DEAMIDATION AND HUMAN BETA CRYSTALLIN STRUCTURE

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DESCRIPTION: A major determinant of the transparency of the lens is the molecular organization of the crystallins. While most of the primary sequences of the crystallins in the beta family are known, the precise tertiary and quaternary structure remains to be determine. Little is known about the crystallin-crystallin interactions of the different beta polypeptides or how these interactions change during normal maturation and aging. One of the most striking observations in the aging human lens is the large amounts of deamidated crystallins. The overall goal of this application is to determine how deamidation affect normal cystallin- crystallin interaction. The hypothesis being tested is that deamidation plays a dual role in the overall lifetime of the lens. During lens maturation, deamidation allows increased pacing of crystallins. However, excessive deamidation causes further collapse and insolubilization of crystallin aggregates in cataract. To test this hypothesize the PI proposes to: 1) Characterize the normal structure of beta crystallins in the human lens with particular reference to those influences by deamidation. 2) Determine the effect of specific sites of deamidation on the secondary structure of beta subunits and on crystallin-crystallin interactions by using site-directed mutagenesis. 3) Determine if deamidation increases or decreases susceptibility of proteins to other post-translational modifications. The modifications to be tested are truncation, oxidation, and disulfide bond formation. These studies are important because they will help to elucidate the role of deamidation in crystallin interaction in the human lens. Site-directed mutagenesis will delineate the role of specific sites of deamidation which the investigator has identified to occur in vivo. A variety of techniques including electron spin resonance spectroscopy (ESR) will be used. ESR is advantageous because it is able to examine all regions within a protein, requires only small amounts of sample, and can be used to identify interactions occurring in solution.