3D ELECTRON MICROSCOPY OF PYRUVATE DEHYDROGENASE

丙酮酸脱氢酶的 3D 电子显微镜

• 批准号: 2842303
• 负责人: JAMES K STOOPS
• 金额: $ 25.42万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2842303
• 关键词: Saccharomyces cerevisiae; X ray crystallography; active sites; binding proteins; chemical cleavage; computer simulation; cryoelectron microscopy; dihydrolipoamide dehydrogenase; dimer; enzyme complex; enzyme structure; image processing; immunoglobulin structure; macroglobulins; methylamines; protease inhibitor; protein structure function; pyruvate dehydrogenase; structural biology

alpha2-Macroglobulin (alpha2M), a general proteinase inhibitor ubiquitous in the plasma of vertebrates, is believed to serve as a general proteinase scavenger thereby protecting blood and tissue proteins from degradation. A better understanding of the structure- function relationships of human alpha2M will result from the determination of the structure of its various complexes by stain and cryo-electron microscopy, and image processing. The native and transformed molecules (formed upon reaction with a proteinase) are comprised of dimers. The determination of the 3-D structure of the native dimer (protomeric unit) will elucidate the structural organization of the native molecule and offer further insight in the mechanism of proteinase entrapment. The 3-D structure of alpha2M with the truncated bait domain compliments this study since this variant alpha2M is not capable of trapping the proteinase. Bait domain cleavage is the initial step in the reaction Of alpha2M with a proteinase. A 3-D structure of anti-bait domain Fab-labelled native alpha2M will locate this important functional site and make it possible to compare it with the location of this epitope in the transformed structure. Further insight into the mechanism of the transformation of native to the activated structure will be obtained by determining the 3-D structures of monoclonal Fab-labelled native, half-transformed and transformed alpha2Ms. Mammalian pyruvate dehydrogenase complex (PDC) plays a critical role in regulating cellular fuel utilization in the heart and, therefore, studies of the structure-function relationships of the PDC may expand the treatment options for heart disease. Saccharomyces cerevisiae PDC activity is regulated by the pyruvate dehydrogenase component (E1). The 3-D structure (determined by cryo-electron microscopy) of E1 and its binding subunit beta associated with dihydrolipoamide acetyltransferase core complex (E2) will elucidate its disposition on the core and may give insight into its structural organization. The disposition of functional sites associated with the PDC will be elucidated by determining the 3-D structure of these sites labelled with a gold cluster. They include the lipoyl domain of the binding protein (BP) and the catalytic sites of the dihydrolipoamide dehydrogenase (E3). 3-D reconstructions of recombinant intact E2 and the E2 core with BP E3 and E2 BP E3 E1 bound should elucidate the structural organization of this large complex. A 3-D reconstruction of the beef kidney intact PDC will permit a comparison of the mammalian and yeast complexes.

α 2-巨球蛋白（α 2 M），一种通用蛋白酶抑制剂 普遍存在于脊椎动物的血浆中，被认为是一种 一般蛋白酶清除剂，从而保护血液和组织 蛋白质降解。 更好地了解结构- 人α 2 M的功能关系将由以下因素产生： 通过染色测定其各种络合物的结构， 冷冻电子显微镜和图像处理。 天然和 转化的分子（在与蛋白酶反应后形成）是 由二聚体组成。 确定了该材料的三维结构， 天然二聚体（原聚体单位）将阐明结构 组织的天然分子，并提供进一步的见解， 蛋白酶截留机制 alpha 2 M的三维结构 截短的诱饵结构域补充了这项研究， α 2 M不能捕获蛋白酶。 诱饵结构域裂解 是α 2 M与蛋白酶反应的第一步。 的3-D 抗诱饵结构域Fab标记天然α 2 M的结构将位于 这个重要的功能网站，使它能够比较， 该表位在转化结构中的位置。 进一步 深入探讨了自然语言向自然语言转化的机制， 通过确定三维结构，将获得激活结构 单克隆Fab标记的天然、半转化和转化 α 2Ms。哺乳动物丙酮酸脱氢酶复合体（PDC）在哺乳动物体内起着重要的作用。 在调节心脏细胞燃料利用方面起关键作用， 因此，研究PDC的结构-功能关系 可能会扩大心脏病的治疗选择。Saccharomyces 酿酒酵母PDC活性受丙酮酸脱氢酶调节 组分（E1）。 三维结构（由低温电子确定 E1及其结合亚基β与 二氢硫辛酰胺乙酰转移酶核心复合物（E2）将阐明其 在核心上的配置，并可能使其结构的洞察力 organization. 与之相关的功能性站点的配置 PDC将通过确定这些网站的3-D结构来阐明 标记着一个金簇。 它们包括硫辛酰结构域的 结合蛋白（BP）和二氢硫辛酰胺的催化位点 脱氢酶（E3）。 重组完整E2的三维重建， E2核心与BP E3和E2 BP E3 E1结合应阐明 这一大型综合体的结构组织。 一个三维重建的 完整的牛肾PDC将允许比较哺乳动物和 酵母复合物