Fig.1 Spectral Overlap of FRET Chromophores

Submitted by Peter W. Murphy
Forster resonance energy transfer, also referred to as fluorescence resonance energy transfer, (FRET) is a distance dependent phenomenon that is used to show close proximity of two molecules by monitoring energy transfer between two chromophores attached to the molecules of interest. The basic jist is that a donor chromophore in its excited state can transfer energy by a nonradiative, long-range dipole-dipole coupling mechanism to an acceptor chromophore in close proximity (typically
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Interesting examples

Submitted by Peter W. Murphy

This paper is interesting and illustrates that sometimes the tagging of proteins with large fluorophores like the GFP variants CFP and YFP will interfere with the normal functioning of the protein in vivo. They are looking at adrenergic receptor activation and the CFP/YFP FRET system disrupted the downstream activation of adenylyl cyclase. The authors get around this problem by replacing YFP with FlAsH, a very small fluorophore, which results in them being able to monitor the activation of the receptor in live cells with all downstream processes intact:

http://www.tsienlab.ucsd.edu/Publications/Hoffmann%202005%20Nature%20Methods%20-%20A%20FlAsH%20Based%20Approach.pdf

This paper reports how the authors developed a three fluorophore FRET system to investigate protein interactions with three different component proteins. They use a traditional CFP/YFP FRET pair along with another fluorophore called mRFP which can be an acceptor for either CFP or YFP. The authors then test the 3-FRET system by tagging known protein complexes in the endosomal compartment of live cells.

http://www.nature.com/nmeth/journal/v1/n3/full/nmeth720.html 

Strengths and Limitations

Submitted by Peter W. Murphy
    Measurement of FRET interactions are commonly done with confocal microscopy but may also be measured with a number of techniques on basically any scale ranging from milliliter samples in cuvette base fluoremeters to the femtoliter volumes used in single molecule microscopy. This makes it a broadly useful technique that can be used in many contexts without specialized equipment.
    FRET gives a measurement of the close proximity of two fluorophores. The strength of this is that it can be used to verify molecular associations of biomolecules. The fluorophores are usually tagging two macromolecules, i.e. a protein and ligand, two single DNA strands, a DNA binding protein and it's target sequence, or even two loci of a single protein to measure conformational changes. To an extent FRET interactions can be used to gauge intermolecular distances. This measurement is limited however due to the way that the fluorophores must be attached to the macromolecules in question. In order to get high enough FRET efficiency, the dipoles of the donor and acceptor must be reasonably aligned. To accomplish this the dye molecules are usually attached by short tether than allows the fluorophore to freely rotate. This means that the precise distance predicted by FRET efficiency measurements may only be a semi-quantitative gauge of the actual association distance of the molecules that are tagged.

Physical Mechanism of Detection

Submitted by Peter W. Murphy
    When measuring FRET, the only the donor fluorophore is excited by a laser and then the emission intensity of both the donor and acceptor fluorophores are measured. When the two fluorophores are not closely associated, the donor fluorophore will emit light normally and the acceptor will not. However, when the two fluorophores are in sufficiently close proximity (1-10nm usually) the emission of the donor will decrease and the acceptor will emit light due to dipole-dipole resonance. The efficiency of the energy transfer depends on the donor-acceptor separation distance, r, with an inverse 6th power law. This adds to the specificity of the technique as FRET can only happen at short distances on the order of nanometers.
   

Fluorescence Resonance Energy Transfer (FRET): Wallrabe, Cur Op Biotech 2005,16:19, Jonathan K. ND

Submitted by Patricia B. O'Hara