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

Abstract

Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in αB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with αA-crystallin. To understand the significance of the RS-2 region in interactions between αA- and αB-crystallins, W60R, F61N, and S66G mutants of αB-crystallin were made and tested for their ability to interact with αA-crystallin. W60R and S66G mutations increased the oligomeric size of αB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of αBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of αBF61N was marginally lower than that of αB, whereas the fluorescence intensity of αBW60R decreased by 40% compared with that of αB. The relative availability of hydrophobic sites in the mutants was in the following order: αBS66G ≫ αB = αBF61N = αBW60R. The far-UV CD profiles for the wild type and αB-crystallin mutants indicated no significant changes in their secondary structures, except for αBS66G, which showed an increase in α-helical content. The near-UV CD profiles of αBW60R and αBF61N were nearly similar to that of wild type αB. On the other hand, αBS66G beyond 270 nm exhibited a signature completely different from that of wild type αB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between αB- and αA-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 αB- and αA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of αB-crystallin and subunit interaction between αB- and αA-crystallins. © 2005 American Chemical Society.