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，导致肌球蛋白的收缩， 肌肉. 为实现这一目标，将开展三项相互关联的工作， 研究项目旨在澄清（1）结构和 肌球蛋白头与肌动蛋白在分子水平上的相互作用;（2） 完整肌原纤维中肌球蛋白丝的功能和结构变化 （3）肌球蛋白分子组装成功能性的肌纤维; 长丝单位和这种长丝的性质。 (1)对肌球蛋白头的拟议研究的具体目的是：i） 阐明肌球蛋白和肌动蛋白上的特定位点在肌动球蛋白中的作用 结合; ii）获得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 涉及多种生物化学和生物物理方法，包括位点特异性 化学修饰，化学交联，选择性蛋白水解， 免疫化学技术、电子显微镜和荧光技术。 (2)功能和结构变化研究的具体目标 在肌球蛋白丝中的作用是：i）确定肌球蛋白与肌动蛋白的结合， ATP的存在，通过使用合成丝系统，肌原纤维， ii）阐明核苷酸类似物（AMPPNP）对肌纤维的影响， 肌原纤维和肌纤维中肌球蛋白的构象; iii）探索 收缩和应力诱导的肌球蛋白变化。 这项工作将利用 蛋白水解方法、化学交联和化学修饰 反应，以测试肌球蛋白上的不同区域，并测量其结合 到actin。 (3)肌球蛋白组装成肌丝的工作的具体目标 是：i）表征细丝组装中的成核步骤; ii） 阐明了丝成核调节中涉及的因素， 增长 这些研究将采用流体力学，光散射，电子 显微镜和光谱技术。 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.