Fig. 3. Functional assays for ATP turnover by WT, R403Q, and R663H human b-cardiac 25-hep HMM and 2-hep HMM.

(A) Actin-activated ATPase of R403Q 2-hep HMM (light blue) and R403Q 25-hep HMM (dark blue). (B) Actin-activated ATPase of R663H 2-hep HMM (orange) and R663H 25-hep HMM (dark red). For (A) and (B), data are combined from two experiments from one protein preparation. Each point is an average, with the error bar as SEM. (C) Fluorescence decay of mant-nucleotide release from WT human ß-cardiac 25-hep HMM in 25 mM KAc (solid black curve). Fitting the traces (dashed cyan curve) to a double-exponential equation yielded the DRX and SRX rates of 0.030 and 0.0034 s⁻¹. The simulated single-exponential orange dashed curve is the SRX rate (0.0034 s⁻¹), and the simulated single-exponential dashed green curve is the DRX rate (0.030 s⁻¹). The simulated orange and green dashed curves act as visual references for the single exponential fits of slow and fast phases, respectively, that derive from fitting the solid black data curves with the best two exponential fits. Thus, the data (black lines) were fit with a combination of these two single exponentials. (D) Fluorescence decay of mant-nucleotide release from R403Q human ß-cardiac 25-hep HMM in 25 mM KAc, where the fast and slow fitting rates were 0.017 and 0.0017 s⁻¹, respectively. (E) Fluorescence decay of mant-nucleotide release from R663H human ß-cardiac 25-hep HMM in 25 mM KAc, where the fast and slow fitting rates were 0.047 and 0.0031 s⁻¹, respectively. (C) and (E) show representative data from one preparation of each protein. (F) Percentage of myosin heads in the SRX (orange) versus DRX (olive green) states calculated from the amplitudes of the double-exponential fits of the fluorescence decays corresponding to the mant-nucleotide release from the 25-hep HMMs shown in (C) to (E). The data are from nine measurements (from six individual protein preparations done on different days) of WT 25-hep HMM, five measurements of R403Q 25-hep HMM (from four individual protein preparations done on different days), and seven measurements of R663H 25-hep HMM (from five individual protein preparations done on different days). Error bars, SEM. **P ≤ 0.01; ****P ≤ 0.0001. WT versus R403Q, P = 0.01; WT versus R663H, P = 0.00003. (G) Homology model of the IHM state of human β-cardiac 25-hep HMM [human sequestered state model from Robert-Paganin et al. (14)] showing the positions of the blocked head (bh) Arg⁴⁰³ residue (dark blue) and the free head (fh) Arg⁶⁶³ residue (light blue) at a head-head interaction site. (H) Blowup of the IHM model showing the positions of the bh Arg⁴⁰³ and fh Arg⁶⁶³ residues at the interface between the blocked head (dark red) and the free head (light brown).