Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis

Abstract

Fluorescence correlation spectroscopy is a potentially powerful tool for measuring protein-protein interactions directly in single living cells. We previously reported on the detection of homodimer formation in cells using molecular brightness analysis. Here, we extend the technique to detect binding between different proteins. Proteins are labeled with the fluorescent markers YFP and CFP. We first determine the coexpression ratio of both proteins by measuring the intensity ratio with a dual-color setup. The effect of fluorescence resonance energy transfer on the intensity ratio is explicitly taken into account. The brightness of cells coexpressing both proteins is measured in a single-color setup. Selecting the laser wavelength of the two-photon light source allows us to either coexcite both proteins or to selectively excite YFP-labeled proteins. This approach enables us to distinguish between homodimer and heterodimer formation. We first present the theory and then demonstrate experimental feasibility using the ligand binding domains of retinoic acid receptor (RARLBD) and of retinoid X receptor (RXRLBD). Both proteins form heterodimers, and RXRLBD also forms homodimers in the presence of its agonist. We explore binding between these proteins in the presence and absence of RXR agonist. Our results demonstrate that brightness analysis offers a quantitative method for determining protein interactions in cells.

FIGURE 1

Intensity ratio r_F as a function of the coexpression ratio r_N = N_YFP/N_CFP. The solid line represents the relationship between the intensity ratio and the coexpression ratio in the absence of FRET. The dashed line was calculated assuming a stoichiometric binding model and a FRET efficiency of 27%. An intensity ratio of 1.25 (dotted arrow) leads to a coexpression ratio of ∼2 (solid arrow) in the absence of FRET, while representing equimolar expression in the presence of FRET (dashed arrow). Each shaded bar indicates the range of intensity ratios selected for further brightness analysis in our experiments.

FIGURE 2

Fluorescence lifetime of the donor RAR-CFP as a function of the intensity ratio of RXR-YFP and RAR-CFP coexpressed in CV-1 cells. The symbols are the experimentally determined fluorescence lifetime values of RAR-CFP, and the solid line is the theoretical predication assuming stoichiometric binding with an apparent FRET efficiency of 27%. The fluorescence lifetime of RAR-CFP is 2.38 ns in the absence of RXR-YFP. The lifetime of RAR-CFP decreases as the concentration of RXR-YFP is raised and reaches a limiting value once all RAR-CFP are bound to RXR-YFP.

FIGURE 3

Relative brightness of RARLBD-CFP and RXRLBD-YFP in CV-1 cells with intensity ratios between 1.15 and 1.35. This range of intensity ratios corresponds to coexpression ratios N_RXRLBD-YFP/N_RARLBD-CFP from 0.9 to 1.2. (A) At an excitation wavelength of 905 nm CFP and YFP are equally bright, and the relative brightness of the heterodimer is ∼2. (B) At 965 nm, only YFP is excited, and the apparent brightness of the heterodimer is reduced to the YFP brightness. The relative brightness of the heterodimer is virtually identical in the absence (▪) and in the presence (▵) of RXR agonist, which indicates that RXRLBD is unable to form homodimers in the presence of equal concentrations of RARLBD. The experimental error in brightness is shown for selected cells.

FIGURE 4

Relative brightness of RARLBD-CFP and RXRLBD-YFP in CV-1 cells at an excitation wavelength of 905 nm. (A) Cells with intensity ratios from 0.7 to 0.9 (N_RXRLBD-YFP/N_RARLBD-CFP from 0.36 to 0.6). Excess of RARLBD leads to an apparent brightness (▪), which is less than that expected for a full dimer. Adding ligand leaves the apparent brightness (▵) unchanged. (B) Cells with intensity ratios from 1.5 to 1.7 (N_RXRLBD-YFP/N_RARLBD-CFP from 1.8 to 2.8). Excess of RXRLBD leads to an apparent brightness (▪), which is less than that expected for a full dimer. Adding ligand restores the apparent molecular brightness (▵) of the mixture to that of a pure dimer population. The dashed lines correspond to the range of relative brightness values in the absence of ligand and are calculated from the simple binding model discussed in the text.

FIGURE 5

Relative brightness of RARLBD-CFP and RXRLBD-YFP in CV-1 cells in the presence of RXR agonist at an excitation wavelength of 965 nm. Cells with an excess of RARLBD (N_RXRLBD-YFP/N_RARLBD-CFP from 0.36 to 0.6) have an apparent brightness equal to monomeric RXRLBD-YFP. Cells with an excess of RXRLBD (N_RXRLBD-YFP/N_RARLBD-CFP from 1.8 to 2.8) have an apparent brightness, which is larger than that of a monomer, but less than that of a dimer. The dashed lines indicate the relative brightness range expected from the binding model described in the text.