DEAMIDATION AND HUMAN BETA CRYSTALLIN STRUCTURE

描述：决定镜片透明度的一个主要因素是 晶体蛋白的分子组织。而大多数初选 贝塔家族中晶体蛋白的序列是已知的，准确的 三元和四元结构尚待确定。鲜为人知 关于不同β-晶状体蛋白相互作用的研究 多肽或这些相互作用如何在正常成熟和 衰老。在老化的人类晶状体中，最引人注目的观察之一是 大量的去胺化晶体蛋白。这个项目的总体目标是 应用是确定去酰胺化如何影响正常的胱蛋白- 晶体蛋白相互作用。正在测试的假设是脱酰胺 在镜片的整个使用寿命中起着双重作用。在镜头中 成熟，去酰胺化可以增加晶体蛋白的步调。然而， 过度的脱酰亚胺导致进一步的坍塌和不溶解 晶状体蛋白在白内障中的聚集。为了检验这一假设，假设PI 建议： 1)描述人类晶状体中β晶体蛋白的正常结构 特别提到去酰胺化的那些影响。 2)确定特定的脱酰亚胺位置对二次 β亚基的结构和晶体蛋白-晶体蛋白相互作用的研究 使用定点突变。 3)确定去酰胺化是否增加或降低了 蛋白质到其他翻译后修饰。对以下内容的修改 被测试的是截断、氧化和二硫键的形成。 这些研究很重要，因为它们将有助于阐明 人类晶状体中晶体蛋白相互作用中的脱酰胺作用。站点定向 突变将描述特定的脱酰胺化位点的作用 研究人员已经确定是在体内发生的。各种技术 包括电子自旋共振波谱(ESR)。ESR是 有利的是它能够检查蛋白质中的所有区域， 只需要少量样品，可用于鉴定 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.