MOLECULAR PROBES OF MYOSIN STRUCTURE AND INTERACTIONS

肌球蛋白结构和相互作用的分子探针

• 批准号: 2079299
• 负责人: DONALD A WINKELMANN
• 金额: $ 19.65万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2079299
• 关键词: Sf9 cell line; actins; adenosinetriphosphatase; chemical stability; chickens; conformation; cryoscopy; epitope mapping; image processing; immunoelectron microscopy; intermolecular interaction; laboratory mouse; laboratory rabbit; microfilaments; molecular cloning; molecular site; monoclonal antibody; myosins; physical model; point mutation; protein folding; protein structure function; recombinant proteins; site directed mutagenesis; structural biology

Actin and myosin are highly conserved proteins found in virtually all eukaryotic cells where they generally exist as labile polymeric structures assembled into the cytoskeleton. They participate directly in determination of cell shape.cellular motility.cytoplasmic streaming.cytokinesis and contractility in muscle cells. The primary role of the interaction of actin and myosin is the generation of force and motion. This occurs as a consequence of the cyclic interaction of the motor domain of myosin with actin filaments and is driven by myosin ATP hydrolysis. Our research has concentrated on the analysis of myosin structure. assembly and function using protein biochemical. immunochemical and ultrastructural techniques. These efforts have lead to the determination of the high resolution structure of the myosin domain responsible for force generation and motion as a result of our work on the structure of myosin S1 crystals. We intend to continue our efforts along these lines by analyzing the conformational transitions within the myosin head which give rise to movement of actin filaments. This will be accomplished using monoclonal antibodies as site specific probes of the interaction of myosin with actin using an in vitro motility assay. The orientation of the myosin head when tightly bound to actin will be examined using cryo-electron microscopy to map the location of specific myosin sequences on rigor complexes of acto-S1 that are labeled with Fab fragments of monoclonal antibodies. These studies will provide landmarks for relating the crystal structure of myosin S1 to the structure of actin. The folding and assembly of the myosin molecule will be analyzed with an in vitro synthesis and assembly assay. These experiments are aimed at defining the role in the folding and stability of the molecule of highly conserved sequences in the myosin head and are derived from the observation that a mutation at one of these sites in the myosin S1 domain is related to a human genetic disease. Familial Hypertrophic Cardiomyopathy (FHC). To analyze the structural and functional consequences of mutations in the S1 domain. We will produce recombinant myosin fragments in eukaryotic expression system. Finally, the design and interpretation of these studies are based on the structure of myosin S1. We will continue our successful collaboration with Dr. Ivan Rayment on the analysis and refinement of the structure of the myosin head with the aim of defining the molecular mechanism used by myosin to generate force and motion.

肌动蛋白和肌球蛋白几乎全部发现了高​​度保守的蛋白质 真核细胞通常作为不稳定聚合物存在 组装成细胞骨架的结构。 他们直接参加 在确定细胞形状时。细胞运动性。 流媒体。肌肉细胞中的细胞动力学和收缩力。 主要 肌动蛋白和肌球蛋白的相互作用的作用是产生力的产生 和运动。 这是由于循环相互作用的结果 肌动蛋白丝的肌球蛋白运动域，由肌球蛋白驱动 ATP水解。 我们的研究集中于肌球蛋白的分析 结构。使用蛋白质生化的组装和功能。 免疫化学和超微结构技术。 这些努力领先 确定肌球蛋白的高分辨率结构 由于我们 在肌球蛋白S1晶体的结构上工作。 我们打算继续我们的 通过分析构象过渡来努力努力 在肌球蛋白头部，引起肌动蛋白丝的运动。 这将使用单克隆抗体作为位点特异性来完成 使用体外运动的肌球蛋白与肌动蛋白相互作用的探针 测定。 紧密绑定到肌动蛋白时，肌球蛋白头的方向 将使用冷冻电子显微镜检查以绘制 标记为Acto-S1的严格复合物上的特定肌球蛋白序列 带有单克隆抗体的Fab片段。 这些研究将提供 将肌球蛋白S1的晶体结构与 肌动蛋白的结构。 肌球蛋白分子的折叠和组装将 用体外合成和组装测定法分析。 这些 实验旨在定义折叠和稳定性中的作用 肌球蛋白头高度保守序列的分子的 从观察到的，即在这些位点之一的突变 肌球蛋白S1结构域与人类遗传疾病有关。 家族 肥厚性心肌病（FHC）。 分析结构和 S1结构域突变的功能后果。 我们会生产 真核表达系统中的重组肌球蛋白片段。 最后， 这些研究的设计和解释是基于结构 肌球蛋白S1。 我们将继续与Dr. Ivan Rayment在分析和改进的结构上 肌球蛋白头的目的是定义由 肌球蛋白产生力和运动。