A–crystallin Exposes Cryptic Substrate Binding Sites on Structural Perturbation

Abstract

Abstract: : Purpose: Previously, we have shown that F71G mutant of αA crystallin fails to prevent the aggregation of proteins at 37^oC but shows significant chaperone–like activity at 55^oC suggesting the activation of "cryptic" functional sites on structural perturbations. The purpose of this study was to delete the chaperone site sequence in αA (70–88) and see if additional sites are available for interaction and suppression of denaturing substrate proteins. Methods: Two deletion mutants, αAΔ70–77 and αAΔ70–88 were prepared using Exsite PCR–based Site–Directed Mutagenesis Kit (Stratagene). The wild–type and mutant proteins were expressed in E.coli BL21(DE3)pLysS cells. The deletion mutants were isolated from the inclusion bodies using urea and precipitated using 30% ammonium sulfate. The precipitate was resolubilized in presence of urea and purified on a reverse phase HPLC fitted with a C18 column. The sample was dried, resuspended in urea buffer and dialyzed extensively against Phosphate buffer. The structure and degree of hydrophobicity of the proteins were analyzed by spectroscopic methods. The chaperone–like activities of the wild–type and mutant proteins were analyzed using citrate synthase (CS) as the substrate. Results: Both deletion mutants after isolation from inclusion bodies and refolding had a molecular mass of >2mDa. However, the wild–type protein isolated from cell extract and purified under identical conditions had a mass of ∼650kDa. Deletions of chaperone site sequence lead to an increase (20–30%) in intrinsic tryptophan fluorescence. Interestingly, deletion of chaperone site in αA, which was earlier identified as a bis–ANS binding site, lead to 50–60% increase in dye binding. The αAΔ70–77 was 40–50% less effective in suppressing the aggregation of CS when compared to wild–type. The αAΔ70–88 completely failed to prevent the aggregation of CS when used at ratio similar to wild–type. However, when used at 5–fold excess, αAΔ70–88 showed 25% protection during CS denaturation assay. Conclusions: The results indicate the exposure of "crytptic" chaperone sites in αA–crystallin on structural perturbation. Marginal chaperone activity of the deletion mutants suggest that the residues 70–88 in αA–crystallin acts a primary chaperone site and contributes to the bulk of the total chaperone–like activity of the protein.

Keywords: chaperones • protein structure/function • crystallins