DYNAMIC EVENTS IN MYOSIN DURING CONTRACTION OF MUSCLE

拟议研究的长期目标是阐明 肌球蛋白中的结构和化学事件，导致肌球蛋白收缩 肌肉。这一目标将通过开展三个相互关联的项目来实现 旨在澄清(1)结构和结构的研究项目 肌球蛋白头部与肌动蛋白在分子水平上的相互作用 肌原纤维中肌球蛋白细丝的功能和结构变化 和肌肉纤维；(3)肌球蛋白分子组装成功能性 长丝单元，以及这种长丝的性能。 (1)建议的肌球蛋白头研究的具体目标如下： 阐明肌球蛋白和肌动蛋白上的特定位点在肌球蛋白中的作用 绑定；ii)派生新的结构信息并定义 肌球蛋白头部的结构-功能关系。这些研究将 涉及不同的生化和生物物理方法，包括特定地点 化学修饰，化学交联，选择性蛋白质分解， 免疫化学技术、电子显微镜和荧光技术。 (二)功能和结构变化研究的具体目标 在肌球蛋白细丝中：i)确定肌球蛋白与肌动蛋白的结合 通过使用合成长丝系统、肌原纤维和 肌纤维；ii)阐明核苷酸类似物(AMPPNP)对 肌球蛋白在肌原纤维和肌纤维中的构象；III)探索 收缩和应激引起的肌球蛋白变化。这项工作将利用 蛋白水解法、化学交联法和化学修饰 测试肌球蛋白不同区域的反应，并测量其结合 到肌动蛋白。 (3)肌球蛋白微丝组装工作的具体目标 是：i)表征灯丝组装中的成核步骤；ii) 阐明影响纤维成核和成核的因素 成长。这些研究将使用流体动力学、光散射、电子 显微镜和光谱技术。 在本申请中提出的研究将带来改进的 了解肌肉和非肌肉细胞的收缩过程。

The long term objective of the proposed research is to elucidate the structural and chemical events in myosin, which lead to the contraction of muscle. This objective will be pursued by undertaking three inter-related research projects aimed at the clarification of (1) the structure and interaction of myosin heads with actin at the molecular level; (2) the function and structural changes in myosin filaments in intact myofibrils and muscle fibers; (3) the assembly of myosin molecules into functional filament units, and the properties of such filaments. (1) The specific aims of the proposed studies on the myosin head are: i) to clarify the role of specific sites on myosin and actin in actomyosin binding; ii) to derive new structural information and define structure-function relationships on the myosin head. These studies will involve diverse biochemical and biophysical methods including site specific chemical modifications, chemical cross-linkings, selective proteolysis, immunochemical techniques, electron microscopy, and fluorescence techniques. (2) The specific aims of the studies on the function and structural changes in myosin filaments are: i) to determine the binding of myosin to actin in the presence of ATP by using synthetic filament systems, myofibrils, and muscle fibers; ii) to clarify the effect of nucleotide analogue (AMPPNP) on the conformation of myosin in myofibrils and muscle fibers; iii) to explore contraction and stress-induced changes in myosin. This work will utilize proteolytic methods, chemical cross-linkings, and chemical modification reactions to test different regions on myosin, and to measure its binding to actin. (3) The specific aims of the work on the assembly of myosin into filaments are: i) to characterize the nucleation step in filament assembly; ii) to clarify the factors involved in the regulation of filament nucleation and growth. These studies will employ hydrodynamic, light scattering, electron microscopy, and spectroscopic techniques. The research proposed in this application will bring an improved understanding of contractile processes in muscle and non-muscle cells.