Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of AEDANS probe attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. To build a realistic model of the FOXO4-14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments.