Influence of IHM/SRX content on motility of WT 2HEP:15HEP mixture. (a) The Velocity Index versus surface occupancy of WT 2HEP, 15HEP, and a mixture of these two constructs. Addition of increasing amounts (0, 25, 50, 75, 100, and 125 nM) of 15HEP WT (76% IHM/SRX) to a constant amount (25 nM) of 2HEP WT (14% IHM/SRX) results in increasing Velocity Index values (filled octagons) that compare favorably to predicted values (open octagons; see Table S1), supporting the model’s predictive nature, which assumes that a given construct has a defined IHM/SRX content and that the Velocity Index reflects actin filament motility generated by only active motors. (b) Data in a were transformed to Velocity Index versus surface occupancy of active heads by replotting the Velocity Index data in a after calculating the surface occupancy of active (DRX) myosin heads from the product of observed total myosin surface occupancy and predicted percent DRX (i.e., 100% − [predicted %IHM/SRX content]). The solid curve represents the 0% IHM/SRX curve (Fig. S4). The 2HEP (open circles), 15HEP (open triangles), and observed mixture (closed hexagons) data, once transformed to represent only active motors, are well fit by the 0% IHM/SRX curve. Data points represent mean values ± SD of three experiments from separate protein preparations.