NANOSECOND ROTATIONAL DYNAMICS OF THIN FILAMENT PROTEINS

这个项目的长期目标是确定分子 肌动球蛋白中力的产生机制及其调控 骨骼肌的收缩系统。该项目旨在实现 这一目标是通过测量行星的纳秒自转运动实现的 肌动球蛋白在三磷酸腺苷收缩过程中的单个蛋白质 在重组的模型系统中进行的水解。旋转动力学 细丝复合体的单个标记蛋白质的 利用荧光的时间分辨偏振各向异性测量 从共价连接到特定巯基上的探针 肌动蛋白、原肌球蛋白、肌钙蛋白C和肌钙蛋白I。 探针包括IAEDANS和PY)。纳秒自转 这些蛋白质中的每一种的动态都将在未来 微丝复合体(F-肌动蛋白+原肌球蛋白+肌钙蛋白)和复合体 与肌球蛋白及其蛋白分解片段(肌球蛋白头部)在 模拟生理上相关的僵硬状态的条件 (-Ca~(2+)，-ATP)，松弛(-Ca~(2+)，+ATP)，收缩(+Ca~(2+)，+ATP)。 这些研究将补充我们正在进行的关于轮换的研究 这些蛋白质在微秒时间尺度上的动力学 磷光发射各向异性。

The long term goal of this project is to determine the molecular mechanism of force generation and its regulation in the actomyosin contractile system of skeletal muscle. The project seeks to achieve this goal through measurements of the nanosecond rotational motions of individual proteins of the actomyosin contractile apparatus during ATP hydrolysis in a reconstituted model system. The rotational dynamics of individually labeled proteins of the thin filament complex will be measured using time-resolved polarization anisotropy of fluorescence from probes covalently attached to specific sulfhydryls in the proteins actin, tropomyosin, troponin C, and troponin I. (Appropriate probes include IAEDANS and pyrene). The nanosecond rotational dynamics of each of these proteins will be monitored in the thin filament complex (F-actin + Tropomyosin + Troponin) and in complexes with myosin and its proteolytic fragments (myosin heads) under conditions that mimic the physiologically relevant states of rigor (-Ca2+, -ATP), relaxation (-Ca2+,+ATP), and contraction (+Ca2+,+ATP). These studies will compliment our ongoing studies of the rotational dynamics of these proteins on the microsecond time scale using phosphorescence emission anisotropy.