Phe71 Is Essential for Chaperone-like Function in αA-crystallin

Abstract

Experiments with mini-␣A-crystallin (KFVIFLD-VKHFSPEDLTVK) showed that Phe 71 in ␣A-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 Phe 71 in rat ␣A-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 ␤ L -crystallin aggregation was observed when mutant ␣A-crystallin was included. These results suggest that Phe 71 contributes to the chaperone-like action of ␣A-crystallin. Therefore we conclude that the 70 -88-region in ␣A-crystallin, identified by us earlier, is the functional chaperone site in ␣A-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 (5-8). 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 (12-15). 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 (22-24). Earlier we were able to map the site in ␣A-and ␣B-crystallin responsible for chaperone-like action using photoactive cross-linkers and hydrophobic probes (25-27). 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-crys...

Deamidation Affects Structural and Functional Properties of Human αA-Crystallin and Its Oligomerization with αB-Crystallin

“…However, such binding caused a decrease in chaperone activity (42). Recent studies in ␣A-crystallin have suggested that factors other than hydrophobicity might also play important roles in the chaperone activity (45)(46)(47). The observation that the hydrophobicity is not the sole determinant of chaperone activity was also supported by our findings.…”

Insights into Hydrophobicity and the Chaperone-like Function of αA- and αB-crystallins

“…There are numerous instances where hydrophobicity could not be directly correlated to the chaperone-like activity of ␣A-and ␣B-crystallins. For instance, replacement of Phe-71 with Gly in ␣A-crystallin results in loss of chaperone activity despite an increase in surface hydrophobicity with no significant alterations in structure (26). In another study, calf lens ␣A-crystallin was found to be more hydrophobic but showed lower chaperone activity than ␣B-crystallin at room temperature (18).…”

Structure and interactions in α-crystallin probed through thiol group reactivity

“…The sequence of α-crystallin is broadly divided into N-terminal region consisting of about 60 residues, the α-crystallin domain consisting of about 80 -90 residues and a C-terminal tail consisting of 25 -30 residues [4,5,8,9]. Numerous studies done on -crystallinsubstrate interaction have implicated parts of the N-terminal region as substrate binding site [7,[10][11][12]. This region has also been reported to control the oligomeric size of the chaperone [13][14][15][16].…”