STRUCTURAL DYNAMICS AT THE ACTO MYOSIN INTERFACE

描述：拟议研究的最终目标是了解 结构/功能关系，使肌球蛋白作为一种分子， 马达，将化学能转换为肌肉运动过程中的机械功 收缩。 本提案的工作重点是阐明动态 肌球蛋白肌动蛋白结合界面的结构变化， 这些动态的结构变化与生物化学和机械状态， 收缩周期 荧光探针将在选定的 在肌动蛋白-肌球蛋白界面内使用化学和光反应 标签技术。 肌球蛋白的上50 kD亚结构域将是 在50/20 kD结附近用甲基香豆素光标记， 肌球蛋白的较低的50 kD亚结构域将用荧光素标记， 赖氨酸553 结合了最先进的光谱学，生物化学， 机械方法将用于严格测试当前的分子模型 肌肉收缩。 具体而言，申请人打算确定 肌球蛋白的上、下50 kD亚结构域在肌球蛋白形成中的作用 弱和强结合复合物与肌动蛋白，检查的作用， 裂解肌球蛋白50 kD结构域介导肌动球蛋白 相互作用，确定肌动蛋白附着肌球蛋白分子的分数 在等长收缩期间及其与肌纤维硬度的关系， 确定是否所有强结合的肌球蛋白跨桥产生 力，并检查肌动蛋白占空比如何随机械力而变化。 即可.

DESCRIPTION: The ultimate goal of the proposed research is to understand structure/function relationships that allow myosin to act as a molecular motor, transducing chemical energy into mechanical work during muscle contraction. Efforts in this proposal are focused on elucidating dynamic structural changes at the actin-binding interface of myosin, and correlating these dynamic structural changes with biochemical and mechanical states of the contractile cycle. Fluorescent probes will be introduced at selected sites within the acto-myosin interface using both chemical and photoreactive labelling techniques. The upper 50 kD subdomain of myosin will be photolabelled with methyl coumarin near the 50/20 kD junction and Lys-553 of the lower 50 kD subdomain of myosin will be lableled with fluorescein at Lys-553. A combination of state-of-the-art spectroscopic, biochemical, and mechanical methods will be used to rigorously test current molecular models of muscle contraction. Specifically, the applicant intends to identify the roles of the upper and lower 50 kD subdomains of myosin in the formation of weakly and strongly bound complexes with actin, examine the role of the cleft splitting the 50 kD domain of myosin in mediating the acto- myosin interaction, determine the fraction of actin-attached myosin molecules during isometric contraction and its relationship to muscle fiber stiffness, determine whether or not all strongly bound myosin cross-bridges generate force, and examine how the acto-myosin duty cycle varies with mechanical load.