A mixed-kinetic model describes unloaded velocities of smooth, skeletal, and cardiac muscle myosin filaments in vitro

Fig. 1. Kinetic scheme, myosin structures, motility assay geometries, and model predictions. (A) Kinetic scheme for myosin (M) attachment to actin (A). D, ADP; T, ATP; Pi, phosphate. Kw, equilibrium constant for weak binding of myosin to actin; kws, forward rate constant for the weak to strong transition. We assume that −kws is insignificant. See text for other rate constants. (B to E) None of the cartoons are to scale. (B) Side polar (smooth muscle) and bipolar myosin filaments (skeletal and cardiac muscle), heads (orange), and tails (black). (C) A/Mm assay with actin filament (green) being moved by myosin heads (orange) attached to coverslip (blue). Only the S2 region of the tail (black) is shown. Before the working step (left), after the working step (middle), and after the fullest possible extension of S2 (right) by other heads undergoing the working steps (not shown for clarity). In this example, d = L = 8 nm. (D) Mf/A assay (5, 6), myosin filament (orange) moves over biotinylated actin (green) attached to PEG brush-coated coverslip (not shown). Pre-working step (left), post-working step (middle), and after full extension of S2 (right) by other working heads (not shown) giving L =…

Fig. 2. Detachment-limited model (Eq. 1) and mixed-kinetic model (Eq. 5) fits to N dependence of filament velocities (V) at saturating [ATP]. All experiments were performed in the observation buffer at 30°C. (A) SMM filaments moving in the Mf/A assay at 0.75 to 2.0 mM ATP from Brizendine et al. (6). Purple triangles, 26% SMM co-filaments, n = 10; orange circles, 51% SMM co-filaments; green triangles, 75% SMM co-filaments; blue diamond, rapidly diluted (short) SMM filaments; each point is the average V and N from 10 individual filament trajectories, and error bars show the SD. Red squares, SMM filaments prepared by dialysis; each point (n = 82) is V of a single filament trajectory. (B) CMM filaments moving in the Mf/A assay at 0.75 to 2.0 mM ATP. Blue diamonds, CMM filaments, n = 95; green triangles, 50% CMM co-filaments, n = 62; orange circles, 30% CMM co-filaments, n = 19. (C) SKM filaments moving in the Mf/A assay at 0.75 to 2.0 mM ATP. Purple triangles, 25% SKM co-filaments, n = 38; orange circles, 50% SKM co-filaments, n = 47; green triangles, 75% SKM co-filaments, n = 41; blue diamonds, SKM filaments prepared by rapid dilution (short), n = 122; red squares, SKM filaments prepared by…

Fig. 3. A/Mm data for SKM. All experiments were performed in the observation buffer at 30°C. (A) Relationship between [myosin] applied to the coverslip (μg ml−1) and N. Red points are averages of two to six independent experiments; error bars show the SD; the red line is a linear fit, slope = 0.34 N ml μg−1. Black squares, data from Harris and Warshaw (17). (B) N dependence of V at 1 mM (red), 100 μM (green), and 20 μM ATP (blue). N values correspond to applied SKM concentrations between 5 and 400 μg ml−1. (C) Contour plot of L versus k−AD showing dependence of R2 values from the fit to 1000 μM ATP data from (B) (see fig. S2 for plots of 20 and 100 μM ATP data). Color scale shows the range of R2 values. (D) ATP dependence of V at N = 34 (100 μg ml−1). For (B) and (D), each point is an average of 40 individual actin filament trajectories, and error bars show the SD. Respective lines show the best fits to Eq. 5.

Fig. 4. ATP dependence of filament V at fixed N from the Mf/A assay. (A) SKM filaments formed by rapid dilution. (B) CMM filaments formed by rapid dilution. For both plots, each point is an average of 40 individual trajectories. Error bars show the SD. Red lines show the fits to Eq. 5 (see Table 2 for results). R2 = 0.95 and 0.96, respectively. All experiments were performed in the observation buffer at 30°C.