FOLDING OF AN ALL-BETA-PROTEIN - INDEPENDENT DOMAIN FOLDING IN GAMMA-II-CRYSTALLIN FROM CALF EYE LENS

γII-crystallin from calf eye lens consists of two homologous domains, each composed of two similar 'Greek key' motifs. As a consequence of the bilobal structure, a biphasic transition is seen upon unfolding by urea at low pH (monitored by circular dichroism, fluorescence emission, and ultracentrifugal analysis). In 3.3 ± 0.5 M urea, a stable intermediate is formed at equilibrium, whereas 5.5 M urea causes maximum denaturation. Unfolding/folding kinetics display a complex pattern characterized by two kinetic phases. Both reactions exhibit strong dependence on the urea concentration; in the range of the respective transition, their rates are extremely slow (k ~ 1 x 10-4 s-1). The kinetic mechanism of unfolding and refolding may be described by a three-state model: native ⇆ intermediate ⇆ denatured. The rate-determining steps are domain folding rather than domain pairing or proline isomerization. Kinetic analysis of the unfolding/folding of the intermediate populated in 3.0 M urea, pH 2.0, reveals that the kinetic and the equilibrium intermediates have similar structures. Limited proteolysis of γII-crystallin by pepsin in 3 M urea, pH 2, allows the NH2-terminal domain of the protein to be isolated. Unfolding/refolding of the fragment parallels the second transition in the above scheme, thus proving that the intermediate contains the COOH-terminal domain in its random state, whereas the NH2-terminal domain is still in its native conformation. In conclusion, folding of γII-crystallin proceeds through the independent sequential structuring of the domains.