence of target protein, and scattering at 360 nm was monitored for 60 min.

Labeling of recombinant αA-, αB-, and αBinvert-crystallin mutants

Purified αA-crystallin was labeled with Alexa fluor 488 (Molecular Probes, Carlsbad, CA), and wild-type αB-crystallin and αBinvert-crystallin mutant were labeled with Alexa fluor 350 (Molecular probes), using a procedure previously described in the literature [8].

Fluorescence resonance energy transfer measurements

The rate of subunit exchange between αB-crystallin (both wild-type and mutant) and αA-crystallin was measured using the fluorescence resonance energy transfer (FRET) technique. Alexa fluor 350-conjugated wild-type αB-crystallin and its mutant αBinvert-crystallin were used as the energy donors, and Alexa fluor 488-conjugated αA-crystallin was the energy acceptor. αB-350 and αBinvert-350, 25 μg each, and 75 μg of αA-labeled with Alexa 488 were taken in PBS (an αA- to αB-crystallin ratio of 3:1 was used to mimic the in vivo situation) and the sample was incubated at 37 °C. Subunit exchange was monitored by exciting the sample at 346 nm (excitation wavelength for Alexa fluor 350) and measuring the emission spectra from 400-600 nm (emission for Alexa fluor 488 is at 520 nm) for 2 h. As the exchange progresses, the fluorescence intensity of the donor decreases while the acceptor fluorescence increases. The rate of subunit exchange was calculated as described based on information in the literature [8].

Results & Discussion

To determine whether the interaction between αA- and αB-crystallin is sequence-specific, we reversed a sequence that comprised a part of the RS-1 site and the intervening sequence between RS-1 and RS-2 sites such that 54FLRAPSW60 became 54WSPARLF60 (Figure 1). Reversal was achieved by site-directed mutagenesis and the effect of inversion of residues 54-60 on the structure-function relationship of αB-crystallin was studied along with the interaction between αB-crystallin and αA-crystallin.

In our study, both the wild-type αB- and αBinvert-crystallin mutant were overexpressed in Escherichia coli BL21(DE3)plysS cells and were purified by gel filtration and ion-exchange chromatography. Nanospray QTOF mass spectrometry revealed that the molecular mass of both wild-type and αBinvert-crystallin mutant was 20,158.9 Da. Dynamic light scattering (Figure 2) demonstrated that wild-type αB-crystallin had an oligomeric size of 572,000 Da, with a polydispersity of 1.011, whereas αBinvert-crystallin exhibits a smaller oligomer of 356,000 Da, with a decreased polydispersity of 1.001. This results in a reduction of estimated number of subunits from 29 in wild-type αB-crystallin to 18 subunits in αBinvert-crystallin mutant and clearly denotes that proper orientation of residues 54-60 is an important determinant of the final oligomeric size of αB-crystallin. This finding also agrees with our previous report that residues in this region contribute toward quaternary organization of αB-crystallin [9].

Tryptophan fluorescence spectra of wild-type αB- and αBinvert-crystallin mutant were significantly different (Figure 3). The fluorescence intensity of αBinvert-crystallin was 50% lower than that of wild-type αB-crystallin. In addition to the reduction in fluorescence intensity, the αBinvert-crystallin mutant also exhibited a 1 nm blue shift in the tryptophan emission maxima from 343 nm for wild-type αB- to 342 nm for the αBinvert-crystallin mutant. Although the blue shift is not significant, its occurrence in conjunction with a decrease in fluorescence intensity of αBinvert-crystallin mutant indicated that inversion of residues 54-60, where W60 becomes W54, caused W54 to be relatively more buried with quenching of its fluorescence than when it was at W60. The degree of fluorescence suppression is similar to that observed when we mutated W60 to R (αBW60R-crystallin mutant) [9].

