AlphaA-crystallin, a member of the small heat-shock protein family (sHsp), is known to play a pivotal role in inhibiting protein aggregation in the lens. This finding has led to the exciting possibility that its chaperone- like properties may be critical in preventing the progression of age-related cataracts. A hallmark of alphaA-crystallin and other sHsps is their tendency to form large oligomers. This unique structural feature of sHsps is thought to be important for their chaperone function. The overall goal of this research proposal is to elucidate the quaternary structure of alphaA- crystallin and to determine the molecular mechanism underlying the assembly of its subunits. Three interrelated research programs are proposed. (1) The specific aim of the first program is to define the subunit organization of the alphaA-crystallin. In this program, site-directed labeling will be employed to determine the orientation of the alphaA- crystallin subunits and the proximity of the N- and C-terminus of neighboring subunits in the oligomeric complex. Knowledge of the subunit organization will provide a starting point to study the accessibility of other important sites. (2) The specific aim of the second program is to uncover the molecular mechanism underlying the formation of the oligomeric complex. A panel of chimeras between alphaA-crystallin and Hsp27 will be constructed. The differences in size, conformation, chaperone activity, and subunit exchange rate between Hsp27, alphaA- crystallin and the chimeric proteins will be used to determine which regions of alphaA-crystallin are involved in oligomer formation. (3) Subunit exchange of alphaA-crystallin is known to involve the breaking of hydrophobic and ionic interactions between neighboring subunits.
The specific aim of the final program is to determine what kind of effects do cationic and anionic detergents have on the subunit dissociation rate constant of alphaA-crystallin. Studies will also be carried out to define how raising the temperature or increasing the chain-length of the cationic detergents may affect the rate of subunit exchange. Together, the findings obtained from these research programs will provide a better understanding of the structure of alphaA-crystallin and its interaction with other lens proteins.
