Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer

We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (α_T) forming complexes, and the average number (n) of those protein molecules in each complex. The method relies both on sensitized acceptor emission and on donor de-quenching (by photobleaching of the acceptor molecules), coupled with full spectral analysis of the differential fluorescence signature, in order to quantify the donor/acceptor energy transfer. The approach and sensitivity limits are well suited for in vivo microscopic investigations. This is demonstrated using a scanning laser confocal microscope to study complex formation of the sterile 2 α-factor receptor protein (Ste2p), labelled with green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP respectively), in budding yeast Saccharomyces cerevisiae. A theoretical model is presented that relates the efficiency of energy transfer in protein populations (the apparent FRET efficiency, E_app) to the energy transferred in a single donor/acceptor pair (E, the true FRET efficiency). We determined E by using a new method that relies on E_app measurements for two donor/acceptor pairs, Ste2p–CFP/Ste2p–YFP and Ste2p–GFP/Ste2p–YFP. From E_app and E we determined α_T≈1 and n≈2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the α-factor pheromone is not necessary for dimerization.