The objective of this research is to improve our understanding of the molecular basis of muscle contraction by studying the three-dimensional structure of myosin subfragment-1 by 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-1, S1) is responsible for the generation of movement through the hydrolysis of ATP and its interaction with actin. During the last funding cycle, the structure of chicken skeletal myosin subfragment-1 was determined to 2.8 Angstroms resolution and refined to a nominal R-factor of 22%. This model provides a description of the secondary and tertiary structure of the molecule and indicates the location of the active site and actin binding site. These results also suggest a role for the light chains and a possible mechanism for how myosin, in conjunction with actin, transduces chemical energy into mechanical force. The goal of the next funding cycle is to test these hypotheses by determining the structural transformations that occur on nucleotide binding and hydrolysis and observing how these effects are communicated to the actin binding site. This will be accomplished in three ways. The first goal will be to complete the structure of chicken skeletal myosin subfragment-1 and extend the resolution of the data in order to produce an accurate model that can serve as the basis for interpreting the genetic, chemical, and kinetic information available for this molecule. Second, the structures of myosin S1 in the presence of nucleotides will be determined in an effort to understand the activated state of the molecule. Concurrent with this, an effort will be made to obtain crystals of the myosin head in the presence of actin peptides or assembly defective actin mutants. Third, the structures of cardiac and scallop myosin subfragment-1 will be determined to understand the differences in kinetic and functional behavior of these molecules and the molecular origin of myosin based regulation.
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