Motility analysis of WT human β-cardiac myosin constructs. (a) The Velocity Index versus surface occupancy for WT S1 and 2HEP constructs showed a dose–response-like characteristic that saturated at ∼2,000 nm/s and were indistinguishable (P = 0.87). (b) The Velocity Index versus surface occupancy for WT 2HEP, 15HEP, and 25HEP constructs. The longer-tailed WT 15HEP and 25HEP constructs had indistinguishable Velocity Indices (P = 0.87), but slower at each myosin surface occupancy compared to WT 2HEP. The WT 2HEP Velocity Index versus surface occupancy data were fitted to a Hill dose–response relationship (solid curve) and then used as the basis for an analytical model (see Results) to predict the IHM/SRX content for each construct. Predicted IHM/SRX percentages (legend in figure) and associated fitted curve (solid curve) are shown. All in vitro motility experiments were performed in low salt (25 mM KCl). Data points are presented as mean ± SD of at least three experiments from separate protein preparations.