Smooth muscle is essential to the normal operation of the body, playing crucial roles in vascular, respiratory, digestive, and other functions. Malfunction of smooth muscle is implicated in major diseases such as hypertension, asthma and arteriosclerosis. Smooth muscle contraction is ultimately a result of the sliding of actin filaments past myosin filaments. The long term objective of this project is to elucidate the structural basis of smooth muscle contraction and its regulation at the molecular level. Exciting new insights into smooth muscle function have recently become possible by solution of the crystal structures of several contractile and regulatory proteins and by recent advances in electron microscopy and image processing. We will take advantage of these advances to carry out the following Specific Aims: (1) To define the three-dimensional molecular architecture and composition of native smooth muscle myosin filaments; (2) To determine the molecular basis of the side-polar structure of smooth muscle myosin filaments and the structural significance of SM1 and SM2 myosin isoforms; (3) To determine the three- dimensional molecular structure of the actin filaments in smooth muscle; and (4) To elucidate the three-dimensional organization of the actin and myosin filaments in smooth muscle cells, and the structural basis of their interaction. Electron microscopy will be used to determine the molecular structure of the actin and myosin filaments, and the structural changes that accompany contraction. Muscle filaments will be preserved in close-to their native states using state-of-the-art techniques, and their three- dimensional structures will be computed by helical or tomographic reconstruction. A near-atomic understanding of filament structure and its relation to function will be gained by fitting the atomic structures of actin and the myosin head to the filament structures determined by electron microscopy. Novel structural insights into molecular assembly, organization and function will be obtained using complementary molecular biological and immunological approaches. Both vascular and gastrointestinal smooth muscle model systems will be studied. The results of this project should provide fundamental insights into the molecular mechanism of smooth muscle contraction and regulation, and into structural changes that may occur in diseased states. Preliminary data are presented showing the feasibility of our goals.
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