Figure 4. Nup159 minimally affects mantADP(±Mg²⁺) dissociation kinetics.

(A) Time courses of irreversible Mg²⁺mantADP dissociation from a pre-incubated solution of Dbp5 (2 μM) and Mg²⁺mantADP (20 μM) upon mixing with 3 mM Mg²⁺ADP and (from lower to upper) 0, 1.5, 4.5, or 10 μM Nup159 in assay buffer containing excess MgCl₂. Concentrations are final after mixing. Smooth lines through the data represent best fits to single exponentials. (B) [Nup159]-dependence of the observed rate constants obtained from A. The line through the data represents the best fit to Equation 4, yielding K_N(+Mg)HmD of 0.3 ± 0.8 μM, k_−mD(+Mg) = 2.7 ± 0.1 s⁻¹ and k_−mD(+Mg),N = 2.0 ± 0.2 s⁻¹. (C) Time courses of FRET signal change after mixing pre-equilibrated solutions of 1 μM Dbp5 and 40 μM mantADP with an equal volume of (lower to upper) 0, 3 or 9 μM Nup159 and 10 mM ADP in assay buffer containing excess EDTA (ca. 27 nM [Mg²⁺]_free after mixing). The concentrations are final after mixing. Continuous lines through the data represent best fits to a single exponential. (D) [Nup159]-dependence of the mantADP dissociation observed rate constants obtained from exponential fits in C. ANOVA analysis suggests that the dependence is insignificant and the average dissociation rate constant is ~ 11 s⁻¹ indicated by a horizontal dashed line. Uncertainty bars represent standard error of the fits and are contained within the data points.