Figure 2.

CapG and CapG-sev binding to actin monomers. (A) Binding to NBD-conjugated monomeric actin. NBD-actin monomers (final concentration 100 nM) were mixed with increasing concentrations of CapG and CapG-sev in modified S2 buffer (see Materials and methods) and the steady-state fluorescence measured. The lines represent the calculated curves for CapG (dashed line) and CapG-sev (solid line). The fitting parameters assumed two non-cooperative binding sites for both CapG and CapG-sev. The dashed line for CapG was fitted using a K_D of 25 nM for the first binding site and 1 μM for the second binding site. The solid line for CapG-sev was fitted using a K_D of 0.1 nM for the first site and a K_D of 220 nM for second site. (B) Binding to pyrene-conjugated monomeric actin. The identical conditions described in (A) were used, except the studies were performed with pyrene actin monomers. The solid line connecting the data points for CapG-sev and the dashed line for CapG were fitted using the parameters shown in the figure, and are almost identical to NBD-actin. (C) Pointed end actin filament assembly in the presence of increasing concentrations of CapG and CapG-sev. Increasing concentrations of the two proteins were added to a 1.5 μM pyrene actin solution. Actin filament assembly was then stimulated by the addition of a final concentration of 40 nM gelsolin capped filamentous actin (see Materials and methods). The rate of rise of fluorescence intensity was monitored over 15 min and these rates plotted versus the final concentrations of CapG and CapG-sev. Note the steep reduction in pointed-end growth rate in the presence of CapG-sev.