R92Q alters the positioning of tropomyosin along the thin filament. (A ) Kinetic scheme for thin filament activation. (B) Measurement of the equilibrium constant K_B using stopped-flow kinetics methods (fluorescence transients are shown). Pyrene-labeled regulated thin filaments were rapidly mixed with myosin at high (pCa 4) or low (pCa 9) calcium, and the rate of myosin binding was measured by quenching of the fluorescence. K_B is calculated as described in Materials and methods. The rate of myosin binding is similar for the WT and R92Q at pCa 4 but much faster for R92Q at pCa 9, consistent with destabilization of the blocked state. K_B for R92Q is significantly larger than the WT. (C) Measurement of the parameters K_T and K_W using equilibrium titrations of myosin with regulated thin filaments. Fitting reveals no significant differences for R92Q for either K_T or K_W compared with the WT. WT data are from Clippinger et al. (2019). Error bars show the standard deviation of five experiments. The average value, 95% confidence intervals calculated from bootstrapping, and P values derived from the distribution of bootstrapped values are shown in the table.