DEAMIDATION AND HUMAN BETA CRYSTALLIN STRUCTURE

脱酰胺和人β晶体结构

• 批准号: 6384753
• 负责人: KIRSTEN Jeanne LAMPI
• 金额: $ 19.4万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6384753
• 关键词: aging; amidation /deamidation; cataract; chemical binding; circular dichroism; conformation; crystallins; disulfide bond; electron spin resonance spectroscopy; electrospray ionization mass spectrometry; gel filtration chromatography; human tissue; lens; mass spectrometry; oxidation; pathologic process; polymerase chain reaction; posttranslational modifications; protein isoforms; protein structure function; site directed mutagenesis

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.

描述：镜头透明度的主要决定因素是 结晶蛋白的分子组织。虽然大多数主要 β家族中的结晶蛋白序列是已知的，精确的 第三和第四纪结构仍有待确定。鲜为人知 关于不同beta的结晶蛋白 - 晶体相互作用 多肽或这些相互作用在正常成熟期间如何变化 老化。衰老的人镜头中最引人注目的观察之一是 大量的脱膜结晶蛋白。总体目标 应用是确定脱氨化如何影响正常的胱胱氨藻蛋白 结晶蛋白相互作用。测试的假设是脱氨酸 在镜头的整体寿命中起双重作用。在镜头期间 成熟，脱氨酸可以增加结晶蛋白的起搏。然而， 过度的脱氨化导致进一步崩溃和不详细化 白内障中的结晶蛋白聚集体。测试这个假设的PI 提议： 1）表征人透镜中β结晶蛋白的正常结构 特别是指通过脱氨基的影响。 2）确定脱氨酸特定位点对次级的影响 Beta亚基的结构和结晶蛋白 - 晶状蛋白相互作用 使用定向诱变。 3）确定脱氨化是否增加或降低 其他翻译后修饰的蛋白质。修改 被测试的是截断，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角色 在人透镜中结晶蛋白相互作用中的脱氨酸。站点定向 诱变将描述脱氨酸特定位点的作用 研究人员已经确定在体内发生。多种技术 将使用包括电子自旋谐振光谱（ESR）。 ESR是 有利，因为它能够检查蛋白质中的所有区域， 仅需要少量样品，可用于识别 溶液中发生的相互作用。