DYNAMIC EVENTS IN MYOSIN DURING CONTRACTION OF MUSCLE

拟议研究的长期目标是阐明 肌肉收缩的结构和化学基础。这一目标将 通过开展四个相关项目，旨在澄清： II）肌球蛋白和肌动蛋白在弱结合和强结合中的相互作用 状态（+/-ATP）;（III）收缩调节;和（IV）动态 肌球蛋白头的转变（S1）。 (I)第一个项目的具体目标是：（一）完成 参与形成的肌动蛋白上带电残基簇的作图 弱结合的肌动球蛋白复合物;（ii）测试的假设， 肌动蛋白的N-末端在弱和 强结合肌动球蛋白状态;（iii）测试离子肌动球蛋白 生理条件下的相互作用;和（iv）改变N- 肌动蛋白末端，用于光谱探针标记。 (II)该项目的具体目标是：（一）制备行动突变体 用于映射强肌球蛋白结合的位点;（ii）用于测试 通过使用肌动蛋白和肌球蛋白突变体的acto-S1结构;以及（iii） 确定特定肌动球蛋白相互作用位点的功能作用。 (III)治理工程的目的是：（一）检验模型预测 原肌球蛋白在肌动蛋白上排列的变化;（ii）阐明 肌钙蛋白I在肌动球蛋白功能调节中的作用。 (IV)最后一个项目的目的是：（一）描述最近的 网囊藻肌球蛋白头的结晶运动结构域（SldC）;和（ii） 阐明S1上的特定位点在肌球蛋白功能中的作用， 结构变化决定功能。 这些项目中提出的研究将联合收割机结合诱变， 用于探测材料免疫化学、肽和化学制备 特定的蛋白质位点。肌球蛋白的结合和功能相互作用 和肌动蛋白突变体将在体外运动试验中进行评估，力 测量，结合和酶活性测量，以及大量的 一系列生物化学和生物物理实验。 本申请中提出的研究将导致更好的 理解力和运动的基本生物过程 家族性心肌病的产生和分子基础 是由β心肌肌球蛋白突变引起的

The long term objective of the proposed research is to elucidate the structural and chemical basis of muscle contraction. This goal will be pursued by working on four related projects aimed at clarifying: (I and II) the interactions of myosin and actin in the weakly and strongly bound states (+/-ATP); (III) regulation of contraction; and (IV) dynamic transitions in myosin heads (S1). (I) The specific aims of the first project are: (i) to complete the mapping of clusters of charged residues on actin involved in the formation of weakly bound actomyosin complexes; (ii) to test the hypothesis on the key role of the N-terminus of actin in the transition between weakly and strongly bound actomyosin states; (iii) to test ionic actomyosin interactions under physiological conditions; and (iv) to alter the N- terminus of actin for labeling with spectroscopic probes. (II) The specific aims of this project are: (i) to prepare action mutants for mapping the sites of strong myosin binding; (ii) to test models of acto-S1 structure by using actin and myosin mutants; and (iii) to determine the functional role of specific actomyosin interaction sites. (III) The aims of the regulation project are: (i) to test model predicted shifts in the arrangement of tropomyosin on actin; and (ii) to clarify the role of troponin I in the regulation of actomyosin function. (IV) The aims of the last project are: (i) to characterize the recently crystallized motor domain of Dictyostelium myosin head (SldC); and (ii) to clarify the role of specific sites on S1 in myosin function and in the structural changes which determine the function. The research proposed in these projects will combine mutagenic, immunochemical, peptide and chemical preparation of materials for probing specific protein sites. The binding and functional interactions of myosin and actin mutants will be evaluated in the in vitro motility assays, force measurements, binding and enzymatic activity measurements, and a large array of biochemical and biophysical experiments. The research proposed in this application will lead to a better understanding of the fundamental biological processes of force and motion generation and the molecular basis of familial cardiac myopathies caused by mutations in beta cardiac myosin.