Myosins are a family of motor proteins ubiquitous in animal cells. The cyclic, ATP-hydrolysis-driven interaction of myosin with filamentous actin drives numerous motile processes in eukaryotic cells including muscle contraction, cytokinesis and cell motility. The importance of this interaction in disease pathogenesis is exemplified by the fact that myosin mutations have been implicated for example in familial hypertrophic cardiomyopathy (FHC), the most common cause of sudden death in otherwise healthy young individuals. Structurally, myosins have one or two heads and a tail. The head is comprised of a motor domain that binds ATP and actin, and a so-called regulatory domain connecting the motor domain to the tail. Although the link between the myosin ATPase cycle and motility has been established through biochemical and mechanical studies, the accompanying structural changes are not completely defined. The myosin regulatory domain is thought to act as a lever arm, amplifying small changes in the motor domain into a large movement of the tail. We propose to study the actin-bound states of smooth and skeletal muscle myosin II. A combination of electron cryomicroscopy (cryo-EM) and image reconstruction in conjunction with real-space difference mapping, structural flexibility analysis, and computer-based fitting of the atomic models into the three-dimensional reconstructions will be used in order to identify conformational and dynamic changes within the complexes. Previous cryo-EM studies on actin-myosin II assemblies provided approximate actomyosin models and an estimation of the movement of the regulatory domain upon MgADP release. The structural studies proposed here will correlate changes within the actomyosin complex to positional changes in the regulatory domain. These studies will provide insights into which regions within the complex are involved in energy transduction. This information will allow us to define structural links between these regions. Finally, the resulting atomic models of the actomyosin complexes will be used to analyze the actomyosin interactions and may reveal how isoforms or mutations in these regions effect normal muscle function. These studies will complement the biochemical and biophysical studies of our collaborators Drs. S. Lowey and K. Trybus at the University of Vermont and D. DeRosier at Brandeis University.
