Unfolding and refolding of a quinone oxidoreductase: alpha-crystallin, a molecular chaperone, assists its reactivation

alpha -Crystallin, a member of the small heat-shock protein family and pres ent in vertebrate eye lens, is known to prevent the aggregation of other pr oteins under conditions of stress. However, its role in the reactivation of enzymes from their non-native inactive states has not been clearly demonst rated. We have studied the effect of alpha -crystallin on the refolding of zeta -crystallin, a quinone oxidoreductase, from its different urea-denatur ed states. Co-refolding zeta -crystallin from its denatured state in 2.5 M urea with either calf eye lens alpha -crystallin or recombinant human alpha B-crystallin could significantly enhance its reactivation yield. alphaB-cry stallin was found to be more efficient than alphaA-crystallin in chaperonin g the refolding of zeta -crystallin. In order to understand the nature of t he denatured state(s) of crystallin that can interact with alpha -crystalli n, we have investigated the unfolding pathway of zeta -crystallin. We find that it unfolds through three distinct intermediates: an altered tetramer, a partially unfolded dimer, which is competent to fold back to its active s tate, and a partially unfolded monomer. The partially unfolded monomer is i nactive, exhibits highly exposed hydrophobic surfaces and has significant s econdary structural elements with little or no tertiary structure. This int ermediate does not refold into the active state without assistance. alpha - Crystallin provides the required assistance and improves the reactivation y ield several-fold.