Figure 3.

Actin-binding properties of the GAB motifs and EVH2 domains of hVASP and DdVASP. (A) Determination of the K_D value of the DdGAB–actin interaction by fluorescence titration of 100 nM OG-actin (left) and 500 nM OG-actin (right) with the DdGAB peptide at the buffer conditions is indicated (for details see Materials and methods section). The solid lines represent calculated binding isotherms. (B) Determination of the K_D value of the hGAB–actin interaction by fluorescence titration of 300 nM OG-actin (left) and 3 μM OG-actin (right) with the hGAB peptide at conditions as in (A). The solid lines represent calculated binding isotherms. (C) Summary of K_D values determined from experiments similar as shown in (A, B) at the conditions indicated. K_D values for the GAB–Ca²⁺–ATP–actin interaction are given in brackets. K_D values obtained under conditions used in TIRF assays (50 mM KCl, Mg²⁺–ATP–actin) are shown in bold. (D) (Left) Kinetics of the DdEVH2–G-actin interaction. Time course of the binding of DdEVH2 to latA-sequestered, Mg²⁺–ATP–OG-actin was monitored by a stopped-flow apparatus at a final actin concentration of 2.5 μM actin and the DdEVH2 concentrations are indicated. Noisy curves represent experimental data, and solid lines are fits for a reversible, bimolecular reaction with K_D=0.6 μM, yielding k_on=53 μM⁻¹ s⁻¹. (Right) k_on values of the interaction of the hEVH2 and DdEVH2 domain with OG-actin. Due to the low affinity of the hGAB, high actin concentrations had to be used (10 μM final), which resulted in more noisy signals (not shown) that could not be fitted as accurately as those for the higher-affinity DdEVH2.