DEAMIDATION AND HUMAN BETA CRYSTALLIN STRUCTURE

描述：决定透镜透明度的主要因素是 晶体蛋白的分子结构。虽然大多数主要 β家族中晶体蛋白的序列是已知的， 三级和四级结构仍有待确定。知之甚少 关于不同β-晶体蛋白之间的相互作用 多肽或这些相互作用在正常成熟过程中如何变化， 衰老在老化的人类透镜中最引人注目的观察结果之一是 大量的脱酰胺晶体蛋白这个项目的总体目标是 应用是确定脱酰胺作用如何影响正常的胱抑素- 晶状体蛋白相互作用正在测试的假设是，脱酰胺 在透镜的整个寿命中起双重作用。在透镜期间 成熟时，脱酰胺允许增加晶体蛋白的起搏。然而，在这方面， 过度的脱酰胺作用会导致 晶状体蛋白在白内障中聚集。为了验证这一假设，PI 建议： 1)表征人透镜中β晶体蛋白的正常结构 特别是脱酰胺作用的影响。 2)确定特定脱酰胺位点对二级结构的影响 β亚基结构和堆叠晶体蛋白相互作用 使用定点诱变。 3)确定脱酰胺是否会增加或降低 其他翻译后修饰。的修改 被测试的是截短、氧化和二硫键形成。 这些研究很重要，因为它们将有助于阐明 在人的透镜中晶状体蛋白相互作用的脱酰胺作用。定点 诱变将描述脱酰胺的特定位点的作用， 研究者已确定在体内发生。多种技术 包括电子自旋共振光谱（ESR）。esr是 因为它能够检查蛋白质内的所有区域， 只需要少量的样品，可以用来识别 在溶液中发生的相互作用。

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.