In the course of aging and cataract formation, loss of lens transparency is associated with aggregation of structural lens proteins (crystallins) and light scattering. These post-translational changes have been observed in both human and bovine proteins of aging and cataractous lenses. They include deamidation, oxidation of tryptophan, oxidation of methionine, selective degradation of polypeptide chains, disulfide bonding (protein-protein, or mixed disulfide) and non-enzymatic glycosylation. However, it is not known whether these modifications induce protein conformational changes and precede protein aggregation and insolubilization. In this proposal, physicochemical measurements (UV-VIS absorption, circular dichroism, fluorescence and laser light scattering) will be employed, to observe (1) whether protein conformations (including secondary and tertiary structures, microenvironments of aromatic amino acids and sulfhydryl groups) have changes as a result of post-translational modifications, and if they do (2) whether conformationally altered lens proteins from aging and cataract lenses give greater light scattering and (3) whether these proteins become more vulnerable to exogeneous stresses (such as UV irradiation and hydrogen peroxide), (4) whether the change in protein conformation has facilitated protein aggregation and insolubilization, and finally (5) whether the enzyme phosphofructokinase also undergoes conformational and postranslational changes similar to crystallins. The significance of these induced changes in lens protein conformation is that it may increase light scattering either through a decreased particle mobility, or through increased protein aggregation and insolubilization. The long-term objective of this proposal is to correlate induced conformational changes to changes in light scattering observed both in protein solutions and intact lenses in vitro.
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