Phe71Is Essential for Chaperone-like Function in αA-crystallin

Experiments with mini-αA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe71 in αA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767–3771). In the present study we replaced Phe71 in rat αA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1′-bi(4-anilino)naphthalene-5,5′-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285–295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of βL-crystallin aggregation was observed when mutant αA-crystallin was included. These results suggest that Phe71 contributes to the chaperone-like action of αA-crystallin. Therefore we conclude that the 70–88-region in αA-crystallin, identified by us earlier, is the functional chaperone site in αA-crystallin.

␣-Crystallins are major refractive proteins in the vertebrate eye lens. When isolated from the lens they exist as polydisperse aggregates having an average molecular mass of Ϸ800 kDa (1,2). ␣-Crystallin is composed of two subunits, ␣A and ␣B, which have considerable sequence homology between them and with other heat shock proteins (3,4). Recently ␣-crystallin subunits were also reported to be present in nonlenticular tissues like heart, brain, and kidney (5)(6)(7)(8). The significance of their presence in nonlenticular tissues is not clear. However, the increased expression of ␣B-crystallin observed in a variety of neurological disorders has drawn significant medical attention (9 -11). Like other small heat shock proteins, ␣-crystallin can sequester certain unfolding proteins in vitro, by preventing their aggregation and insolubilization (12)(13)(14)(15). Both subunits of ␣-crystallin show chaperone-like activity to different extents (16 -18). Complex formation with ␤ L -and ␥-crystallin and decreased chaperone-like function during aging has indicated the importance of ␣-crystallin in maintaining the transparency of the lens (2, 19 -21). During chaperone-like action, hydrophobic surfaces in ␣-crystallin interact with specific sites in non-native target proteins (22)(23)(24). Earlier we were able to map the site in ␣Aand ␣B-crystallin responsible for chaperone-like action using photoactive cross-linkers and hydrophobic probes (25)(26)(27). Our studies with bis-ANS 1 and the hydrophobic protein mellitin have shown that there is an overlapping of chaperone site and hydrophobic site in ␣A-crystallin. Further, using a synthetic peptide (mini-␣A-crystallin), we were able to demonstrate the importance of sequence 70 -88 in the chaperonelike action of ␣A-crystallin (28). The experiments with truncated forms of mini-␣A-crystallin had suggested that Phe 71 in ␣A-crystallin might be critical for chaperone-like function. In the present study, we did a site-directed mutagenesis of Phe 71 to Gly in ␣A-crystallin and compared the structural and functional properties of this mutant protein with the wild-type protein.
Recombinant ␣Aand ␣B-crystallins show similar structural and functional properties to crystallins isolated from lens tissues and are widely used in the characterization of the protein.
Site-directed mutations of recombinant protein provide an excellent means of studying the role of constituent amino acids in the functional properties of the protein. Earlier, several sitedirected mutations were conducted on ␣-crystallin either to identify the region responsible for chaperone-like function or to explain the role of ␣-crystallin in hereditary cataracts and certain other diseases (29 -38). The majority of these studies report either no change in chaperone-like function or a partial loss of this function. We report here, for the first time, a complete loss in the functional property of a mutant ␣A-crystallin at and slightly above physiological temperatures. The results also indicate the presence of additional sites in ␣-crystallin that become available at elevated temperatures. We conclude here that the region identified by us earlier as chaperone site contributes to the chaperone-like activity of ␣A-crystallin.

