Chaperone-like activity of alpha-crystallin. The effect of NADPH on its interaction with zeta-crystallin.

alpha-Crystallin, a major structural protein of the ocular lens of vertebrates, has been characterized recently as a molecular chaperone (Horwitz, J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10449-10453 and Jakob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520). While alpha-crystallins prevent the aggregation of various proteins denatured by heat or chaotropic agents, neither the mode of interaction between target proteins and alpha-crystallin nor the specific conformational requirements, if any, of the target protein are known. Here, we demonstrate that the ability of alpha-crystallin to prevent thermally induced aggregation of zeta-crystallin/NADPH:quinone oxidoreductase, an abundant crystallin of guinea pigs and camelids, is strongly dependent on the presence of the obligate cofactor (NADPH) of the target enzyme. zeta-crystallin in the absence of NADPH is readily aggregated at 41 degrees C, and alpha-crystallin added at a 1:1 (w/w) ratio offers very little protection. In contrast, in the presence of NADPH zeta-crystallin remains stable to 45 degrees C and with the addition of alpha-crystallin (1:1 (w/w)) is protected from aggregation even at 55 degrees C. Cibacron blue 3GA, a nonmetabolized pyridine nucleotide analog, which has very high binding affinity to zeta-crystallin had similar effects. NADH and NAD+, which are not bound by zeta-crystallin, had no such effect. Complex formation between alpha-crystallin and non-native zeta-crystallin was demonstrated in the presence of either cibacron blue 3GA or NADPH. Circular dichroism spectroscopy of zeta-crystallin in the presence and absence of NADPH or cibacron blue indicated that nucleotide binding was accompanied by a change in the protein's aromatic amino acid environment but that the secondary structure was unaffected. The data suggest that subtle change in the conformation of denaturing proteins can markedly affect the ability of alpha-crystallin to protect them from aggregation.