Does Conserved Histidine Residue (H83) Has Any Role in the Chaperone-like Action of Alpha B Crystallin?

Abstract

Abstract: : Purpose: The H83 in alpha B crystallin is highly conserved in most vertebrate species and is localized in the target protein binding region. The aim of this study was to determine the role of conserved H83 residue in chaperone-like function of alpha B crystallin by replacing it by Ala and analyzing the structural and functional properties of the mutant protein. Methods: The H83 in human alpha B crystallin was replaced by Ala by site-directed mutagenesis. The mutant protein was expressed in E.coli BL21(DE3)pLysS cells. The cells were lysed using bugbuster reagent (Novagen) and the protein was purified by ion-exchange and gel filtration chromatography. The purity was checked by SDS-PAGE and the mutation was confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone-like activity of wild-type and mutant were compared using several substrates. Results: Both wild-type and mutant alpha B crystallin showed similar elution profile during gel filtration chromatography with a molecular mass of 3.6 x 10₅ daltons. The mutant protein showed two-fold increase in tryptophan fluorescence intensity and an increased binding of bis-ANS when compared to wild-type. The H83A mutant was more effective in suppressing the aggregation of denaturing proteins when compared to wild-type. Conclusion: The higher chaperone-like action of the mutant can be due to increased unfolding and exposure of additional hydrophobic surfaces compared to wild-type protein. The results indicate that despite highly conserved, H83 in alpha B crystallin is not a critical residue for chaperone-like action of the protein.

Keywords: 343 chaperones • 338 cataract • 480 mutations