EXPERIMENTAL PROCEDURES
Preparation of the Mutant Clone-Rat ␣A-crystallin cDNA cloned in pET21b was kindly donated by Dr. Suraj Bhat (UCLA). ␣AF71G mutant was constructed using a QuikChange site-directed mutagenesis kit (Stratagene). The following set of primers were used: 5Ј-CTGACCGG-GACAAGGGTGTCATCTTCTTGG-3Ј and 5Ј-CCAAGAAGATGACAC-CCTTGTCCCGGTCAG-3Ј. The mutation was confirmed by automated DNA sequencing.
Expression and Purification of Wild-type and Mutant ␣A-crystallin-The proteins were expressed in Escherichia coli BL21(DE3) cells (Novagen) as described by Horwitz et al. (39). The proteins were isolated from the cell pellet using Bugbuster protein extract reagent (Novagen). In brief, 1 g of cells was suspended in 5 ml of reagent at room temperature and vortexed gently. Protease inhibitor mixture set III (Novagen) was then added. The cell suspension was treated with 1 l (25 units) of benzonase/ml of Bugbuster reagent and incubated at room temperature on a shaking platform for 30 min. The extract was centrifuged at * This work is supported in part by National Institutes of Health Grant EY11981 and a grant-in-aid from Research to Prevent Blindness. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Mason Eye Inst., Dept. of Ophthalmology, 1 Hospital Dr., University of Missouri, Columbia, MO 65212. E-mail: Sharmak@health.missouri.edu. 17,000 ϫ g for 2 h, and the supernatant was filtered through a 0.2-m filter. The filtrate was loaded onto a Bio-Rad High Q anion exchange column and eluted using a linear gradient of NaCl (0 -1 M) in 20 mM Tris-HCl (pH 8) at a flow rate of 2 ml/min. The fractions containing the recombinant crystallin (determined by SDS-PAGE) were pooled and concentrated. The concentrated protein was purified further on a C18 reverse phase HPLC using a water-acetonitrile gradient containing 0.1% trifluoroacetic acid. The peaks corresponding to wild-type and mutant ␣A-crystallin were pooled, dried on a Speedvac, resuspended in 6 M urea, and dialyzed extensively against 0.05 M phosphate (PO 4 ) buffer containing 0.15 M NaCl, with several changes, for a period of 2 days. The purity of the proteins was checked by SDS-PAGE, and the mass was determined by mass spectrometry.
Molecular Size Determination-Size determination was carried out using a Amersham Biosciences Hiload 16/60 Superdex 200 gel filtration column equilibrated with 0.05 M PO 4 buffer containing 0.15 M NaCl (pH 7.4). The mass was calculated from the calibration curve generated by using Sigma molecular weight marker standards.
Tryptophan Fluorescence Measurements-The intrinsic fluorescence spectra of the wild-type and mutant ␣A-crystallin were recorded using a Jasco spectrofluorometer FP-750. Protein samples of 200 g/ml in 0.05 M PO 4 buffer containing 0.15 M NaCl were used. The excitation was set to 295 nm, and the emission was recorded between 300 and 400 nm.
bis-ANS Fluorescence Measurement-To 100 g of wild-type and mutant protein taken in 0.05 M PO 4 containing 0.15 M NaCl (pH 7.4) was added 20 l of 10 mM bis-ANS dissolved in ethanol. The sample was excited at 365 nm, and the fluorescence spectrum was measured between 400 and 600 nm using a Jasco spectrofluorometer.
Circular Dichroism Studies-Protein secondary and tertiary structural changes were investigated by far and near UV CD measurements using an AVIV circular dichroism spectrometer. The concentration of the proteins used was 1.5 and 0.35 mg/ml for near and far UV CD, respectively. The reported CD spectra are the averages of four scans.
Chaperone-like Activity-The ability of the wild-type and mutant proteins to prevent protein aggregation was determined using several substrates. The extent of aggregation was measured by monitoring the light scattering at 360 nm in a Shimadzu spectrophotometer. Insulin

RESULTS
Characterization of Recombinant ␣A-crystallin-In the present study, the recombinant proteins were purified by a combination of ion exchange and reverse phase HPLC. The purified proteins were dissolved in urea and refolded by extensive dialysis. The proteins thus obtained were highly pure (Fig. 1). The ES mass spectrometry analysis revealed molecular masses of 19,799 and 19,709 daltons, which would be expected for the wild-type and mutant ␣A-crystallin respectively. Like the ␣-crystallin subunits isolated from eye lens, recombinant proteins exist in oligomeric form. For analysis of the molecular mass of the homoaggregates, the purified recombinant proteins were chromatographed on a Superdex-200 column. Both wildtype and ␣AF71G mutant proteins showed similar elution profiles, corresponding to an oligomeric mass of 7.1 ϫ 10 5 daltons (Fig. 2). This is slightly higher than the earlier published values for rat ␣A-crystallin (37). The discrepancy can be attributed to the different buffer conditions used in analysis, because it is known that the mass of the purified protein varies depending on the buffer condition (40,41). During these studies we also observed a similar mass for reconstituted homopolymers of bovine lens ␣A-crystallin.
The structural differences between the wild-type and mutant proteins were analyzed by spectroscopic methods. Tryptophans of the protein have a fixed solvent accessibility, and any change in their environment leads to an altered fluorescence emission pattern and intensity. Our results show no change in the tryptophan region of the recombinant proteins, as evidenced by the similar fluorescence emission maximum (340 nm) and intensity (Fig. 3). Unfolding of proteins increases the exposure of hydrophobic surfaces that can be probed with bis-ANS fluorescence (26). We see an increase in bis-ANS binding to the ␣AF71G mutant (Fig. 4), indicating an increased hydrophobicity compared with wild-type protein.
The secondary and tertiary structures of wild-type and mutant ␣A-crystallin were determined by far and near UV CD spectral analysis. The far UV profile showed a characteristic ␤-sheet conformation with a slight increase in the negative intensity of the mutant protein (Fig. 5). Both proteins showed similar amounts of ␣-helix, ␤-sheet, and random coil (42). Near

