Recognition sequence 2 (residues 60-71) plays a role in oligomerization and exchange dynamics of αB-crystallin

Previously, using the peptide scan method, we have determined that residues 42-57 and 6071 in alpha B-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with alpha A-crystallin. To understand the significance of the RS-2 region in interactions between alpha A- and alpha B-crystallins, W60R, F61N, and S66G mutants of alpha B-crystallin were made and tested for their ability to interact with alpha A-crystallin. W60R and S66G mutations increased the oligomeric size of alpha B-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of alpha BS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of alpha BF61N was marginally lower than that of alpha B, whereas the fluorescence intensity of alpha BW60R decreased by 40% compared with that of alpha B. The relative availability of hydrophobic sites in the mutants was in the following order: alpha BS66G >> alpha B = alpha BF61N = alpha BW60R. The far-UV CD profiles for the wild type and alpha B-crystallin mutants indicated no significant changes in their secondary structures, except for alpha BS66G, which showed an increase in alpha-helical content. The near-UV CD profiles of alpha BW60R and alpha BF61N were nearly similar to that of wild type alpha B. On the other hand, alpha BS66G beyond 270 nm exhibited a signature completely different from that of wild type alpha B. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between alpha B- and alpha A-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between alpha B- and alpha A-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of alpha B-crystallin and subunit interaction between alpha B- and alpha A-crystallins.