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 present in vertebrate eye lens, is known to prevent the aggregation of other proteins under conditions of stress. However, its role in the reactivation of enzymes from their non-native inactive states has not been clearly demonstrated. We have studied the effect of alpha-crystallin on the refolding of zeta-crystallin, a quinone oxidoreductase, from its different urea-denatured 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. alpha B-crystallin was found to be more efficient than alpha A-crystallin in chaperoning the refolding of zeta-crystallin. In order to understand the nature of the denatured state(s) of zeta-crystallin that can interact with alpha-crystallin, 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 state, and a partially unfolded monomer. The partially unfolded monomer is inactive, exhibits highly exposed hydrophobic surfaces and has significant secondary structural elements with little or no tertiary structure. This intermediate does not refold into the active state without assistance. alpha-Crystallin provides the required assistance and improves the reactivation yield several-fold.
Goenka
S
S
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India.
Raman
B
B
Ramakrishna
T
T
Rao
C M
CM
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins; D005456:Fluorescent Dyes; D009249:NADP; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea