Motility is one of the characteristic features of all living organisms and involves the transduction of chemical into mechanical energy. It now appears that nature has evolved three primary classes of molecules that accomplish this task; the myosin, dynein, and kinesin super families of molecular motors. The objective of this proposal is to provide a molecular basis for understanding muscle contraction and myosin-based motility by studying the three-dimensional structure of myosin with a combination of single crystal x-ray diffraction and site- directed mutagenesis. There are five specific aims. The immediate goals are 1) to understand the molecular mechanism of ATP hydrolysis by myosin and how this is structurally coupled to the contractile cycle. 2) to trap myosin in its conformation at the start of the power stroke. 3) to trap the molecule in a structural state that resembles the end of the power stroke when it is tightly bound to actin. The first goal will be achieved by determining the contribution that the adenine binding pocket and switch I and II make to the contractile cycle. Switch I and II are loops that are analogous to those found in the G-proteins and are believed to participate in the transmission of the conformational change associated with ATP hydrolysis to C-terminus of the myosin head. The second and third goals will be achieved by introducing pairs of cysteine residues between domains that are believed to move during the contractile cycle. The immediate goals will be accomplished by studying genetically truncated fragments of Dictyostelium discoideum myosin II. The long term goals of the project are 4). to crystallize an actomyosin complex and 5. to crystallize several non-muscle myosins to identify the basis for the differences in motility, efficiency and power-output between these motor proteins.
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