Insights into hydrophobicity and the chaperone-like function of αA- and αB-crystallins

Crystallin, composed of two subunits, alpha A and alpha B, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alpha A- and alpha B-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alpha A- and alpha B-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alpha B showing a higher number of sites than alpha A at 30 degrees C. In agreement with the higher number of hydrophobic sites, alpha B-crystallin demonstrated higher chaperone activity than alpha A at this temperature. Thermodynamic analysis of ANS binding to alpha A- and alpha B-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alpha A and alpha B at 15 degrees C, alpha A was more potent than alpha B in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alpha A and alpha B for ANS upon preheating, there was an increase in the number of low affinity sites of alpha A and alpha B. Preheated alpha A, in contrast to alpha B, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.