Figure 3. Single molecule optical trapping reveals that danicamtiv reduces the size of myosin’s working stroke without altering detachment kinetics.

Black = DMSO control. Pink = 10 µM danicamtiv. A) Cartoon schematic of the optical trapping assay. An actin filament is strung between two optically-trapped beads and lowered onto a pedestal bead sparsely bound with myosin. B) Optical trapping data traces showing the stochastic binding of myosin to actin. Binding interactions are shown in grey or pink and detached states are shown in black. C) Time forward ensemble averages of myosin’s working stroke reveal a ~50% reduction in the size of myosin’s total working stroke in the presence of danicamtiv. D) The cumulative distribution of the total working stroke displacements at 10 µM ATP is well fit by a single cumulative Gaussian function (dotted lines) with average values of 4.9 ± 9.7 nm versus 3.0 ± 9.0 nm for DMSO control and 10 µM danicamtiv, respectively (P < 0.001 using a two-tailed T-test). N = 2076 binding interactions for control and 4776 binding events for 10 µM danicamtiv. E) The cumulative distributions of attachment durations at 10 µM ATP. Single exponential functions were fit to the distributions using maximum likelihood estimation. 95% confidence intervals were calculated using bootstrapping methods. There is no statistical difference between control and 10 µM danicamtiv, 23 (−2.5/+2.5) s⁻¹ vs. 24 (−0.8/+0.9) s⁻¹ (P = 0.48). For all trapping values, see Table 2.