Figure 5
PI(4,5)-P2-binding of profilin mutants. A. Microfiltration of profilin-PI(4,5)-P2 complexes. 4 μM profilin is incubated with increasing concentrations of PI(4,5)-P2 as indicated and applied to a filter with MWCO of 30.000. Non-bound profilin passes through the filter upon centrifugation. The flowthrough is analyzed by SDS-PAGE and is shown here for wild type profilin, R135D, R136D and R135A/R136A. B. Examples of gel filtration experiments. Profilin (10 μM) was pre-incubated with increasing concentrations of PI(4,5)-P2 and run over a SMART Superdex75 gel filtration column. Free profilin elutes at 1.62 ml, while the profilin-PI(4,5)-P2 complex elutes in the void (0.96 ml). The profilin peak shifts to the void fraction upon binding to PI(4,5)-P2. Elution pattern of wild type profilin alone (black line), profilin with 40 μM PI(4,5)-P2 (dark grey line) and profilin with 150 μM PI(4,5)-P2 (light grey line) are shown. We calculated the peak surface of free profilin to determine the percentage of bound profilin for different PI(4,5)-P2 concentrations. These data were then plotted in curves as shown in C. C. Percentage of bound profilin in function of PI(4,5)-P2 concentration as determined from the gel filtration curves. Wild type profilin (closed circle), R136D (open circle), R88A (closed triangle), R88A/R136D (open triangle) and R88E/R136D (closed square) in the gel filtration experiment. The concentration of PI(4,5)-P2 where 50% of profilin is bound to the micelles was derived from these curves and is given in Table 3 for the different mutants.