Rapid refolding studies on the chaperone-like alpha-crystallin. Effect of alpha-crystallin on refolding of beta- and gamma-crystallins.
alpha-Crystallin, a multimeric protein present in the eye lens, is shown to have chaperone-like activity in preventing thermally induced aggregation of enzymes and other crystallins. We have studied the rapid refolding of alpha-crystallin, and compared it with other calf eye lens proteins, namely beta- and gamma-crystallins. alpha-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded alpha-crystallin has native-like secondary, tertiary, and quaternary structures as revealed by circular dichroism and fluorescence characteristics as well as gel filtration and sedimentation velocity measurements. On rapid refolding, beta- and gamma-crystallins aggregate and form turbid solutions. The presence of alpha-crystallin in the refolding buffer marginally increases the recovery of beta- and gamma-crystallins in the soluble form. However, unfolding of these crystallins together with alpha-crystallin using 8 M urea and subsequent refolding significantly increases the recovery of these proteins in the soluble form. These results indicate that an intermediate of alpha-crystallin formed during refolding is more effective in preventing the aggregation of beta- and gamma-crystallins. This supports our earlier hypothesis (Raman, B., and Rao, C. M. (1994) J. Biol. Chem. 269, 27264-27268) that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.
Raman
B
B
Centre for Cellular and Molecular Biology, Hyderabad, India.
Ramakrishna
T
T
Rao
C M
CM
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000494:pharmacology; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D018832:Molecular Chaperones / Q000737:chemistry*; D015394:Molecular Structure; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012995:Solubility; D013050:Spectrometry, Fluorescence
D003459:Crystallins; D046911:Macromolecular Substances; D018832:Molecular Chaperones