Box 1 | Light resonance energy transfer approaches 

Light resonance energy transfer approaches are based on the non-radiative transfer of excitation energy between the electromagnetic dipoles of an energy donor and acceptor. In the case of fluorescence resonance energy transfer (FRET), both the donor and acceptor are fluorescent molecules, whereas for bioluminescence resonance energy transfer (BRET), the donor is bioluminescent and the acceptor is fluorescent. A prerequisite for these processes is that the emission spectrum of the donor and the excitation spectrum of the acceptor must overlap and that the donor and acceptor be in close proximity.

BRET⁹⁵ is a phenomenon occurring naturally in several marine animals such as the sea pansy Renilla reniformis and the jellyfish Aequorea victoria. In R. reniformis, the luminescence resulting from the catalytic degradation of coelenterazine by luciferase (Rluc) is transferred to green fluorescent protein (GFP), which, in turn, emits fluorescence at its characteristic wavelength on dimerization of the two proteins. The strict dependence on the molecular proximity between donors and acceptors for energy transfer makes it a system of choice to monitor protein–protein interactions in living cells.

As shown in the figure, one can take advantage of this phenomenon to study dimerization of G-protein-coupled receptors (GPCRs). Fusion proteins that link GFP and Rluc to the carboxyl terminus of individual GPCRs are constructed and coexpressed. In the absence of dimerization, the addition of coelenterazine H should lead to a characteristic broad bioluminescence signal with an emission peak at 470 nm, consistent with the spectral properties of Rluc. If homodimerization occurs, the energy transfer between Rluc and GFP (resulting from the proximity between the bioluminescent and the fluorescent fusion proteins) should lead to the appearance of an additional fluorescence signal with an emission peak at 530 nm that is characteristic of the GFP used (namely the red-shifted YFP)⁵⁴.

FRET can be used in the same way, using GPCRs fused to GFPs that have overlapping spectral properties (typically the CFP and the YFP). In this case, the initial energy is provided by direct excitation of the fluorescent donor with a light source⁴¹. Both the fluorescence emission of the acceptor and the quenching of the fluorescence of the donor can be used to quantitate the energy transfer. Antibodies⁵⁷ or ligands⁵⁸ that bind to the receptors can also be coupled to fluorophores that can be used as FRET pairs. Other variations of the FRET technique that have been used to study GPCR dimerization include photo-bleaching FRET⁵⁷ and time-resolved FRET⁵⁵. In photo-bleaching FRET, the efficacy of energy transfer is indirectly determined by measuring the photo-bleaching time of the energy donor (upon sustained excitation) in the presence and absence of the energy acceptor. The energy transfer between the donor and the acceptor results in a slowing down of the photo-bleaching. Time-resolved FRET takes advantage of the long-lived fluorescence of fluorophores such as the lanthanide chelate Europium³⁺, which allow delayed FRET measurements while reducing the background resulting from the short-lived autofluorescence⁹⁶.

Please close this window to return to the main article.

Note: some figures may render poorly in a web browser. In such cases, please see the associated PDF file.