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

Abstract

We describe a fluorescence resonance energy transfer (FRET)-based method for finding in living cells the fraction of a protein population (alpha(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 alpha-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 alpha(T) approximately 1 and n approximately 2 for Ste2 proteins. Since the Ste2p complexes are formed in the absence of the ligand in our experiments, we conclude that the alpha-factor pheromone is not necessary for dimerization.

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

“…The direct excitation of acceptors was even lower, however, as the efficiency of excitation at 458 nm is only 9% of that at 514 nm (i.e. max for YFP) (37). For cells expressing low levels of acceptor relative to donor, it is likely that most of the acceptor-tagged receptors are in donor-containing complexes and undergo FRET; moreover, the likelihood of such complexes increases with the size of the oligomer.…”

“…The absorbance of the donor (a D ) was obtained from Equation 19, in which Q D is the quantum yield of eGFP taken as 0.64 (37). Equation 19 incorporates a correction for the FRET efficiency as determined by donor dequenching (1 Ϫ E app ddq ) and for the difference between excitation at 458 nm and excitation at 488 nm (e D 458 ).…”

“…The individual contributions of donor and acceptor to each spectrum were determined by deconvolution (37) and expressed as k DA(j) and k AD(j) , respectively, which represent the emission from each fluorophore at its respective peak after the jth round of photobleaching.…”

Oligomerization of G-Protein-Coupled Receptors: Lessons from the YeastSaccharomyces cerevisiae

“…Because receptor dimers versus oligomers could not be distinguished by simple FRET experiments used to show that ␣-factor receptors interact, other approaches have been required to address this question. Among such methods, quantitative FRET imaging and donor dequenching upon acceptor photobleaching have been applied recently to coexpressed CFP-and YFP-tagged ␣-factor receptors (86). Remarkably, these studies showed that in the absence of agonist, the entire ␣-factor receptor population on the plasma membrane or the endoplasmic reticulum (ER) apparently is, on average, dimeric.…”

ExiFRET: flexible tool for understanding FRET in complex geometries

“…Analytical methods have been used to detect and characterize microdomains in membranes, [11][12][13] monitor lipid and protein interactions, 14,15 and to investigate proteins that form oligomeric complexes. [16][17][18][19] One of the advantages of analytical expressions is that they uniquely describe the FRET efficiency for a specific fluorophore distribution. Yet, this advantage also becomes a limitation as these models often show poor transferability to other systems.…”