Förster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ) labeled with “wild-type” CFP and YFP is compared with that of labeled with “monomeric” A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced - interaction is similar for labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for - interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of - interaction but, when brought close together via - interaction, FP dimerization modestly improves FRET measurement.
