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FORMATION OF ISOASPARTATE IN PEPTIDES AND PROTEINS

肽和蛋白质中异天冬氨酸的形成

基本信息

批准号：
2267594
负责人：
DANA WILLIAM ASWAD
金额：
$ 10.9万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-05-01 至 1995-04-30
项目状态：
已结题

项目摘要

Recent findings indicate that formation of isoaspartate (isoAsp) via deamidation of labile Asn-X sequences or direct isomerization at certain Asp-X sequences is a major source of spontaneous protein damage at physiological pH and temperature and that this process may have important consequences for protein function and turnover. This proposal seeks to understand the role of protein sequence and structure on formation of isoaspartate and to explore the effects of isomerization on protein function. Four experimental approaches are proposed. First, we will determine the sites of isoAsp formation in synapsin 1, tissue plasminogen activator, triosephosphate isomerase and the major intrinsic protein of the eye lens. All four of these proteins appear to generate isoAsp at significant rates at physiological pH and temperature. Second, we will synthesize a series homologous Asn- and Asp-containing pentapeptides in which the amino acids neighboring the Asn or Asp are varied in a systematic way. Substitutions will be designed to fill in several critical gaps in our knowledge of how neighboring amino acids influence the propensity of Asn or Asp to isomerize. Knowledge of the sites of isoAsp formation in the above proteins and synthetic peptides will be used to strengthen or refute our current paradigm, which emphasizes the nature of the amino acid linked to the Asn/Asp-carboxyl, together with regional flexibility, to predict sites of isoAsp formation. Our third aim is to determine to what extent isoAsp effects several functional attributes of synapsin 1, including its ability to be phosphorylated by the cAMP- and type II calcium/calmodulin-dependent protein kinases, its ability to bind to small synaptic vesicles, and its ability to bind F-actin. Finally, we will investigate the possibility that protein sequences which are prone to forming isoaspartate may also be prone to crosslinking. These studies should provide important new information on basic mechanisms which govern the stability and integrity of proteins in vitro and in vivo.

最近的研究结果表明，异天冬氨酸 (isoAsp) 的形成通过不稳定 Asn-X 序列的脱酰胺或在一定条件下直接异构化 Asp-X 序列是自发蛋白质损伤的主要来源生理 pH 值和温度，这个过程可能具有重要意义对蛋白质功能和周转的影响。该提案旨在了解蛋白质序列和结构对形成的作用异天冬氨酸并探讨异构化对蛋白质的影响功能。提出了四种实验方法。首先，我们将确定突触蛋白 1（组织纤溶酶原）中 isoAsp 形成的位点激活剂、磷酸三糖异构酶和主要内在蛋白眼睛的晶状体。所有这四种蛋白质似乎都会在以下位置产生 isoAsp 在生理 pH 值和温度下具有显着的变化率。其次，我们将合成了一系列同源的含 Asn 和 Asp 的五肽其中与 Asn 或 Asp 相邻的氨基酸在系统化的方式。替代品将被设计来填补几个我们对邻近氨基酸如何影响的知识存在重大差距 Asn 或 Asp 异构化的倾向。网站的知识上述蛋白质和合成肽中的 isoAsp 形成将用于加强或反驳我们当前的范式，该范式强调连接到 Asn/Asp-羧基的氨基酸的性质，以及区域灵活性，预测 isoAsp 形成的位点。我们的第三个目的是确定 isoAsp 对多种功能的影响程度突触蛋白 1 的属性，包括其被磷酸化的能力 cAMP 和 II 型钙/钙调蛋白依赖性蛋白激酶，其与小突触囊泡结合的能力及其结合能力 F-肌动蛋白。最后，我们将研究蛋白质的可能性易于形成异天冬氨酸的序列也可能易于形成交联。这些研究应该提供重要的新信息控制蛋白质稳定性和完整性的基本机制体外和体内。