STRUCTURAL ANALYSIS OF THE DYNEIN ATPASE

动力蛋白ATP酶的结构分析

• 批准号: 3280579
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 14.69万
• 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/3280579
• 关键词: Tetrahymena; X ray crystallography; adenosinetriphosphatase; binding proteins; cilium /flagellum motility; cytoskeleton; dynein ATPase; electron microscopy; enzyme linked immunosorbent assay; enzyme structure; gel electrophoresis; ion exchange chromatography; laboratory mouse; monoclonal antibody; protein structure; 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 以及动力蛋白ATP酶在产生力 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，ATP酶位点， 和微管结合位点的三头花束， 22 S动力蛋白;（2）评估连接22 S动力蛋白的链的柔韧性。 （3）确定动力蛋白头的排列 在微管表面晶格和相互作用的性质， （4）检查构象的ATP依赖性变化;和 (5)继续研究14 S动力蛋白的结构和定位 ATP酶 这些问题将通过技术的组合来解决 包括：透射电子显微镜（TEM）定位 单克隆抗体和多克隆抗体;扫描透射电子显微镜 动力蛋白片段和微管-动力蛋白复合物的显微镜（STEM）; 对分离动力蛋白的流体动力学和荧光各向异性测量; 未染色、未固定、冷冻水合标本或复制品的TEM分析 旋转阴影制备;和溶液X射线散射研究 微管-动力蛋白复合体和完整的轴丝。 目前的工作建立在我们以前的结构和 纤毛四膜虫动力蛋白的ATP酶途径。 这些研究 预计将建立动力蛋白的详细结构，并指定 偶联ATP所需构象变化的结构基础 动力蛋白桥与微管的相互作用 以产生纤毛运动的力。 这项工作也提供了基础 用于分析其他微管系统中的动力蛋白样ATP酶， 染色体运动或膜结合的细胞内转运 粒子