The lens contains three major types of structural protein: alpha-, beta-, and gamma-crystallins. Previous biochemical and spectroscopic studies indicate the possibility of interactions between alpha-crystallin and beta- or gamma-crystallin. Our recent studies using a mammalian two-hybrid system confirm that there are interactions among crystallins and that these interactions are altered by some congenital cataract crystallin mutations. The two-hybrid system can detect not only inter-molecular interactions but also intra-molecular interactions and is especially useful in studying alpha- (alphaA- and alphaB) crystallin. Mapping interface domains is one of the major applications of the two-hybrid system. alphaA- and alphaB-crystallins are oligomers whose three-dimensional structures have not been determined because of their inability to be crystallized for x-ray diffraction study. In this proposal, it is hypothesized that inter-subunit reaction domains can be mapped by the two-hybrid system assay with the aid of site-specific mutation, and that interface domains consist mainly of beta-strands and can be targeted for mutations. Once the interface beta-strands are identified, small peptides mimicking the beta-strands can be designed and synthesized. The reactions between peptides and alphaA- or alphaB-crystallin will prevent formation of dimers and oligomers, and native monomers will be available for structural studies. The first specific aim will be preliminary studies with known structural beta-and gamma-crystallins; the second specific aim will be mapping interface domains of alphaA- and alphaB-crystallins, followed by studying peptide-protein interactions. The methodology includes cloning, subcloning, site-specific mutagenesis, cell culture, protein expression and purification, small peptide design and synthesis, and two-hybrid system assay. The long-term objective is to understand the mechanisms of dimerization and oligomerization, and the results may help us to understand the function of alpha-crystallin in normal lens and in cataract formation.
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