VASP enhances barbed end polymerization in the presence of profilin–actin. (A) Interaction between Cy3-VASP^1-240aa and human profilin I measured by sedimentation equilibrium. Equilibrium traces from left to right: 5 µM Cy3-VASP^1-240aa plus 30, 60, or 90 µM hProI. A predicted size of 24.8 kD for Cy3-VASP^1-240aa alone closely matched the observed molecular weight of 24.5 kD (not depicted). Based on a global fit of all traces, Cy3-VASP^1-240aa interacts with two hProI proteins (15 kD per hPro1). (B) Barbed end growth rates measured in the presence of 1, 1.5, 2 µM profilin–actin (10% Alexa Fluor 488), plus 50 nM VASP. Mutations made to profilin or VASP are indicated with yellow stars. From left to right: (1) hPro1-Actin alone, (2) hPro1^H133S-Actin, (3) hPro1-Actin-VASP, (4) hPro1^H133S-Actin-VASP, (5) hPro1-Actin-VASP^PPPLPAP-8A, (6) hPro1-Actin-VASP^{GAB*(LIL-AAA)}, (7) hPro1^H133S-Actin-VASP^{GAB*(LIL-AAA)}, and (8) hPro1-Actin-VASP^{FAB*(RRRK-4E)}. (C) Barbed end filament growth rates in the presence of 2 µM profilin–actin, 100 mM KCl, and 0–50 nM VASP. (D) Barbed end dwell times for Cy3-VASP measured in the presence hPro1-actin and hPro1^H133S-actin. (E) The number of actin monomers delivered to the barbed end equals the product of the polymerization rate (B) and dwell time (D).