Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells.
Forster 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, ERalpha) labeled with "wild-type" CFP and YFP is compared with that of ERalpha labeled with "monomeric" A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced ERalpha-ERalpha interaction is similar for ERalpha labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERalpha-ERalpha interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERalpha-ERalpha interaction but, when brought close together via ERalpha-ERalpha interaction, FP dimerization modestly improves FRET measurement.
Kofoed
Eric M
EM
University of California, San Francisco, Diabetes Center and Department of Medicine, S-1230, 513 Parnassus, San Francisco, California 94143-0540, USA.
Guerbadot
Martin
M
Schaufele
Fred
F
D016477:Artifacts; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D011960:Receptors, Estrogen / Q000378:metabolism*; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D047628:Estrogen Receptor alpha; D008164:Luminescent Proteins; D011960:Receptors, Estrogen; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins