All the experiments were performed using freshly prepared myosin at 23°C as described previously (7). WT and R663H sS1, 2-hep HMM, and 25-hep HMM mutant proteins were prepared simultaneously to minimize the effect of preparation variability. Myosin concentration was measured using eGFP absorbance. G-actin was prepared as described previously (7). F-actin free of ATP was prepared by extensively dialyzing G-actin into ATPase buffer to remove any residual ATP. Actin concentration was then measured using absorbance at 290 nm in a spectrophotometer. This eliminates any contribution from the nucleotide at 280 nm.

The steady-state actin-activated ATPase activities of the human β-cardiac myosin preparations were determined using a colorimetric assay to measure inorganic phosphate production at various time points (0 to 30 min) from a solution containing myosin (0.01 mg/ml), ATP and increasing amounts of actin filaments (0 to 100 μM), and ATPase buffer [10 mM imidazole (pH 7.5), 5 mM KCl, 1 mM DTT, and 3 mM MgCl2]. The time-dependent rate for each actin concentration was calculated by fitting the phosphate signal as a function of time to a linear function. The slope was then converted to activity units normalized to a single myosin head. The kcat was extracted from the data by fitting the activity at each actin concentration to the Michaelis-Menten equation to determine maximal activity using the curve fitting toolbox in MatLab. The errors in the fitted values were determined using 100 bootstrap iterations.

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