Figure 5.
Estimation of EC for FRET pairs. In vivo titration of YFP-Cdc42(V12), YFP-Rac1(L61), and YFP-Rac2(V12) with CFP-PBD. Populations of cells were imaged by FRET stoichiometry and then average cellular EA (A, C, and E) or ED (B, D, and F) of entire cells were plotted against Log(1/Ratio) or Log(Ratio), respectively. At high 1/Ratio, CFP-PBD was present in excess and the plateau of EA indicated the characteristic FRET efficiency (EC). Conversely, at high Ratio, YFP-GTPase was present in excess and the maximal ED indicated EC. Data were fit to single site equilibrium binding equations and the parameters determined as indicated (see MATERIALS AND METHODS). Cells expressed CFP-PBD plus YFP-Cdc42(V12) (A and B), YFP-Rac(L61) (C and D), or YFP-Rac2(V12) (E and F).