F71G Mutant of ␣A-crystallin
UV CD spectra showed a slight increase in the negative intensity of the mutant protein (Fig. 6). Although significant portions of the near UV spectra for the two proteins were similar, only minor changes were seen in the 285-295 nm region of the spectra, suggesting some differences in the tyrosine and/or tryptophan microenvironments of the mutant protein compared with the wild-type ␣A-crystallin. Surprisingly enough, there was no alteration in the signal caused by phenylalanine in the 250 -270-nm region. In summary, the data in Fig. 6 do not suggest a significant difference in the tertiary structure between wild-type and mutant ␣A-crystallin.
The Chaperone-like Activity of F71G ␣A-crystallin-The consequence of mutation on recombinant crystallin chaperone-like activity was determined under different conditions. Reduction of insulin results in the separation of the subunits and precipitation of B chain that can be followed by measurement of light scattering. The presence of ␣-crystallin subunits in the assay prevents the aggregation of insulin B chain, and the solution remains clear. Fig. 7 shows the dithiothreitol-induced aggregation kinetics of insulin in the presence of both wild-type and mutant ␣A-crystallins. The wild-type protein showed suppression of insulin B chain aggregation that increased with the concentration of the protein in the assay tube. However, the mutant ␣A-crystallin completely failed to prevent the formation of light-scattering aggregates. In fact, a marginal increase in light scattering was observed in some assays. Higher concentrations of mutant protein had no effect on the aggregation of polypeptide. The chaperone-like activity of the recombinant proteins was also investigated at different temperatures.

F71G Mutant of ␣A-crystallin
shows the thermal aggregation of CS in the presence of wildtype and mutant proteins. Although the wild-type protein (50 g) completely suppressed the aggregation of CS (75 g), the mutant protein, as with insulin, failed to prevent the aggregation of denaturing CS. We also analyzed the ability of recombinant proteins to suppress the aggregation of ADH at 45°C. The wild-type ␣A-crystallin showed increased suppression of denaturing protein aggregation with increasing concentration (Fig. 9A). Although the mutant protein appeared to suppress the aggregation of ADH at initial time points, the aggregation at 80 min was comparable with ADH by itself (Fig. 9B). Increasing the concentration of mutant protein had no effect on the aggregation of ADH. We also compared the abilities of mutant and wild-type ␣A-crystallin to prevent the heat-induced aggregation of ␤ L -crystallin at 55°C (Fig. 10). Unlike other substrates, mutant ␣A-crystallin showed a significant protection of ␤ L -crystallin with increasing concentration. However, compared with the wild-type ␣A-crystallin, the mutant ␣A-crystallin was 6 -10-fold less effective in suppressing ␤ L -aggregation. DISCUSSION ␣A-crystallin subunit has been categorized into three domains: an N-terminal domain containing residues 1-66, a Cterminal or ␣-crystallin domain (central core) comprising residues 64 -105, and an extended C-terminal including residues 106 -173 (43)(44)(45). Most of the mutational studies on ␣A-crystallin were conducted either on the N-terminal domain or the C-terminal extension. Derham and Harding (46) have reviewed the mutations conducted by different laboratories on ␣-crystallin and have recently reanalyzed the chaperone-like activity of several mutants (35). In the present study, we produced an ␣A-crystallin mutant by substituting Phe 71 in the core region with a neutral amino acid Gly. This residue is highly conserved in ␣A-crystallin and is located in the region identified as the chaperone site of ␣A-crystallin (28). Biophysical characterization of the recombinant protein revealed no change in the oligomer size or tryptophan fluorescence. Because the ␣-crystallin molecule has only one tryptophan at position 9, the intrinsic tryptophan fluorescence data may be of limited value

