Muscle contraction is fundamentally driven by an interaction between two proteins: actin and myosin. Myosin is a molecular motor, and assumes the active role in this relationship, coupling energy from hydrolysis of ATP with conformational changes to generate force on actin. In the context of a muscle fiber, this force causes filaments of myosin and actin to slide past one another in an ordered lattice, drawing the ends of individual contractile units (called sarcomeres) together. Concerted shortening of sarcomeres along the length of a fiber results in large-scale shortening of the entire fiber. Although muscle myosin has been the focus of intense study for many years, crucial details regarding its mechanism remain unknown. In particular, few structures of actin and myosin together have been reported—this is largely due to the inherent difficulties of handling large, filamentous protein complexes in traditional methods for structure determination. Myosin's interactions with actin are absolutely essential for macroscopic function, and this lack of structural information has created a knowledge gap: there is an abundance of functional and kinetic data for myosin in both normal and pathological states, but often no direct insight into the underlying structural causes for the observed behavior. In the present work, I seek to address this knowledge gap by providing high-resolution insight into the structural states of actin-bound myosin. My work is based on the hypothesis that allosteric coupling in myosin's catalytic domain (the domain responsible for actin binding, ATP hydrolysis, and initiation of force-generating conformational change) is accomplished via subtle internal rearrangements of individual structural elements. Furthermore, I hypothesize that these changes can be detected and quantified by innovative applications of site-directed spectroscopy. In Chapter 4, I establish a method using electron paramagnetic resonance (EPR) of a bifunctional spin label to probe nucleotide-dependent changes in the actomyosin complex. In Chapter 5, this method is expanded to include two complementary EPR techniques, ultimately providing sufficient constraints for direct modeling of nucleotide-dependent changes. Following these results, Chapter 6 addresses the ongoing development and further application of these methods within myosin and other protein systems.
University of Minnesota Ph.D. dissertation. November 2016. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: David Thomas. 1 computer file (PDF); xiv, 152 pages.
Elucidating the Structural Dynamics of Muscle Myosin Using Novel Methods in Electron Paramagnetic Resonance.
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