StructuraL Dynamics of Actomyosin Motility

项目摘要 本研究的总体目的是了解分子机制。 肌动球蛋白运动系统将化学能转化为机械功，并获得 分子中的机械、生化和结构事件之间的精确关联 水平。新方法将应用于非肌肉肌球蛋白分子马达，以探测 收缩蛋白的生化反应与基础机械的关系 跨桥旋回的阶跃和相应的构造运动。双功能，双- 砷和量子棒荧光探针将以已知的方向稳定地结合到 马达的结构域、轻链亚单位和马达的尾巴。空间方向和 这些组件的平移位置将以高时间分辨率由NOVICE 单分子偏振荧光、全内反射(PolTIRF)显微镜 确定肌动蛋白转位过程中特定蛋白质结构变化的动力学 在机械载荷作用下。提高了最近实现的测量时间分辨率 旋转、平移和热摆动运动，并将使详细事件 在稳定驻留期间的短暂分子步进期间被检测到。一台红外线 光学陷阱，具有高速反馈以将肌动蛋白夹在适当的位置并快速测量 肌球蛋白工作卒中后肌动蛋白附着，将用于确定特异性 ATPase产物的释放与磷酸盐和ADP的关系 肌动球蛋白键，转变为力生成状态，并倾斜，导致运动 货物。反馈光学陷阱将与单分子聚TIRF显微镜相结合，以 直接评估机械应力、应变和灵活性对步进率的影响 与化学-机械转导有关的蛋白质取向变化。能量学和 肌动蛋白亚基靶点选择的统计将根据方向和力来确定 结构域角、生化状态和步长的依赖关系。这些实验将是 对从重组表达系统中分离的非肌肉肌球蛋白进行了研究。结果来自 这个项目应该会大大提高对细胞运动过程的了解，从而带来 对神经元和感觉神经的正常和病理状态有更多的了解 发育和许多其他类型的细胞运动。

Project Summary The overall aims of this research are to understand the molecular mechanism by which actomyosin motility systems convert chemical energy into mechanical work, and to obtain a precise correlation between the mechanical, biochemical and structural events at the molecular level. Novel methods will be applied to non-muscle myosin molecular motors to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. Bifunctional, bi- arsenical and quantum rod fluorescent probes will be stably bound with known orientation to the motor domains, light chain subunits, and tails of the motors. The spatial orientation and translational position of these components will be monitored at high time resolution by novel single-molecule polarized fluorescence, total internal reflection (polTIRF) microscopy to determine the dynamics of specific protein structural changes during translocation along actin and under mechanical load. Increased time resolution recently achieved for measuring the rotational, translational, and thermal wobbling motions and will enable detailed events to be detected during the brief period of molecular stepping between stable dwell periods. An infrared optical trap, with high-speed feedback to clamp the actin in place and to rapidly measure the myosin working stroke after actin attachment, will be used to determine the specific relationships between release of ATPase products, phosphate and ADP, strengthening of the actomyosin bond, transition into force generating states, and tilting, resulting in movement of the cargo. The feedback optical trap will be combined with single-molecule polTIRF microscopy to directly evaluate the influence of mechanical stress, strain, and flexibility on stepping rates and protein orientation changes that relate to chemo-mechanical transduction. The energetics and statistics of actin subunit target selection will be determined from the orientation and force dependence of the domain angles, biochemical states and step sizes. The experiments will be carried out on non-muscle myosins isolated from recombinant expression systems. Results from this project should significantly advance knowledge of cell motility processes and thus bring a greater understanding of both normal and pathological states of neuronal and sensory-neural development and many other types of cell motility.