StructuraL Dynamics of Actomyosin Motility

项目摘要 这项研究的总体目标是了解分子机制， 肌动球蛋白运动系统将化学能转化为机械功，并获得 分子水平上的机械、生物化学和结构事件之间的精确相关性 水平 新的方法将被应用于非肌肉肌球蛋白分子马达，以探测肌球蛋白分子马达。 收缩蛋白的生化反应、基本力学性质、 跨桥周期的步骤和相应的结构运动。 双功能，双- 砷和量子棒荧光探针将以已知的取向稳定地结合到 马达结构域、轻链亚基和马达的尾部。 空间方向和 这些组件的平移位置将通过新颖的方法以高时间分辨率监测。 单分子偏振荧光，全内反射（polTIRF）显微镜， 确定特定蛋白质结构变化的动力学过程中，移位沿着肌动蛋白 并且在机械负载下。 最近实现的测量时间分辨率提高 旋转，平移和热摆动运动，并将使详细的事件， 在稳定停留期之间的短暂分子步进期间检测到。红外 光学陷阱，具有高速反馈以将肌动蛋白夹在适当位置并快速测量 肌球蛋白工作行程后，肌动蛋白的附件，将用于确定具体 ATP酶产物、磷酸盐和ADP的释放， 肌动球蛋白键，过渡到力产生状态，并倾斜，导致运动的 货物.反馈光学陷阱将与单分子polTIRF显微镜相结合， 直接评估机械应力、应变和柔性对步进速率的影响， 与化学机械转导相关的蛋白质方向变化。能量学和 肌动蛋白亚基靶选择的统计学将从方向和力确定 依赖的域角度，生化状态和步长。实验将是 在从重组表达系统分离的非肌肉肌球蛋白上进行。结果 该项目将大大推进细胞运动过程的知识，从而带来 更好地理解神经元和感觉神经的正常和病理状态 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.