a-d TIRF-microscopic images (110 x 100 pixels) are shown. Deac-aminoADP was imaged at 442 nm (a-c); Alexa-Fluor-568-MyoV-HMM was imaged at 568 nm (d). Two-dimensional intensity profiles from each white square in a-d are shown in e-h.a,e, Deac-aminoADP bound directly on the coverslip, with maximum camera gain (1000). b,f, Deac-aminoADP bound directly on the coverslip surface at a gain of 400 on camera. c,g, Deac-aminoADP bound to MyoV-HMM at a gain of 400. d,h, Alexa-Fluor-568- MyoV-HMM bound to surface at a gain of 400. All data were taken with an iXon+ camera (DV897, Andor technology) with at 10 MHz readout at a constant laser power. The background level was fixed at about 750 (arbitrary units, a.u..) intensity. Scale bar, 2 μm

a-c, Histograms of the lifetimes prior to deac-aminonucleotide dissociation at 100 nM (n = 261 steps, 44 MyoV-HMM molecules), 200 nM (n = 262 steps, 38 myosin-V molecule), and 400 nM (n = 310 steps, 35 MyoV-HMM molecules) deac-aminoATP. The solid lines represent the exponential fit of the dwell time distribution. The fitted lifetimes at 100 nM (a), 200 nM (b), and 400 nM (c) deac-aminoATP are 0.85 ± 0.06 s (r² = 0.98), 0.88 ± 0.07 s (r² = 0.98), and 0.77 ± 0.04 s (r² = 0.98; all mean ± S.D.), respectively, corresponding to rate constants of 0.82 ± 0.06 s^-1, 0..79 ± 0.07 s^-1 and 0.90 ± 0.05 s^-1. d-f, Histograms of the lifetimes of deac-amino ATP binding. The fitted lifetimes at 100 nM (n = 296); d), 200 nM (n = 267); e) and 400 nM (n = 309); f) deac-aminoATP are 1.32 ± 0.17 s (r² = 0.98), 1.08 ± 0.11 s (r² = 0.98), and, 0.68 ± 0.08 s (r² = 0.98) (mean ± S.D.), respectively. Note that the number of spots is the same as a-c. This corresponds to rate constants of 0.53 ± 0.07 s^-1, 0.64 ± 0.06 s^-1, and 1.02 ± 0.12 s^-1, respectively. Statistical analysis (t-test) between each experimental points revealed that the data of ADP dissociation rate from three conditions are not significantly different (P(T≤t) = 0.68, 0.09, 0.16 of 100 vs. 200, 100 vs 400, and 200 vs. 400 nM deac-aminoATP, respectively). In contrast, P values of ATP binding rates are significantly different as shown (P(T≤t) = 0.01, 0.028, and 0.02 for 100 vs. 200, 100 vs 400, and 200 vs. 400 nM deac-aminoATP, respectively). g, Concentration dependence of the rates of deac-aminoADP dissociation (open circles) and deac-aminoATP binding (filled circles). The deac-aminoATP binding data were fit by linear regression which gave a slope corresponding to a second order rate constant of 1.67 μM^-1s^-1. Note the nonzero intercept which is also present in the solution kinetic measurements of deac-aminoATP binding to acto-HMM in Supplementary Fig. 9. A non-zero intercept is predicted by modeling the kinetic mechanism and is seen in some published studies (Rief et al5) but is readily because of the large extrapolation from the high nucleotide values typically used. The horizontal line through the deac-aminoADP dissociation data represents the average of the mean value for the three nucleotide concentrations (0.86 s^-1).

Images of Alexa-Fluor-568-MyoV-HMM and deac-aminoATP fluorescence were acquired simultaneously with a Dual-View system. The excitation/emission wavelengths of Alexa Fluor 568 are distinct from those of deac-aminoATP, allowing simultaneous visualization of the MyoV-HMM and the nucleotide. The photons from the spots were acquired in 330 ms integrations and the point spread function from each spot was fit with a two-dimensional Gaussian to determine the location of the fluorophor(es) at each time point18. a, The protein fluorescence data shows ∼36 nm steps, which are marked by red vertical dotted lines at 200 nM deac-aminoATP. Red double-ended arrows delineate the dwell time of a step. b, The deac-aminonucleotide stepping events are marked by alternating red and blue vertical dotted lines. Red vertical dotted lines are steps of both MyoV-HMM and deac-aminonucleotide, whereas blue vertical dotted lines show only stepping of deac-aminonucleotide. Individual spots move in a stepwise manner in the same direction as the MyoV-HMM. Dwell times are marked by blue and green double arrows. Red horizontal lines mark the average position of the spot during a pause. c, Normalized intensity of the deac-aminonucleotide fluorescence. d, Step-size histogram for the movement of Alexa-Fluor-568-MyoV-HMM (n = 145 steps, 38 MyoV-HMM molecules). The curve represents the fit to a Gaussian, (mean ± S.D.: 36.3 ± 7.2 nm.) e, Step-size histogram for the movement of the deac-aminonucleotide (n = 267 steps, 38 MyoV-HMM molecules). The red curve represents the fit to the sum of two Gaussians (shown individually in black lines) (mean ± S.D: 17.5 ± 7.1 nm, 36.0 ± 8.6 nm). F, Model for correlation of movement of Alexa-Fluor-568-MyoV-HMM and deac-aminonucleotide binding and dissociation. The red and blue arrows show the position of the centroid of the Alexa-Fluor-568-MyoV-HMM and of the deac-aminonucleotide fluorescence, respectively. See text for description of the model.

The mechanism shown by intermediates (1) through (4) is the main pathway of stepping during a MyoV-HMM processive run. Termination of runs would occur via pathway (1) to (A) to (B) or more rarely by (C) to (D) to (E) to (A) to (B). Red spots represent ADP and green spots represent P_i.