Synthesis and characterization of a peptide identified as a functional element in alphaA-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 partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to alpha-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in alpha-crystallin. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we determined the melittin-binding sites in alpha-crystallin by cross-linking studies. Localization of melittin-binding sites in alpha-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of alphaA-crystallin and FSVNLDVK of alphaB-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 alphaA-crystallin. We show that this peptide has characteristics similar to that of alphaA-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the beta3 and beta4 region present in the alpha-crystallin domain of sHSP 16.5. We hypothesize that the alpha-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.
Sharma
K K
KK
Mason Eye Institute, Department of Ophthalmology, University of Missouri, Columbia, Missouri, 65212, USA. sharmak@health.missouri.edu
Kumar
R S
RS
Kumar
G S
GS
Quinn
P T
PT
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry*; D006360:Heat-Shock Proteins / Q000737:chemistry; D008555:Melitten / Q000737:chemistry*; D018832:Molecular Chaperones / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D008555:Melitten; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000426:Alcohol Dehydrogenase