F71G Mutant of ␣A-crystallin
to describe the structural changes in the central core or Cterminal domain as a consequence of the mutation. However, it will be a valuable tool for analyzing the stability of the Nterminal domain of the mutant protein. When the mutant protein was heated up to 60°C, we did not observe any aggregation or shift in tryptophan fluorescence emission wavelength or intensity (data not shown), suggesting that the heat stability of the protein was not affected by the mutation.
It has been hypothesized that hydrophobic sites in ␣-crystallin are responsible for chaperone-like activity (22,23). However, this is not free of controversy (29,47). In the present study we see a complete loss in the chaperone-like function of mutant ␣A-crystallin at and slightly above physiological temperatures despite an increase in hydrophobicity. Smulders et al. (29) observed an increase in the chaperone-like activity of an ␣AF74N mutant with a slight decrease in ANS binding. They concluded that there is no correlation between surface hydrophobicity and chaperone-like activity. Experiments with super-␣A-crystallin have indicated that the disappearance of chaperone-like activity may be independent of hydrophobicity (47). Further, Reddy et al. (18) have shown recently that hydrophobicity is not the sole determinant of chaperone-like activity in ␣-crystallin. Recently, the studies with mini-␣A-crystallin showed that both hydrophobicity and ␤-sheet conformation of the functional element are essential for chaperone-like activity (48). Although we see increased exposure of hydrophobic surfaces in the mutant, it is quite unlikely that all exposed hydrophobic patches would be involved in suppressing the substrate protein aggregation. We, as well as others, have observed bis-ANS binding to residues other than those necessary for chaperone activity (27,49). Taking these observations together, one can conclude that although hydrophobicity is important, the extent of hydrophobicity does not reflect the chaperone-like activity of the protein.
The ␣AF71G mutant has similar secondary structure to that of the wild type. However, the tertiary structure shows some minor changes around the 285-295-nm region. The signal in this region is produced by tyrosine or tryptophan residues. Because we did not observe any change in the tryptophan fluorescence intensity, it is possible that the alteration is in the tyrosine region. Interestingly, two tyrosine residues in ␣Acrystallin are found near the bis-ANS-binding region 50 -54 (27). This may explain the increased bis-ANS binding of the mutant protein. However, it is unlikely that such a minor difference in the near UV CD signal would completely abolish the chaperone-like activity of the molecule. The ␣AR116C mutant, with structural alterations at many levels, showed only a 25% decrease in chaperone-like activity (37,38). Further, it has been shown that ␣-crystallin could preserve its chaperone function despite some irreversible structural changes (50).
We have measured the chaperone-like function of the ␣AF71G mutant under different conditions and observed a complete loss in the activity of the mutant up to 45°C. However, at elevated temperatures the mutant showed some suppression of ␤ L -crystallin aggregation. It has been shown that ␣-crystallin undergoes a structural transition around 55°C, resulting in the exposure of more hydrophobic patches (23,24,26). Our study on the stabilization of restriction enzyme (51) as well as studies conducted by others (35,52) indicates the presence of multiple sites in ␣-crystallin for chaperone function. Based on the experiments with mini-␣A-crystallin (28) and the complete loss of chaperone-like function of the mutant protein at physiological temperatures in this study, we conclude that the region identified by us earlier (residues 71-88) contributes to the chaperone-like function.
Plater et al. (30) reported that the F27R mutation in the N-terminal domain of ␣B-crystallin completely abolishes its chaperone-like activity at higher temperatures, which led them to conclude that this conserved residue is vital for chaperone function. Their report is controversial because later studies have shown that the mutant F27R is fully active (34,35). Earlier, work showed that proteins resulting from mutation of V72N and F74N in the core region of ␣A-crystallin had normal activity (29). However, their conclusion was based on a single assay conducted at 58°C. We also found some activity of the mutant with ␤ L -crystallin around this temperature. Also, unlike Phe 71 , the Val 72 and Phe 74 residues show variations in different vertebrate lens species. The conserved Phe 71 residue appears to be important for suppressing the aggregation of proteins. Other factors like charge, hydrophobicity, and structural integrity may influence the functional property to different extents. Recently, Kumar and Rao (53) produced a chimeric ␣-crystallin by swapping the domains of ␣Aand ␣B-crystallin and tested its effect on the chaperone-like activity. Interestingly, the ␣ANBC chimeric protein which contained residues 1-79 of ␣A-crystallin, including a part of the functional site in ␣A-crystallin, was completely inactive in suppressing the aggregation of insulin. However, the ␣BNAC chimeric protein containing the complete ADH binding sequence (25) and a part of the functional site in ␣A-crystallin had enhanced chaperonelike activity. This suggests that other residues in the chaperone site of ␣A-crystallin are also important in suppressing the aggregation of proteins. Therefore it would be interesting to study the role of other conserved residues on chaperone-like action.