To examine whether the observed changes in oligomeric size and intrinsic fluorescence is associated with a change in surface hydrophobicity, we measured the binding of hydrophobic probe bis-ANS to wild-type and mutant proteins. Bis-ANS binding increased only marginally (about 10%) for the αBinvert-crystallin mutant (data not shown), suggesting that the total available hydrophobic sites remain unaltered after inversion of residues 54-60 in αB-crystallin.

Secondary structural analysis of wild-type αB- and αBinvert-crystallin mutant revealed changes in the far-UV CD spectra for the αBinvert-crystallin mutant (Figure 4). The αBinvert-crystallin mutant showed negative ellipticity greater than that of wild-type αB-crystallin. Negative ellipticity is a characteristic of helical structures. However, estimation of the secondary structural elements in αB- and αBinvert-crystallin proteins using a self-consistent method of analysis [23] showed an insignificant increase in α-helical content (1.4%) of the αBinvert-crystallin mutant. In addition, a minor 1.0% increase in random coil, a 1.0% decrease each in β-sheet and β-turn contents were observed in the αBinvert-crystallin mutant as compared to wild-type αB-crystallin.

The near-UV CD spectra were almost similar for the wild-type αB- and αBinvert-crystallin mutant, except for an increase in positive ellipticity for the αBinvert-crystallin mutant (Figure 5). Both wild-type αB- and αBinvert-crystallin mutant exhibited maxima at 259 and 265 nm that are characteristic of Phe fine structure, but the αBinvert-crystallin mutant showed greater amplitude. The transitions that arise between 270 and 290 nm are contributed by Tyr and Trp residues. These transitions were exhibited by both wild-type and mutant αB-crystallins, although the αBinvert-crystallin mutant exhibited an increase in positive ellipticity. As a result of inversion, the positions of seven amino acids were changed, thereby affecting the interactions of these amino acids with the others in the core region. Negative transitions at 293 nm are characteristic of Trp residues, and both wild-type and mutant exhibited at similar amplitude. The change observed in Trp fluorescence spectra of the αBinvert-crystallin mutant may not translate to a decrease in 293 nm signal because, as reported earlier, the near UV CD spectra of αB-crystallin is not clearly discernible beyond 270 nm [24]. In a previous study, we have also observed that when the Trp residue was replaced by Arg in αBW60R-crystallin, there was no change in the 293 nm band in the near UV CD spectra [9].

The stability of wild-type αB- and αBinvert-crystallin mutant was determined by measuring light scattering of the proteins at 37 °C and 43 °C, the temperatures used in the chaperone assays. At both these temperatures, αBinvert-crystallin mutant behaved like wild-type αB-crystallin by not showing any light scattering (data not shown). This indicates that the inversion of residues 54-60 does not affect the stability of αB-crystallin at these temperatures.

To investigate the role of residues 54-60 in chaperone-like function of αB-crystallin, we examined the chaperone-like activity of wild-type αB- and αBinvert-crystallin mutant by using two different substrates, ADH and CS. Figure 6A shows the EDTA-induced aggregation of ADH at 37 °C in the presence of both wild-type and mutant proteins at different concentrations. The αBinvert-crystallin protein showed chaperone-like activity similar to that of wild-type αB-crystallin. The results were similar when thermal aggregation of CS was monitored in the presence of wild-type and mutant proteins (Figure 6B). This finding suggests that inversion of residues 54-60 in αB-crystallin does not disrupt the chaperone site nor modulate chaperone-like activity. These results confirm our earlier observation that the chaperone site and the subunit interaction site are distinct [8,9].

