CHEMISTRY OF MUSCLE PROTEINS

描述：当前理论的力量产生的步骤， 收缩周期涉及肌球蛋白头部相对于肌球蛋白头部的重新定向。 肌动蛋白丝在滑动。 无法检测到任何大的 肌球蛋白的球状催化结构域的构象变化 集中的兴趣在颈部区域，其中包括一个长的α-螺旋 通过调节（RCL）和必需的稳定化重链片段 ELC轻链 有人提出，小的结构变化， 马达结构域用于旋转该轻链结合结构域。 这 假设将通过以下方式进行检验：（1）使用体外运动性测定， 测量单个肌球蛋白分子的工作行程和单位力， RCL或ELC已通过生物化学方法去除，或 LC结合位点已通过重组技术被删除。 的 单头肌球蛋白的机械性能将与 双头物种 (2)头部内和头部之间的轻链相互作用 将通过用荧光标记突变RLC和ELC来确定头部 probes. 与肌球蛋白结合的荧光LC之间的距离的变化将是 通过共振能量转移测量，以及 纤维中的LC结合区域将通过瞬态荧光来测量 极化 (3)轻链和重链同种型对力和 移动将通过（a）分析标记的 用荧光光谱法测定ELC与肌动蛋白的N-末端区域， cro-电子显微镜，和（B）肌球蛋白中的突变可变区 同种型，以确定负责偶联的头部序列 ATP水解为运动。

DESCRIPTION: Current theories of the force generating step in the contractile cycle involve a reorientation of the myosin head relative to the actin filament during sliding. The inability to detect any large conformational changes in the globular, catalytic domain of myosin has focused interest on the neck region, which consists of a long alpha-helical heavy chain segment that is stabilized by the regulatory (RCL) and essential ELC light chains. It has been proposed that small structural changes in the motor domain serve to rotate this light chain binding domain. This hypothesis will be tested by: (1) Using an in vitro motility assay to measure the working stroke and unitary force of single myosin molecules from which the RCL or ELC has been removed by biochemical methods, or from which the LC-binding site has been deleted by recombinant techniques. The mechanical properties of single-headed myosin will be compared to the two-headed species. (2) Light chain interactions within a head and between heads will be determined by labelling mutant RLCs and ELCs with fluorescent probes. Changes in distance between fluorescent LCs bound to myosin will be measured by resonance energy transfer, and changes in orientation of the LC-binding region in fibers will be measured by transient fluorescence polarization. (3) The effect of light and heavy chain isoforms on force and movement will be determined by (a) analyzing the interaction of a labelled N-terminal region of ELC with actin by fluorescence spectroscopy and cro-electron microscopy, and (b) mutating variable regions in myosin isoforms to identify sequences in the head that are responsible for coupling ATP hydrolysis to movement.