FIGURE 7.

Mg²⁺ binding to Trp-239 DdMII motor domain·ADP complex. A, fluorescence (arbitrary units, AU) transients upon mixing ADP-bound DdMII with increasing concentrations of Mg²⁺. Mixing conditions were as follows: 4 μm DdMII Trp-239, 1 mm ADP, 120 mm NaCl, 0.5 mm EDTA or 0–40 mm MgCl₂, and 0–120 mm NaCl; both syringes contained 20 mm HEPES, pH 7.3, at 13 °C. Double exponential decays were fitted to the averages of 3–5 measurements. B, Mg²⁺ concentration dependence of the fitted observed rate constants is shown. The rate of fluorescent decrease step is independent of Mg²⁺ concentration. The rate constants for Mg²⁺ binding and unbinding, k_+Mg and k_−Mg, are calculated from the Mg²⁺ dependence of the observed rate constant of the fluorescent increase and are summarized in Scheme 2 (the Mg affinity for free ADP was determined by Cahours et al. (71)). C, Mg concentration dependence of the fitted amplitudes of the fluorescent transients.