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

• Published in: The Journal of biological chemistry Vol. 275; no. 6; pp. 3767 - 3771
• Main Authors: Sharma, K.Krishna, Kumar, R.Senthil, Kumar, G.Suresh, Quinn, P.Thomas
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 11.02.2000
• Subjects: alcohol dehydrogenase; amino acid sequences; binding; binding sites; cattle; crystallins; eye lens; melittin; peptides; synthetic peptides
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.6.3767

Eye lens α-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 α-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in α-crystallin. To investigate the mechanism of the interaction between α-crystallin and substrate proteins, we determined the melittin-binding sites in α-crystallin by cross-linking studies. Localization of melittin-binding sites in α-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of αA-crystallin and FSVNLDVK of αB-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 αA-crystallin. We show that this peptide has characteristics similar to that of αA-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the β3 and β4 region present in the α-crystallin domain of sHSP 16.5. We hypothesize that the α-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.