Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human αB-crystallin

Human alpha B-crystallin is a small heat-shock protein that functions as a molecular chaperone. Recent studies indicate that deletion of a peptide ((54)FLRAPSWF(61)) from its N-terminus makes it a better chaperone, and this particular sequence is thought to participate in substrate interaction and subunit exchange with alpha A-crystallin. To determine whether the positive charge on arginine 56 (R56) influences these functions, we prepared human alpha B-crystallin mutants in which R56 was deleted (Delta R56) or replaced by alanine (R56A). To determine if the effects are specific to R56, we generated two additional mutant proteins in which the two neighboring amino acids were deleted (Delta L55 and Delta A57). Dynamic light scattering studies suggested that none of the mutations affected the oligomeric mass of the protein. Far-ultraviolet circular dichroism (UV CD) spectra revealed greater helicity in the secondary structures of R56A and Delta R56 compared to that of the wild-type (Wt) protein. Near-UV CD spectra showed that the tertiary structure is perturbed in all mutants. Insulin and citrate synthase aggregation assays showed 38 and 30% improvement of chaperone function in Delta R56 compared to that of the Wt. In contrast, the R56A mutant lost most of its chaperone function. Deletion mutants, Delta L55 and Delta A57, showed no significant changes in the chaperone function compared to that of the Wt. The Delta R56 mutant had a higher surface hydrophobicity than the Wt, but the R56A mutant had a lower hydrophobicity. Our data show paradoxical effects of the deletion and substitution of R56 and imply that the chaperone function of human alpha B-crystallin is dictated not only by the positive charge on R56 but also by the conformational change that it bestows on the protein.