STRUCTURAL ANALYSIS OF THE DYNEIN ATPASE

动力蛋白ATP酶的结构分析

• 批准号: 3280576
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 13.21万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280576
• 关键词: Tetrahymena; X ray crystallography; adenosinetriphosphatase; cilium /flagellum motility; cytoskeleton; electron microscopy; enzyme linked immunosorbent assay; enzyme structure; gel electrophoresis; ion exchange chromatography; monoclonal antibody; proteolysis; scanning electron microscopy

The overall goals of the proposed research are to establish the structure and mechanism of action of the dynein ATPase in generating a force for ciliary and flagellar movement. The specific goals of these studies can be divided into five parts: (1) Localize the polypeptides, the ATPase sites, and the microtubule-binding sites in terms of the three-headed bouquet of the 22S dynein; (2) evaluate the flexibility of the strands connecting the dynein heads to the base; (3) determine the arrangement of the dynein heads on the microtubule surface lattice and the nature of the interaction in the rigor complex; (4) examine the ATP-dependent changes in conformation; and (5) continue studies on the structure and localization the 14S dynein ATPase. These problems will be addressed by a combination of techniques including: transmission electron microscopic (TEM) localization of monoclonal and polyclonal antibodies; scanning transmission electron microscopy (STEM) of dynein fragments and the microtubule-dynein complex; hydrodynamic and fluorescence anisotropy measurements on isolated dynein; TEM analysis of unstained, unfixed, frozen-hydrated specimens or replicas of rotary shadowed preparations; and solution x-ray scattering studies of the microtubule-dynein complex and intact axonemes. The current work builds upon our previous results on the structure and ATPase pathway of dynein isolated from Tetrahymena cilia. These studies are expected to establish the structure of dynein in detail and to specify the structural basis for changes in conformation necessary to couple ATP hydrolysis to the interaction of the dynein crossbridge with microtubules to produce a force for ciliary movement. The work also provides a basis for analysis of dynein-like ATPases in other microtubule systems such as chromosome movement or the intracellular transport of membrane bound particles.

拟议研究的总体目标是建立结构 和动力蛋白ATPase在产生压力中的作用机制 纤毛和鞭毛运动。这些研究的具体目标可以是 分为五个部分：(1)定位多肽、ATPase位点、 以及微管结合部位，就三头花束而言 22S动力蛋白；(2)评价连接股的柔韧性 动力蛋白头部朝向底部；(3)确定动力蛋白头部的排列 微管表面晶格及其相互作用的本质 Rigor Complex；(4)检测依赖于ATP的构象变化； (5)继续研究14S dynein的结构和定位 ATPase。这些问题将通过多种技术的组合来解决 包括：透射电子显微镜(TEM)定位 单抗和多克隆抗体.扫描透射电子 动力蛋白片段和微管-动力蛋白复合体的显微镜观察； 分离的动力蛋白的流体力学和荧光各向异性测量； 未染色、未固定、冷冻水合的标本或复制品的电子显微镜分析 旋转阴影制剂；和溶液x射线散射研究 微管-动力蛋白复合体和完整的轴突。 目前的工作建立在我们之前关于结构和 纤毛四膜虫动力蛋白的ATPase途径。这些研究 预计将详细建立动力蛋白的结构并具体说明 连接三磷酸腺苷所必需的构象变化的结构基础 动力蛋白交叉桥与微管相互作用的水解性 产生纤毛运动的力量。该工作也为以后的研究提供了依据 用于分析其他微管系统中的动力蛋白样ATPase，例如 染色体运动或膜结合的细胞内运输 粒子。