Smooth muscle actomyosin motor activity is activated by the phosphorylation of the 20,000 dalton regulatory light chain (LC20) of myosin and this initiates smooth muscle contraction. The long-term objectives are to elucidate the regulation and function of myosin in smooth muscle cells so as to understand the regulation and characteristics of smooth muscle contraction. While it has been known for years that myosin phosphorylation activates actomyosin ATPase activity, it is poorly understood how the smooth muscle myosin motor activity is regulated by the phosphorylation of LC20. The hypothesis is that the phosphorylation changes the conformation of LC20 which alters the myosin heavy chain at the head-rod hinge region. This information is transmitted to the distal motor domain of myosin via the long a-helix shaft of heavy chain at the C-terminal part of S-1. The PI will verify this hypothesis by employing the methods of molecular genetics, protein biochemistry, and structural biology. Various mutant proteins will be made by use of molecular genetic strategy. The recombinant proteins will be functionally expressed and analyzed by ATPase activity, in vitro motility assay and structurally analyzed by 19F-NMR, solution X-ray diffraction and X-ray crystallography. The itemized specific aims are: 1) Elucidation of the structure of 20,000 dalton light chain critical for the phosphorylation induced activation of myosin motor; 2) Identification of the function of 17,000 dalton light chain on the motor activity of myosin; 3) Identification of heavy chain structure responsible for the regulation and the characteristics of smooth muscle myosin function; XXXXchain structure critical for high force production/slow velocity of smooth 4) To investigate the conformational change of smooth muscle myosin subfragments using solution X-ray diffraction; 5) To determine the 3D structure of smooth muscle myosin S1.
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