Fig. 5: HCM mutations Y115H and E497D increase the number of active human β-cardiac myosin heads as measured by the actin-activated ATPase LSAR assay.
Actin-activated ATPase curves for 8-hep and 15-hep constructs of a WT, b Y115H, and c E497D myosin. For each mutant, a set of WT 8-hep, mutant 8-hep and mutant 15-hep proteins was grown and processed for purification in parallel to reliably capture the differences in kcat due to the mutations. Raw data is combined from 12 experimental replicates from 4 independent protein preparations for WT and 9 experimental replicates from 3 independent protein preparations for mutants. Each data point represents the average across all replicates (mean ± SD is plotted). The data are fit to the Michaelis–Menten equation (solid line) to determine kcat and Km for each myosin (shaded areas indicate the 95% CI of the fit). ATPase curves in each panel are normalized to the respective 8-hep kcat (see Supplementary Table 2 for raw values and statistics). Downward arrows in a-c indicate the average % drop in kcat of 15-hep constructs relative to 8-hep controls. All numbers including exact p-values from multiple pairwise comparisons shown here are given in Supplementary Table 2. d Actin-activated ATPase curves with the Michaelis–Menten fits for the 15-hep constructs of WT (kcat = 2.9 ± 0.2 s−1), Y115H (kcat = 4.4 ± 0.1 s−1) and E497D (kcat = 5.8 ± 0.2 s−1) proteins from one tandem protein preparation; each data point denotes mean ± SD from three independent measurements at each actin concentration. Upward arrows indicate % increase in kcat of mutant 15-hep constructs relative to WT 15-hep protein. Source data are provided as a Source Data file.
