Physical-Chemical Basis of the Contractile Mechanism

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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. ***