Single molecule, optical trap analysis of Myo5 step size and kinetics. (A) Cartoon schematic of the three-bead optical trapping setup. A biotinylated actin filament is tethered between two neutravidin-coated beads that are trapped in a dual-beam optical trap. This bead-actin-bead “dumbbell” is lowered onto pedestal beads that have been sparsely coated with His6 antibody to attach Myo5-motor/lever-Avi-Tev-His9. (B–D) Single Myo5 displacements of a single bead position and covariance traces, calculated using both beads, showing single molecule interactions acquired in the presence of 1 µM (B) 10 µM (C), and 1,000 µM ATP (D). Blue bars indicate attachment events as identified by covariance (gray) decreases. The threshold of event detection by the covariance traces are indicated by dashed gray lines. (E) Schematic of displacement traces depicting the two-step nature of actomyosin displacements in the optical trap. (F–H) Binding events were synchronized at their beginnings (left) or ends (right) and averaged forward or backward in time, respectively. The measured total displacement of Myo5 was 5.0 nm at 10 µM ATP, with the first substep contributing a 4.8 nm displacement (arrow 1 in G) and the second substep contributing a 0.2-nm displacement (arrow 2 in G). (F–H) Left: Forward-averaged ensembles synchronized at the beginnings of events. Right: Reverse-averaged ensembles synchronized at the ends of events. Black and gray lines are single exponential fits in the forward and reverse ensembles, respectively. (I) Cumulative distributions of attachment durations for Myo5 at 1, 10, and 1,000 µM ATP. Blue lines show the cumulative frequency of attachment durations at the indicated ATP concentrations, and the red, yellow, and green lines indicate fitted exponential distributions at 1, 10, and 1,000 µM ATP, respectively. 1 and 10 µM ATP were fit well to single exponentials, and the 1,000 µM ATP data were best described by the sum of two exponentials. (J) Summary of rates at 1, 10, and 1,000 µM ATP calculated from F–H. Blue boxes are the fitted exponential distributions from I, black diamonds are forward ensemble fits from F–H (left), and gray diamonds are reverse ensemble fits from F–H (right). At lower concentrations of ATP (1 and 10 µM), the rate of detachment is limited by ATP association, corresponding to the reverse ensemble fits, while at saturating ATP concentration (1,000 µM), the detachment rate is limited by the rate of ADP dissociation, corresponding to the forward ensemble fits. (K) Summary of rates determined via single-molecule optical trapping. Errors for detachment rates are 95% confidence intervals. Errors for forward and reverse ensemble fits are standard errors of the fits. *Detachment rates at 1,000 µM ATP were best fit to the sum of two exponents. The major component of the fit (67.8 s–1) comprises 92.1% of the total with the remaining 7.9% having a rate of 11.6 s–1.
