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Review
. 2016 Sep 14;16(9):1488.
doi: 10.3390/s16091488.

A Guide to Fluorescent Protein FRET Pairs

Bryce T Bajar  1 , Emily S Wang  2 , Shu Zhang  3 , Michael Z Lin  4 , Jun Chu  5
Affiliations
Review

A Guide to Fluorescent Protein FRET Pairs

Bryce T Bajar et al. Sensors (Basel). .

Abstract

Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.

Keywords: biosensors; fluorescence resonance energy transfer (FRET); fluorescent proteins.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
The principle of fluorescence resonance energy transfer (FRET). (A) Spectral overlap between mClover3 and mRuby3. The spectral overlap integrand (a product of fd(λ), εA and λ4 in the Equation (2)) is indicated by the black dashed line; (B) FRET efficiency (FRET E) versus distance. The FRET E varies with the sixth power of distance between donor and acceptor. The Förster radius (r0) is the distance at which 50% FRET occurs. Compared to ECFP-EYFP, mClover3-mRuby3 exhibits a larger FRET E change because of the larger r0 at which the given FRET biosensor operates; (C) Two types of FRET biosensors: intramolecular and intermolecular FRET biosensors. The sensing domains undergo conformational changes (intramolecular) or inter-domain interactions upon biochemical changes, leading to the change in FRET E; (D) The relationship between the intensity ratio of acceptor to donor (IA/ID) and FRET E. The ratio of peaks of the emission spectrum acquired by a sensitivity-normalized spectrum-scanning device is non-linearly related to the actual FRET E. However, it is important to note that ratios taken through filter cubes and cameras are not equivalent to ratios derived from a spectrum-scanning device, as filter cubes pass different amounts of light depending on the transmission spectra and cameras exhibit wavelength-dependent sensitivity.
Figure 2
Figure 2
Normalized excitation (or absorbance) and emission spectra of FPs of representative two-color FRET pairs. (A) mTurquoise2-mCitrine, a CFP-YFP pair; (B) mClover3-mRuby3, a GFP-RFP pair; (C) eqFP650-iRFP, an FFP-IFP pair; (D) mAmetrine-tdTomato, a LSS-FP based pair; (E) mEGFP-sREACh, a dark FP-based pair; (F) EYFP-rsTagRFP, an optical highlighter FP-based pair.
Figure 3
Figure 3
Normalized excitation (or absorbance) and emission spectra of FPs of representative four-color FRET pairs: (A) mTagRFP-sfGFP and mVenus-mKOκ pairs, two FRET pairs with two excitations; and (B) ECFP-cpVenus and LSSmOrange-mKate2 pairs, two FRET pairs with single excitation.
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