The hypertrophic cardiomyopathy mutation G768R makes cardiac myosin a high duty ratio motor.

β-cardiac myosin is the primary motor protein in the human heart responsible for force generation by converting chemical energy from ATP hydrolysis to mechanical work. It binds actin, produces force through its powerstroke, and releases actin, and must complete this cycle several times a second within each heartbeat. Like other muscle myosins, cardiac myosin has a low duty ratio of ~5%, the fraction of time in the actin-bound force-producing state. Here we present a hypertrophic cardiomyopathy (HCM) causing mutation, G768R, that increases cardiac myosin's duty ratio to >60%, an unprecedented >10x increase unmatched by any previously studied muscle myosin mutation. In recombinantly expressed human β-cardiac myosin subfragment-1 (sS1), G768R dramatically decreases the load-sensitive actin-detachment rate and step size of single molecules measured by optical tweezers. The ~15x longer actin-bound time combined with only a modest change in overall ATPase rate predicts a duty ratio of >60%. Motility velocity is also severely slowed, as expected given the long bound time and decreased step size. All-atom molecular dynamic simulations of the pre-powerstroke and post-rigor states predict that the mutation alters lever arm priming and reduces ADP pocket opening, providing a possible structural mechanism to corroborate the experimental observations. Finally, ATPase experiments on 2-headed heavy meromyosin (HMM) constructs suggest that G768R destabilizes the autoinhibited state of myosin. Our findings of high duty ratio and reduced autoinhibition provide molecular mechanisms of cardiac hypercontractility and impaired relaxation. A muscle myosin in the heart that stays bound to actin for over half its period is bound to have significant functional and clinical consequences.