Fig. 3: Increased actin-gliding velocity of MYO6^S267E in vitro is achieved by faster nucleotide-exchange rates without change in the W/S of the motor.

a Scheme of the in vitro actin-gliding assay. Monomeric MYO6, immobilised on a nitrocellulose-coated glass surface, translocates rhodamine-phalloidin labelled F-actin in the presence of ATP. b, c Actin-gliding velocity (2 mM ATP, 22 °C) increased 4-fold from 45 nm s⁻¹ (MYO6^WT) and 56 nm s⁻¹ (MYO6^S267A) to 172 nm s⁻¹ for MYO6^S267E. 3 independent experiments from 3 protein purifications were performed; Statistical analysis was performed using an unpaired two-sided t-test. ***P < 0.01. MYO6^WT n = 567, average 0.045 µm/s SD ± 0.008283, MYO6^S267A n = 557, average 0.0557 µm/s SD ± 0.010488, MYO6^S267E n = 569, average 0.1725 µm/s SD ± 0.0345.; d Scheme of the inverted in vitro motility assay. Dimerised MYO6 translocates on fascin-stabilised actin bundles. e Fraction of processive runs of MYO6^WT (48.6%), MYO6^S267A (36.2%) and MYO6^S267E (24%). f Median run-length of MYO6^WT, MYO6^S267A and MYO6^S267E was determined in the inverted motility assay using 3 different flow cells for each protein. Statistical analysis was performed using the Kruskal-Wallis test by ranks, a non-parametric method for testing whether samples originate from the same distribution. It is used for comparing two or more independent samples of equal or different sample sizes. ***P < 0.01. MYO6^WT n = 7926, average 0.22 µm SEM ± 0.0047, MYO6^S267A n = 3981, average 0.148 µm SEM ± 0.0058 and MYO6^S267E n = 2367, average 0.083 µm SEM ± 0.0061. g Actin-gliding velocity (2 mM ATP, 22 °C) for the double mutants MYO6^S267A/E plus MYO6^T406A/E were determined by the mutation at S267 and independent of the mutation at T405 (S267A+T406A 50 ± 14 nm s⁻¹; S267A+T405E 41 ± 17 nm s⁻¹; S267E+T405A 134 ± 25 nm s⁻¹; S267E+T405E 133 ± 35 nm s⁻¹ mean ± SD, N = 51 filaments for each double mutant, ***P < 0.01, unpaired two-sided t-test; 3 experiments from 3 different protein preparations, Fig. 4). h Scheme of single-molecule mechanical experiments using optical tweezers. i Raw data traces of single MYO6^S267A and MYO6^S267E molecules interacting with F-actin (single trap-stiffness κ_trap ~ 0.02 pN nm⁻¹; 100 µM ATP). MYO6 binding events to actin were detected by changes in the variance of thermal motion. Grey bars indicate actin-attached dwell times. j To determine the working stroke (W/S) for the MYO6^S267A and MYO6^S267E the displacement distribution of actin-binding events was analysed at 100 µM ATP. k Characterisation of the apparent duty ratio and apparent on-rate of actin binding for the MYO6 A-and E-mutants. Source data are provided as a Source Data file.