Fig. 6

Effect of Ca²⁺ and CaM on single-molecule processivity and ensemble kinetics of myosin-5B. Dependence of myosin-5B velocity (a) and number of processive runs (b) on Ca²⁺ and CaM in the single-molecule motility assay. In this particular set of measurements, we observed 0.0125 runs s⁻¹ with an average velocity of 559 ± 64 nm s⁻¹ in the absence of Ca²⁺ and CaM. In the presence of 0.1 mM Ca²⁺, no significant movement (NSM) was observed. In the presence of 0.1 mM Ca²⁺ and 2 μM CaM, myosin processivity and velocity were partially rescued to 0.0055 runs s⁻¹ and 383 ± 93 nm s⁻¹ at [ATP] = 1000 μM (n = 12, p value = 0.02). Error bars, s.d. No error bars are reported in b because data are from single experiments. c Dependence of myosin-5B velocity on Ca²⁺ and CaM in the gliding assay. Myosin-5B translocated actin filaments with a speed of 413 ± 128 nm s⁻¹ (n = 417) in the in vitro gliding assay. Filaments movement stalled (NSM) in the presence of 0.2 mM Ca²⁺ but was partially restored (264 ± 92 nm s⁻¹, n = 439, p value = 4E−17) in the presence of 5 µM CaM. Error bars, s.d. Student’s t test applied on a and c, *P < 0.1, ****P < 0.0001. d Actin-activated steady-state ATPase activity of myosin-5B yielding the kinetic parameters k_cat = 6.96 ± 0.14 s⁻¹ and K_app = 1.34 ± 0.16 µM (n = 3). e ATP-dependence of the steady-state ATPase activity of myosin-5B at 30 µM [F-actin] (k_cat,ATP = 6.41 ± 0.14 s⁻¹; K_app,ATP = 59.07 ± 6.95 µM) (n = 3). f Increasing [Ca²⁺] does not affect the steady state ATPase of myosin-5B at a constant F-actin concentration of 30 µM (n = 3). g Quantification of CaM dissociation. Top, F-actin cosedimentation assays show that increasing concentrations of Ca²⁺ partially dissociate CaM from myosin-5B. The abbreviations S and P refer to supernatant and pellet fraction (full-length gel image in Supplementary Fig. 6). Bottom, densitometric analysis shows that on average 25% of the six CaM molecules dissociate with a K_D < 10 µM Ca²⁺ per myosin-5B heavy chain (n = 3)