Determining the atomic level mechanism by which muscle proteins generate force and motion remains one of the fundamental questions in physiology. The recent determinations of the x-ray structures of different conformations of the motor protein, myosin, have provided new hypotheses as to its mechanism of function. However, a unifying framework defining the interaction of the nucleotide, ATP, with myosin to generate force and motion remains undetermined. Nucleotide (ATP) analogs allow one to perturb the interaction of the substrate with the protein, and correlate the perturbations with modulation of contractile activity. This can serve as a probe of the mechanism by which nucleotide hydrolysis drives the conformational changes in myosin that result in motility. We will use molecular dynamics (MD) simulations to investigate the interaction of ATP and nucleotide analogs with myosin x-ray structures as a probe of the mechanism by which nucleotide hydrolysis drives the conformational changes in myosin that generate force and motion. Our fundamental working hypothesis is that quantitative analyses of the x-ray structures can yield insights into function that other approaches have failed to discern. The simulation of nucleotide analogs at the active site will perturb the nucleotide-protein patterns normally associated with ATP binding, and allow further structure- function correlations to be drawn regarding the interaction of protein and substrate. A major goal will be to relate the simulation analyses to existing experimental data, and to identify further experimental challenges to the hypotheses generated by our modeling studies. Additional MD simulations will investigate the thermodynamic stability of the myosin dimerization domain and other alpha-helical coiled coil structures. The stability of the dimerization domain and the implications for myosin head-head interactions and the regulation of function remain unresolved, with some models suggesting a melting of the coiled coil as crucial to function. Quantitative MD simulations will be employed to analyze the relative stabilities of the coiled-coil domains of different myosin isoforms and their relationship to function. In addition to myosin II, we will investigate myosin V and myosin VI. In the latter isoform, the presence of an unstable coiled-coli domain has been hypothesized to be crucial for function. The protocols will be extended to study mutations in the coiled-coil regulatory protein, tropomyosin, which are associated with familial hypertrophic cardiomyopathy (FHC). An atomic level understanding of muscle protein function can be expected to lead to more rational therapies for FHC and other muscle diseases. Improved understanding of the function of myosin V and myosin VI can be anticipated to improve therapies for Griscelli syndrome and myosin Vl-based sensorineural hearing loss in humans. ? ? ?
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