Figure 5. Danicamtiv increases myosin’s attachment rate in a single turnover stopped flow assay.

Black = DMSO control. Pink = 10 μM danicamtiv. A) Schematic conceptually describing the single turnover assay used to measure myosin’s attachment and detachment rates. In this assay, myosin and pyrene labeled actin are pre-incubated and then mixed with a sub-saturating concentration of ATP. The pyrene fluorescence increases as myosins detach from actin, and the increase in fluorescence reports the rate of detachment of myosin from actin (k_det). Myosin then reattaches to actin, quenching the fluorescence and reporting the attachment rate (k_att). B) Fluorescence transients from the single turnover assay. Data were fitted as described in the Supplemental Methods. C) The average second-order rate of detachment (k_det) was similar with and without 10 μM danicamtiv (3.8 ± 0.9 vs. 4.2 ± 0.8 s⁻¹; P = 0.23). D) The second-order rate of attachment (k_att) increased with the addition of 10 μM danicamtiv (0.0040 ± 0.0002 vs 0.0063 ± 0.0007 μM⁻¹·s⁻¹; P = < 0.001). For C and D, the thick lines show the average values, and the error bars show the standard deviation. The individual points are the fitted second-order rates to individual transients collected across three experimental replicates. Statistical testing was done using a two-tailed T-test after passing a normality test.