STRUCTURAL DYNAMICS AT THE ACTO MYOSIN INTERFACE

描述：拟议研究的最终目标是了解 允许肌球蛋白作为分子的结构/功能关系 马达，在肌肉过程中将化学能转化为机械功 收缩。这项提案的努力集中在阐明动态 肌球蛋白肌动蛋白结合界面的结构变化及其相互关系 这些具有生化和机械状态的动态结构变化 收缩周期。荧光探头将在选定的地点推出 利用化学和光反应在acto-myosin界面内的位置 标签技术。肌球蛋白的上50kD亚区将是 甲基香豆素在50/20kD连接附近的光标记和Lys-553 肌球蛋白较低的50kD亚区将用荧光素标记在 莱斯-553。结合了最先进的光谱、生化和 将使用机械方法严格测试当前的分子模型 肌肉收缩。具体地说，申请人打算识别 肌球蛋白上下50kD亚区在肌球蛋白形成中的作用 与肌动蛋白弱结合和强结合的复合体，研究 裂解肌球蛋白50kD结构域介导肌球蛋白活化 相互作用，决定肌动蛋白附着的肌球蛋白分子的比例 在等长收缩及其与肌肉纤维僵硬的关系中， 确定所有强结合的肌球蛋白交叉桥是否生成 力，并检查acto-myosin占空比如何随机械力变化 装填。

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.