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.

Figure 1. FRET in cells expressing Ste2p–GFP and Ste2p–YFP

(A) Cells that co-express Ste2p–GFP and Ste2p–YFP were excited by the 458-nm line of an Argon-ion laser. Emission images (6 out of the 16 sampled) at the indicated wavelengths (in nm) were obtained before (top panels) and after (bottom panels) bleaching by 60 scans (lasting about 30 s) using a 514-nm laser. Arrowheads indicate an area with more pronounced GFP fluorescence after bleaching of YFP. (B) Enlarged emission images at 499 nm from (A) before (left-hand panel) and after (right-hand panel) bleaching of the yellow fluorescent protein. (C) Images showing the fluorescent emission of Ste2p–YFP at 523 nm upon excitation at 514 nm before (left-hand panel) and after (centre panel) bleaching, and the cells in DIC (right-hand panel) after photobleaching to show the cells are intact.

Figure 2. Excitation and emission spectra of GFP variants used in this study

Normalized fluorescence emission spectra of CFP- (circles), GFP- (squares) and YFP-tagged (triangles) Ste2 proteins in yeast cells measured by a confocal microscope (see Experimental section) before (filled symbols, solid lines) and after (open symbols, dashed lines) 60 scans (approx. 30 s) by 514-nm light (photobleaching). Data are means±S.D. over n=21 (for CFP), n=23 (GFP) and n=52 (YFP) cells. Lines serve as guides to the eyes only.

Figure 3. Spectral deconvolution of composite fluorescence spectra of cells expressing Ste2p–CFP and Ste2p–YFP

Fluorescence spectra of a typical yeast cell containing two distinct populations of Ste2p–CFP and Ste2p–YFP (top panel). The data were acquired by a confocal laser scanning microscope under excitation at 458 nm before (●) and after (▲) bleaching of YFP (by ∼60 scans with 514 nm laser radiation) under the same microscope. The solid and dashed lines, are spectra simulated by Equation 1, before and after photobleaching respectively. Simulated spectra (top panel) decomposed into cyan and yellow components by plotting the first and second term of Equation 1 individually (bottom panel). Solid lines, spectra before bleaching; dashed lines, spectra after bleaching. Note an increase in the spectral area for CFP following YFP bleaching, as a result of the FRET interaction in the system.

Figure 4. Spectral deconvolution of composite fluorescence spectra of cells expressing Ste2p–GFP and Ste2p–YFP

The details are as in Figure 3 legend, but with GFP replacing CFP.

Figure 5. Correction for donor photobleaching effects when using the gradual photobleaching method

Effect of acceptor photobleaching by 514-nm light on the donor fluorescence intensity (top panel), expressed as scaling factor of the GFP spectrum (i.e. k_D in Equation 1). The apparent FRET efficiency (bottom panel) uncorrected for donor photobleaching (●) appears to decrease with acceptor bleaching degree. The FRET efficiency corrected for donor bleaching (■) as described in the text does not change with the acceptor bleaching degree. Lines serve as guide to the eyes only.

Figure 6. Determination of the number of oligomers in a complex

Means (●) and S.D. (bars) of α_D/α_A for each image (containing typically three cells) were plotted against their corresponding means±S.D. of [A]_T/[D]_T (see text for details). The solid line is obtained from simulations with Equation 16 and the n value (2.27±0.43) that minimizes the χ² of the fit divided by the number of degrees of freedom (0.5).