Myosin molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in the myosin family of molecular motors. A prime example is inherited familial hypertrophic cardiomyopathy (HCM), which leads to hyper-contractility of the heart. HCM results from mutations in various cardiac muscle proteins, with mutations in ?-cardiac myosin and its associated thick filament protein myosin binding protein C (MyBPC) accounting for about 80% of these cases. HCM is not rare, affecting as many as 1 in 200 people. Current therapeutic interventions for cardiomyopathies are limited to symptomatic relief, in large part because the molecular underpinnings of the disease ? how mutations affect the biomechanical interaction of myosin with its sarcomeric partners, and thus sarcomeric force, velocity, and power output ? are not well understood. Studies using human ?-cardiac myosin have shown that mutation-induced changes in the basic biochemical and biomechanical parameters of the myosin motor do not adequately account for the cardiac hypercontractility that is a clinical hallmark of HCM. Rather, it has recently been shown that HCM-causing mutations in the myosin motor domain disrupt intramolecular interactions that stabilize a folded-back, off state of myosin. This results in an increase in the number of heads functionally accessible to interact with actin, which in turn may lead to hypercontractility. In this proposal, the effects of HCM-causing point mutations in different regions of human ?-cardiac myosin will be explored, including the motor domain and both the proximal and distal portions of the alpha-helical coiled coil tail that allows myosin to form bipolar thick filaments. The interaction of myosin with MyBPC has also been implicated in regulation of the folded-back state of myosin. The effects of potential physiological regulators of this interaction, including phosphorylation and calcium binding, will be assessed using binding and functional assays. The effects of point mutations in different regions of myosin and in different domains of MyBPC will also be determined. Finally, a variety of structural approaches will be employed to determine the structure of the folded-back state of myosin in the absence and presence of MyBPC. Both negative stain and cryo-electron microscopy will be used to study the folded-back form of myosin and the myosin-MyBPC complex, and this work will be supplemented by cross-linking mass spectrometry to define interfacial residues. FRET probes will be placed on the human ?-cardiac myosin to observe its transition between the on-and-off states. This measurement will be critical in the HCM mutant myosins which have been shown to disrupt the off-state of myosin. Transient time-resolved FRET measurements will enable us to measure the nanosecond dynamics of myosin as it undergoes the on-to-off transition. Thus, we will use this more complex reconstituted system and an array of assays to determine the role of ?-cardiac myosin and MyBPC on the sequestration of myosin heads under physiological and pathophysiological conditions.
Inherited familial cardiomyopathies affecting 1 out of 500 people result from missense mutations in various cardiac muscle proteins, with mutations in beta-cardiac myosin and myosin binding protein C accounting for the vast majority of this disease. Current therapies for cardiomyopathies are limited to symptomatic relief, in large part because it is not understood how these mutations affect the ability of the heart to contract at its most fundamental level. Our research will significantly enhance our understanding of the effects of disease causing mutations on the fundamental contractile properties of the heart.
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