Dynamic behavior is a characteristic of both homo- and heterooligomers of sHSPs [4]. The minimal oligomeric state required for the exchange of subunits is a tetramer in the case of αA-crystallin, as dimers and trimers fail to exchange subunits [5]. However, in nonmammalian sHSPs, even dimers can exchange subunits with their counterparts [25]. By creating point mutations, we previously showed that RS-1 and RS-2 regions in αB-crystallin have an important role in interaction with αA-crystallin [8,9]. In the current study, inverted residues in the RS-1 and intervening sequence between RS-1 and RS-2 sites made it imperative to investigate the dynamics of subunit exchange of the αBinvert-crystallin mutant with αA-crystallin. While wild-type αB-crystallin exchanged subunits with αA-crystallin at a rate of 11.06x10^-4 sec^-1, the αBinvert-crystallin mutant exchanged slowly with αA-crystallin at a rate of 5.98x10^-4 sec^-1 (Figure 7). Thus, inversion of residues 54-60 significantly affects the dynamics of αB-crystallin, and the interaction between αB- and αA-crystallin appears to be sequence-specific. Although the exact role of this slower/faster subunit exchange is not known, it appears that it affects the formation of an efficient functional heterooligomer, most probably by contributing to the overall stability [14,26,27].

Studies of α-crystallin structure and function have not yet delineated fundamental properties required for subunit exchange. Distinct reports exist regarding correlation between oligomeric size, dynamics of α-crystallin subunits, and chaperone function. COOH-terminally truncated αA-crystallin, αA-crystallin(1-168), forms smaller oligomers (18.5% reduction) than wild-type αA-crystallin and has chaperone-like activity similar to that of wild-type, but exchanges subunits at a two to three fold slower rate [26]. Therefore, no correlation apparently exists between chaperone function and subunit dynamics [26]. On the contrary, arginine hydrochloride (Arg.HCl)-induced dissociation of α-crystallin subunits leads to enhanced subunit exchange and increased chaperone function [28]. In the case of Hsp 16.3, a highly monodisperse dodecameric form is the inactive storage form and its dissociation is a prerequisite for chaperone function [29,30]. But this inactive storage form exhibits dynamic dissociation/reassociation even at 4 °C [30]. Recently, our lab showed that αBS66G-crystallin mutant forms large oligomers that exchange subunits with αA-crystallin at a 100% accelerated rate but exhibit normal chaperone function like wild-type αB-crytsallin [9]. The αBinvert-crystallin mutant in the current study formed oligomers that were 38% smaller than their wild-type counterparts and showed no difference in the chaperone efficacy as compared to wild-type αB-crystallin. Subunit dynamics, however, were slowed by two fold. Inversion of 54-60 residues apparently does not disrupt the chaperone site. It appears that mechanisms involved in subunit exchange and substrate recognition during chaperone function may be different, as no common rule/platform applies to the afore mentioned proteins.

Inversion of a stretch of seven residues, 54-60 of αB-crystallin with four residues from RS-1 region (42-57 residues), two residues which form the intervening sequence between RS-1 and RS-2 sites, and one residue from RS-2 region (60-71 residues), alters the interaction of αB-crystallin with contact points in αB- (homooligomer) and αA-crystallin. In this way, inversion affects the oligomeric size and subunit dynamics. The overall effect of inversion of residues 54-60 is different from that of the single-residue mutation we previously created in this region [9], as interaction of 7 amino acids is affected at the same time. We feel that studies involving inversion of residues are a better indicator of the role of a stretch of amino acids rather than the experiments following deletion of those residues, for the following reasons: (1) the inversion of residues does not affect the molecular size of the subunit; and (2) it is unlikely to affect the structure of strands or helices away from the point of inversion. Therefore, our study, for the first time, pinpoints the significance of proper orientation of residues contributing to RS-1 and RS-2 sites of αB-crystallin for interaction with αA-crystallin.

Acknowledgements

We thank Sharon Morey for help in the preparation of this manuscript. We acknowledge the technical assistance of Jing Wang and Elizabeth Cheney. We also thank Dr. Santhoshkumar Puttur and Dr. Lixing Reneker for helpful discussions in the course of this study. This work was supported by NIH grants EY 11981 and EY 14795 awarded to KKS and a grant-in-aid from research to prevent blindness (RPB), awarded to University of Missouri Department of Ophthalmology.

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