I will use a combination of experimental and theoretical methods to define the conformational changes at the nucleotide site of myosin associated with the interactions with nucleotides and with actin, along with the relationship of these conformational changes to the motility cycle. Myosin x-ray structures in the presence of actin remain elusive, and must instead be extrapolated from actin-free structures. A central problem in defining the molecular mechanism of myosin remains to determine how the structure of myosin changes when it binds either weakly or strongly to actin, and whether additional important structures occur in the cycle that have not yet been resolved. To address this question, I will use paramagnetic probes at the nucleotide site to monitor nucleotide-state dependent conformational changes in myosin and those associated with the weakly and strongly bound actomyosin states. This information will be combined with distance measurements acrossthe nucleotide site using spectroscopic probes bound to the protein, along with biochemical and mechanical observations of actomyosin function, as a further monitor of structuralchanges. In particular, myosin II, myosin V, and myosin VI have been crystallized in structures with very open nucleotide sites that are widely hypothesized to represent the actin-bound states of myosin. These myosins will be investigated. Spin labeled nucleotides will also be bound to the crystal forms and the properties compared to in vitro observations, above, as an additional probe of whether the crystal structures, in fact, represent the actin bound forms, and whether they occur in the motor cycle. An initial analysis of in vitro spectroscopic and biochemical data suggests that they do not. Kinesin-family motors will be investigated as a model for myosin. A major effort will be devoted to developing more quantitative interpretations of EPR spectra in terms of protein structure using molecular dynamics modeling. Together these data will lead to a more detailed picture of how conformational changes at the nucleotide site are involved in force generation.
