AGE & CATARACT RELATED CHANGES IN LENS PROTEIN STRUCTURE

在老化和白内障形成的过程中，透镜透明度的丧失是 与结构透镜蛋白（晶状体蛋白）的聚集有关， 光散射 这些翻译后的变化已经被观察到， 人类和牛的老化和白内障晶状体蛋白。 他们 包括脱酰胺、色氨酸氧化、甲硫氨酸氧化 多肽链的选择性降解，二硫键 （蛋白质-蛋白质，或混合二硫化物）和非酶促糖基化。 然而，尚不清楚这些修饰是否诱导蛋白质 构象变化和蛋白质聚集之前， 不溶解 在该提案中，物理化学测量（UV-VIS 吸收、圆二色性、荧光和激光散射） 将采用，以观察（1）是否蛋白质构象（包括 二级和三级结构，芳香胺的微环境 酸和巯基）由于翻译后 修改，如果他们这样做（2）是否构象改变透镜 来自老化和白内障晶状体的蛋白质产生更大的光散射， (3)这些蛋白质是否更容易受到外源压力的影响 (such如紫外线照射和过氧化氢），（4）是否改变 蛋白质构象促进了蛋白质聚集， 不溶解，最后（5）磷酸果糖激酶 也经历了类似的构象和翻译后变化， 晶体蛋白。 这些诱导的透镜蛋白质构象变化的意义在于 它可以增加光散射， 流动性，或通过增加蛋白质聚集和不溶解。 该提案的长期目标是将诱导的 构象变化的光散射观察到的变化， 蛋白质溶液和完整的晶状体。

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.