Physical-Chemical Basis of the Contractile Mechanism

本研究的目的是通过两种方法来检验肌肉收缩的“旋转过桥模型”：(1)利用冷冻电子显微镜和图像分析来分析重组肌凝蛋白头片段修饰的肌动蛋白丝的分子结构。通过将肌动蛋白和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图三维重建相结合，有可能看到肌球蛋白与肌动蛋白结合时是否发生构象变化。(2)利用多维核磁共振（NMR）研究调控轻链在自由和束缚状态下的结构，以及它们在磷酸化过程中的相互作用。轻链及其靶肽将在大肠杆菌中表达，用于同位素标记。将骨骼肌轻链的结构与平滑肌轻链进行比较，以便更好地了解这两种主要肌肉类型的调节机制。关于肌肉如何收缩，最被广泛接受的理论涉及到当肌动蛋白丝相互滑动时，肌凝蛋白头相对于肌动蛋白丝的重新定向。“旋转交叉桥模型”的一个基本问题是无法检测到肌球蛋白分子内任何大的构象变化，而这种变化可能解释了连接的交叉桥产生张力的大距离。这里提出的研究旨在测试基于最近解决的肌凝蛋白头部晶体结构的肌肉收缩模型。＊＊＊

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. ***