The lens proteins become more susceptible to aggregation with age and cataract. The increased susceptibility may be related to the fact that they are in less stable states, either intrinsically or by posttranslational modifications. To test the hypothesis, a quantitative analysis of the stability will be made on the main lens crystallins, either in unmodified or modified states. The stability is usually expressed by the vague and imprecise term conformation or conformational change. In this proposal, the values of standard free energy, deltaGH20, and activation energy, deltaGplusplusH20, will be obtained by studying the folding reactions using recombinant crystallins, which are pure and unmodified. The relative stability will be correlated to their susceptibility to aggregation, i.e., to determine whether a less stable protein or a protein that becomes less stable by modification will be more susceptible to aggregation. A comparison of stability among crystallins, recombinant alphaA-, betaB2-, and gammaC-crystallins, will be studied; they are the major crystallins in the human lens. For the study of modifications, nonenzymatic glycation, mixed disulfide formation, and C-terminal degradation will be used as models. To further understand the effect of modification on conformational and kinetic stability, site-directed mutation will be performed. Site-directed mutation is more specific; the effect of a small change in amino acid sequence can be studied. Another factor that affects protein stability is the binding of a small or large ligand to alpha-crystallin; small molecules, such as Ca2+ and ATP, and large molecules, such as partially unfolded beta- and gamma-crystallins. The main techniques that will be used include equilibrium and kinetic analysis by fluorescence and circular dichroism, and detection of aggregation by FPLC liquid chromatography and light scattering. Since lens opacity is caused by protein aggregation, the underlying mechanisms that induce protein aggregation are fundamentally important in cataract research. The knowledge obtained from this proposal may provide insight on cataract formation on a molecular level and may help in formulating a strategy for developing anticataract agents.
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