Figure 4.

Measurements of actin activation of the power stroke in M2β-S1. The rate constants of the actin-activated power stroke were measured by monitoring the fluorescence enhancement of Alexa 488 during actin-activated product release. Sequential-mix stopped-flow experiments were performed by mixing 0.25 μM M2β-S1 A488RLC with 0.2 μM Cy3ATP, aged for 10 s for hydrolysis to occur, and then mixed with varying concentrations of actin (5–30 μM). The fluorescence transients were best fitted by a double-exponential function. (A) The rate constants of the fast phase were plotted as a function of actin concentration and fitted to a hyperbolic function. The slow phase was linearly dependent on actin concentration and has a rate of 1.5 s⁻¹ at 30 μM actin. Data points at each actin concentration represent the average ± SEM of three to seven experiments from separate protein preparations. (B) Representative fluorescence transient in the presence of 30 μM actin (average of two transients) is shown fitted to a double-exponential function. The donor-only and acceptor-only controls are shown for comparison. (C) Experiments in (A) were performed as a function of temperature, and the rate constants of the fast and slow phases of the power stroke were plotted together with the actin-activated ATPase activity (10–35°C). (D) Eyring plots of the power-stroke rate constants and corresponding ATPase activity demonstrate the temperature dependence of the different rate constants (slopes, k_Fast, −17.03 ± 0.76; k_Slow, −18.25 ± 1.85; ATPase, −15.53 ± 0.51).