Cardiac myosin toggles between force-producing ON conformations and autoinhibited OFF states that include the interacting-heads motif (IHM). We examined two complementary IHM FRET sensors: (1) AF488 on the regulatory light chain (RLC) paired with Cy3-ATP in the active site (lever-arm docking), and (2) Cy3-ATP paired with a C-terminal GFP fusion on the heavy chain (free-head docking). Human beta-cardiac heavy meromyosin with a 15-heptad tail (15HP), as well as controls that cannot form the IHM (2-heptad, 2HP, and subfragment 1, S1), were assayed at varying ionic strengths using stopped-flow and steady-state FRET. Both sensors in the 15 HP construct report higher FRET at low ionic strength and lower FRET at high ionic strength, consistent with stabilization and disruption of OFF conformations, respectively. In low-salt conditions with both FRET sensors, the binding of Cy3ATP to 15HP results in two phases, a fast phase similar to ATP binding (2 μM -1 ∙s -1 ) and a slow phase of unknown origin. In high salt conditions, there is a significant slowing of the FRET signal upon Cy3ATP binding that remains linearly dependent on Cy3ATP concentration (0.4–0.7 μM −1 ∙s −1 ). We monitored the tryptophan fluorescence enhancement upon ATP binding and hydrolysis to determine if the slower transition could be a slowing of ATP binding and hydrolysis. However, we found that the tryptophan fluorescence signal was insensitive to ionic strength in the 15HP as well as in the 2HP and S1 constructs. Our data fit a model in which the transition into the IHM is dependent on ATP binding and hydrolysis, at low ATP concentrations, as well as the equilibrium between the ON/OFF states. Overall, this work adds to our understanding of the kinetic mechanism of the ON/OFF transitions in beta-cardiac myosin.
Ge et al. (Sun,) studied this question.