R92Q does not change the rate of unloaded actomyosin dissociation or calcium binding affinity to troponin C. (A) In vitro motility assays using cardiac myosin and reconstituted regulated thin filaments. The speed of motility was measured as a function of calcium. R92Q shows a shift toward submaximal calcium activation. WT data are from Clippinger et al. (2019). Error bars represent SDs from three separate experiments. (B) The rate of ADP release from myosin attached to regulated thin filaments was measured using stopped-flow kinetics (fluorescence transients are shown). Myosin bound to ADP and pyrene-labeled regulated thin filaments was rapidly mixed with ATP, and the fluorescence increases as myosin dissociates from the thin filament. The rate of actomyosin dissociation was unchanged by the R92Q mutation. WT data are from Clippinger et al. (2019). Curves show the average of five technical repeats. (C) The affinity of calcium binding to the troponin complex. IAANS-labeled troponin C was reconstituted into regulated thin filaments. Titrations with increasing calcium were conducted, and there is no difference in calcium binding affinity between WT and R92Q. Error bars show the SD of five experiments. Values derived from fits, standard errors in the fits, and P values are shown. P values were calculated using a two-tailed Student’s t test.