Sensitive detection of p65 homodimers using red-shifted and fluorescent protein-based FRET couples

Abstract

Background: Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET.

Methodology/principal findings: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP.

Conclusions/significance: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells.

Figure 1. Schematic overview of the tandem fluorescent protein constructs used in this study.

The red fluorescent protein, abbreviated as RFP, is either mRFP1, mStrawberry or mCherry. Single letter abbreviations are used for the amino acids that comprise the linker.

Figure 2. FLIM data of living cells expressing the mStrawberry-SYFP2 tandem.

Experiments were performed either 1 day (a–d) or 2 days (e–h) after transfection. The panels show the fluorescence intensity of SYFP2 (a, e), the phase lifetime map of SYFP2 (b, f), the histogram of the lifetime distribution (c, g) and the 2D histogram of the lifetime distribution versus the fluorescence intensity (d, h). The false color representation of the lifetime map corresponds to the colors used in the lifetime histogram. The width of the images corresponds to 112 µm.

Figure 3. FLIM data of cells expressing mKO or mOrange.

The FLIM was done before (a–d) and after (e–h) exposing the cells to 436 nm light. The panels show the fluorescence intensity (a, e), the modulation lifetime map (b, f), the histogram of the modulation lifetime distribution (c, g) and the 2D histogram of the phase lifetime versus the modulation lifetime (d, h). The false color representation of the lifetime map corresponds to the colors used in the lifetime histogram. The width of the images corresponds to 85 µm.

Figure 4. Homodimerization of p65 can be detected by FLIM.

The lifetime data of multiple cells is summarized (a), by plotting the modulation lifetime against the phase lifetime for cells expressing ECFP-p65 (circles), ECFP-p65 and EYFP-p65 (squares), mKO-p65 only (diamonds) or cells expressing mKO-p65 and mCherry-p65 (triangles). FLIM images of cells expressing mKO-p65 in absence (b–d) or presence (e–g) of mCherry-p65. The panels show the fluorescence intensity of two merged representative nuclei (b, e), the modulation lifetime map (c, f) and the histogram of the modulation lifetime distribution (d, g). The reduced lifetime observed for cells expressing both mKO-p65 and p65-mCherry is due to FRET, indicating homodimerization of p65. The width of the images corresponds to 28 µm.