CHEMISTRY OF MUSCLE PROTEINS

描述：当前关于力产生步骤的理论 收缩周期涉及肌球蛋白头部相对于 滑动过程中的肌动蛋白细丝。无法检测到任何大型 肌球蛋白球形催化结构域的构象变化 注意力集中在颈部区域，该区域由一个长的阿尔法螺旋组成 由调节性(RCL)和必需的重链片段稳定 ELC轻链。有人提出，经济结构上的微小变化 运动域用于旋转该轻链结合结构域。这 假说将通过以下方式进行检验：(1)使用体外运动试验 测量单个肌球蛋白分子的工作行程和单位作用力 RCL或ELC已通过生化方法去除，或 通过重组技术，LC结合位点被删除。这个 单头肌球蛋白的机械性能将与 双头物种。(2)头部内部和头部之间的轻链相互作用 头部将通过用荧光标记突变的RLC和ELC来确定 探测器。结合肌球蛋白的荧光LC之间的距离变化将是 通过共振能量转移和方向的变化来测量 将用瞬时荧光法测量纤维中的LC结合区 极化。(3)轻、重链异构体对力和 移动将通过以下方式确定：(A)分析标记的 荧光光谱分析ELC与肌动蛋白结合的N-末端区域 CRO电子显微镜，和(B)肌球蛋白中突变的可变区 识别头部负责偶联的序列的异构体 将三磷酸腺苷的水解度转化为运动。

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.