Fig. 4: Kinetics from single molecule mechanical experiments and gliding filament assays on the monomeric MYO6 phospho-mutants.

Kinetics from single molecule mechanical experiments and gliding-filament assay on the monomeric MYO6 phospho-mutants. a Scheme of the chemo-mechanical ATPase cycle of a single motor head. b Rate constants for single molecule MYO6 interactions with F-actin measured using optical tweezers at 22 °C, single trap stiffness ktrap 0.02 pN nm⁻¹. The cumulative dwell time distributions were fitted with a double exponential function, with f(t) = A₁ exp(−k₁ t) + A₂ exp(−k₂ t). StdErr = standard error, N = number of binding events in each condition, R² = regression coefficient for the fit function. The ensemble average displacement data and stiffness data were fitted by single exponentials. The load dependence of the rates k₁ and k₂ was obtained by fitting, using k = k_o exp(−Fd /k_BT), with ko rate at zero load, F force, d distance parameter and k_BT thermal energy. All single molecule mechanical data were obtained from at least 10 different motor heads in all different conditions. For comparison, values measured here and previously for MYO6^WT in single molecule mechanical experiments (SMM) and in bulk solution (stopped-flow, SF) were included in the ‘Literature’ section of the table; (a) Lister et al.³¹, (b) Altman et al.³³, (c) DeLaCruz et al.²³, (d) Polypenko et al.⁷. Gliding filament assay for monomeric double mutants S267A/E plus T405A/E; mean velocity and standard deviation SD.