Stepwise dissociation/denaturation and reassociation/renaturation of bovine α-crystallin in urea and guanidine hydrochloride: Sedimentation, fluorescence, near-ultraviolet and far-ultraviolet circular dichroism studies

Abstract

Quaternary, tertiary and secondary structure changes of calf α-crystallin were studied as a function of urea and guanidine hydrochloride concentration at pH 7·3 and 20°C. Sedimentation velocity analysis shows that native α-crystallin (19 s, 850 000 M r) dissociates in a step-wise fashion to single subunits (1·4 s, 20 000 M r). Complete dissociation is reached above 5 m-urea or 2·5 m-guanidine hydrochloride. The most stable dissociation intermediate (12 s) is observed around 2–3 m-urea or 1–1·5 m-guanidine hydrochloride. Fluorescence and near-ultraviolet circular dichroism analyses indicate that the exposure of aromatic residues is correlated with step-wise dissociation. Far-ultraviolet circular dichroism analysis shows that unfolding of subunits occurs over a very wide range of denaturant concentrations. Unfolding begins as subunits are released from the 12 s dissociation intermediate and may not be complete even in 8 m-urea or 6 m-guanidine hydrochloride. Dissociation of native α-crystallin to 12 s intermediate is irreversible, but subsequent dissociation steps are reversible. Dissociated and denatured subunits can be reassembled in a 12 s ‘reassociated α-crystallin’ molecule which is identical to the 12 s dissociation intermediate in nearly every aspect of secondary, tertiary and quaternary structure. The results are discussed in relation to our earlier proposed three-layer subunit model of calf α-crystallin and are found to support this model.