Browsing by Subject "heart failure"

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    Myosin structural dynamics: mechanistic insights and therapeutic technology developments
    (2019-03) Rohde, John
    A major focus in molecular biophysics is to understand how protein structural isomerizations correspond to cellular and organismal physiology. The heart generates force to perfuse the body with oxygenated blood through contractile units in myocytes called sarcomeres. The primary force-generating protein in this contractile apparatus is myosin. Our lab has developed a strategic tool called transient time-resolved FRET, (TR)2FRET, to measure directly, with sub-nanometer and sub-millisecond resolution, the structural and biochemical kinetics of muscle myosin. This tool allows us to directly determine how myosin’s power stroke is coupled to the thermodynamic drive for force generation—the entropically-favored dissociation of inorganic phosphate. My research revealed that actin initiates the force-generating power stroke before phosphate dissociation, revealing how power output and efficiency are regulated by the distribution of myosin’s structural states. (TR)2FRET is also a powerful tool to examine small-molecule perturbations of structural transitions within myosin’s kinetic cycle. Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. My results demonstrate that OM stabilizes myosin’s pre-powerstroke structural state and significantly slows the actin-induced powerstroke. I also used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and prospective therapeutic for hypertrophic cardiomyopathy. I found that mavacamten stabilizes an auto-inhibited state of two-headed cardiac myosin, not present in the single-headed myosin motor fragment. From these results, we predicted that cardiac myosin is regulated by an interaction between its two heads and the thick filament, and proposed that mavacamten stabilizes this state. I also investigated two mutations in the converter domain of myosin V to examine how point mutations alter specific structural transitions in the myosin motor’s ATPase cycle. Transient kinetics analyses and FRET-based experiments demonstrated that one mutation slowed the recovery-stroke rate constants, while a second mutation enhanced these steps. These mutations correspond to human mutations that give rise to dilated or hypertrophic cardiomyopathies, respectively. Together these experiments reveal new and important mechanistic insights into myosin’s structural dynamics and provide proof-of-concept results for developing therapeutic technology.