Physical-Chemical Basis of the Contractile Mechanism

9513898 Lowey本研究的目的是通过两种方法来测试肌肉收缩的“旋转横桥模型”：（1）冷冻电子显微镜和图像分析将用于分析用重组肌球蛋白头部片段修饰的肌动蛋白丝的分子结构。 通过结合肌动蛋白和S1的X射线晶体结构与计算机生成的冷冻水合修饰的F-肌动蛋白的电子显微照片的三维重建，它可能会看到是否构象变化发生在肌球蛋白结合肌动蛋白。 (2)多维核磁共振（NMR）将用于探索游离和结合状态下调控轻链的结构及其在磷酸化后的相互作用。 轻链及其靶肽将在E. coli进行同位素标记。 骨骼肌轻链的结构将与平滑肌轻链进行比较，以更好地理解这两种主要肌肉类型的调节机制。 关于肌肉如何收缩，最广为接受的理论是肌球蛋白头相对于肌动蛋白丝的重新定位，因为肌动蛋白丝相互滑动。 “旋转横桥模型”的一个基本问题是未能检测到肌球蛋白分子内任何大的构象变化，这些变化可以解释连接的横桥可以产生张力的大距离。 这里提出的研究旨在测试模型的肌肉收缩的基础上最近解决的晶体结构的肌球蛋白头。 ***

9513898 Lowey The goal of the research is to test the "rotating crossbridge model" for muscle contraction by two approaches: (1) cryo-electron microscopy and image analysis will be used to analyze the molecular structure of actin filaments decorated with recombinant myosin head fragments. By combining the X-ray crystal structures of actin and S1 with the computer generated three-dimensional reconstructions from electron micrographs of frozen-hydrated decorated F-actin, it may be possible to see whether conformational changes occur in myosin upon its binding to actin. (2) Multi-dimensional nuclear magnetic resonance (NMR) will be used to explore the structure of the regulatory light chain in the free and bound state, and its interactions upon phosphorylation. The light chain and its target peptide will be expressed in E. coli for isotope labeling. The structure of the skeletal muscle light chain will be compared to the smooth muscle light chain for a better understanding of the mechanism of regulation in these two major muscle types. %%% The most widely accepted theory of how muscles contract involves a reorientation of the myosin head relative to the actin filament as the filaments slide past one another. A fundamental problem with the "rotating crossbridge model" has been the failure to detect any large conformational changes within the myosin molecule that could account for the large distance over which an attached crossbridge can develop tension. The studies proposed here are designed to test models for muscle contraction based on the recently solved crystal structure of the myosin head. ***