Synthesis and characterization of a peptide identified as a functional element in alpha A-crystallin

Eye lens alpha-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 p artially unfolded proteins, We have observed that prior binding of the hydr ophobic protein melittin to alpha-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence o f mutually exclusive sites for these proteins in alpha-crystallin, To inves tigate the mechanism of the interaction between alpha-crystallin and substr ate proteins, we determined the melittin-binding sites in alpha-crystallin by cross-linking studies. Localization of melittin-binding sites in alpha-c rystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLT VK of alpha A-crystallin and FSVNLDVK of alpha B-crystallin as the chaperon e sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified ea rlier as 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS)-bind ing hydrophobic sites. Here we also report the synthesis and characterizati on of the peptide, KFVIFLDVKHFSPEDLTVK, having the melittin as well as bis- ANS-binding sequence of alpha A-crystallin. We show that this peptide has c haracteristics similar to that of alpha A-crystallin by in vitro thermal ag gregation assay, gel filtration study, CD spectroscopy, and bis-ANS interac tion studies. The peptide sequence corresponds to the beta 3 and beta 4 reg ion present in the cu-crystallin domain of sHSP 16.5. We hypothesize that t he alpha-crystallin domain in other sHSPs may have a similar function and w ould likely possess the anti-aggregation property even when separated from the native protein.