Actin is the most ubiquitous and conserved eukaryotic protein, and plays a central role in motility and the control of cell form. Understanding the structure and function of actin will have an enormous impact in both the study of normal cellular processes, as well as in the study of diseases such as myopathies. While crystal structures exist for monomeric, G-actin, our structural knowledge of filamentous F-actin has come in large part from electron microscopy. Given that the active form of actin is the filament, understanding the structure and dynamics of the filament in terms of the atomic structure of the monomer has great relevance in such areas as cell motility and muscle contraction. This proposal is aimed at understanding the role of different structural states of actin, as well as the role of these states in the interaction of actin with other proteins, such as myosin, dystrophin and nebulin. Previous evidence shows that the state of an actin filament may be determined in part by the protein nucleating the filament, so studies of F-actin nucleated by the smooth muscle protein fesselin are expected to have important biological relevance. Significant advances in understanding the nature of actin filament states will only come from higher resolution reconstructions of F-actin, and a large thrust will be to use cryo-EM in a Field Emission Gun EM to help achieve this goal. These images, combined with a new algorithm for image analysis of helical polymers, should extend the resolution to the point that domain-domain motions and conformational changes from existing crystal structures can be described with high confidence. An important constraint in generating and interpreting such higher-resolution reconstructions will be to use engineered disulfides between adjacent subunits in the filament. Since a growing body of evidence suggests that conformational changes in actin are necessary for actomyosin motility, insight that will be gained from these studies is likely to have a very direct bearing on understanding muscle contraction.
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