One focus of 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 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 of force generation—the entropically-favored dissociation of inorganic phosphate.
My graduate research revealed that actin initiates the force-generating power stroke before phosphate dissociation and elucidated how power output and efficiency are regulated by the distribution of myosin’s structural states. Time-resolved FRET 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 demonstrated 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 (mava), 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 that is not present in the single-headed myosin fragment. From these results, we concluded that cardiac myosin is regulated by an interaction between its two heads and the thick filament; and proposed that mavacamten stabilizes this state.
As a post-doc working in collaboration with the lab of Dr. Chris Yengo at Penn State, I am investigating mutations in expressed human b-cardiac myosin and myosin V to examine how point mutations alter specific structural transitions in the ATPase cycle of these motor proteins. Some of these mutations cause dilated or hypertrophic cardiomyopathies (DCM, HCM) in humans. These experiments reveal new and important mechanistic insights into myosin’s structural dynamics and provide proof-of-concept results for developing therapeutic technology.
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