The objective of this research is to improve our understanding of the molecular basis of muscle contraction by determining the three-dimensional structure of myosin subfragment-l using single-crystal X-ray diffraction. Myosin is the major protein in muscle, in which it plays both a structural and enzymatic role. The myosin rod forms the backbone of the thick filament, whereas the myosin head (subfragment-l) is responsible for the generation of movement through the hydrolysis of ATP and its interaction with actin. Crystals of pectoralis myosin subfragment-l which diffract to at least 3 angstrom resolution have been grown reproducibly. The subfragment which was crystallized contains both the essential and regulatory classes of light chains. The determination of the structure of these crystals is in progress. In the next grant period it is planned to complete the data collection for the native crystals and four heavy atom derivatives using synchrotron radiation and then to solve the structure using a combination of multiple isomorphous replacement and solvent flattening methods. At an early stage this study should reveal the domain structure of the head and localize the position of the light chains. The second part of the study will be directed towards understanding the functional aspects of the molecule by determining the structure of the myosin head in the presence of nucleotide analogues. This will yield the location of the active site and should suggest how the energy derived from the hydrolysis of ATP is transferred into mechanical force.
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