Rapid refolding studies on the chaperone-like α-crystallin effect of α-crystallin on refolding of β- and γ-crystallins

Abstract

α-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 α-crystallin, and compared it with other calf eye lens proteins, namely β- and γ-crystallins. α-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded α-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, β- and γ-crystallins aggregate and form turbid solutions. The presence of α-crystallin in the refolding buffer marginally increases the recovery of β- and γ-crystallins in the soluble form. However, unfolding of these crystallins together with α-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 α-crystallin formed during refolding is more effective in preventing the aggregation of β- and γ-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 α-crystallin is more pronounced in its structurally perturbed state.