Fig. 1. Srv2 binds directly to profilin (K_D=1.3 µM). (A) Profilin binding to GST-Srv2_253-526 on glutathione agarose beads measured by supernatant depletion pull-down assays. Reactions contained 2 µM wild-type profilin (Pfy1) or mutant profilin with impaired binding to polyproline (Pfy1-14) and variable concentrations of Srv2 (0-20 µM). The amount of Pfy1 in the supernatant was examined by SDS gel electrophoresis. First lane, no beads; second lane, controlbeads; lanes 3-6, 3, 6, 12 and 20 µM Srv2 immobilized on beads. (B) Quantification of five independent pull-down assays comparing Pfy1 with Pfy1-14. Srv2 efficiently depleted Pfy1 from the supernatant, but failed to deplete Pfy1-14. Standard deviations are indicated with error bars. (C) Tryptophan fluorescence assay for determining the dissociation constant of the Srv2-profilin complex. Reactions contained 1 µM profilin. Pfy1 binds to Srv2_253-373 with a K_D of 1.3 µM, whereas Pfy1-14 shows no detectable affinity for Srv2_253-373.