Synthesis and Characterization of a Peptide Identified as a Functional Element in aA-crystallin*

Eye lens a-crystallin is a member of the small heat shock protein (sHSP) family and forms large multimeric structures. Earlier studies have shown that it can act like a molecular chaperone and form a stable complex with partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to a-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in a-crystallin. To investigate the mechanism of the interaction between a-crystallin and substrate proteins, we determined the melittin-binding sites in a-crystallin by cross-linking studies. Localization of melittin-binding sites in a-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of aAcrystallin and FSVNLDVK of aB-crystallin as the chaperone sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified earlier as 1,1*-bi(4-anilino) naphthalene-5,5*-disulfonic acid (bis-ANS)-binding hydrophobic sites. Here we also report the synthesis and characterization of the peptide, KFVIFLDVKHFSPEDLTVK, having the melittin as well as bis-ANS-binding sequence of aA-crystallin. We show that this peptide has characteristics similar to that of aA-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the b3 and b4 region present in the a-crystallin domain of sHSP 16.5. We hypothesize that the a-crystallin domain in other sHSPs may have a similar function and would likely possess the anti-aggregation property even when separated from the native protein.