Fluorescence Resonance Energy Transfer


Fluorescence resonance energy transfer is a valuable tool for determining intra‐ and intermolecular distances in the range 10–100 Å. It is particularly valuable for measuring changes in molecular distance, such as conformational changes in proteins.

Keywords: protein chemistry; molecular ruler; green fluorescent proteins; properties of fluorescent probes; molecular distance determinations

Figure 1.

The airy disk (shaded blue) scans across a specimen irradiating and exciting fluorophores with the appropriate absorption spectra. As the airy disk scans across a specimen, the fluorophore that is initially not emitting light (green spot) becomes progressively brighter, then darker as the nonuniform airy disk crosses the fluorophore. In this way, a confocal image is formed one point at a time.

Figure 2.

In this situation, two probes are separated by less than approximately 200 nm. The airy disk excites both fluorophores simultaneously and their fluorescent peaks are not so well resolved. Consequently, they appear as one single point of emitting light.

Figure 3.

Jablonski diagram illustrating electronic transitions. The bold horizontal lines represent the S0 and S1 electronic energy levels, and the thin horizontal lines represent different vibrational levels within the S1 state. Irradiation of a fluorophore elicits a transition from the ground S0 state to a higher energy S1 state to any vibrational level (10−15 s). IC represents the rapid internal conversion to the lowest vibrational level in the excited state (10−10 s). From this point energy is lost relatively slowly ((10−8 s) by nonradiative decay (NRD) and by emission of a photon (fluorescence, F).

Figure 4.

FRET is possible if a donor fluorophore is in molecular contact with an acceptor fluorophore with sufficient spectral overlap. FRET provides an additional deactivation pathway for an excited donor probe.

Figure 5.

Cartoon illustrating overlap between the donor emission and acceptor absorption spectra. The shaded area is the overlap integral (J).

Figure 6.

Demonstrating protein interaction using acceptor photobleaching FRET. (a) and (b) Distribution of two binding partners labelled with donor and acceptor probes, respectively, (c) and (d) Same distribution following spot bleaching of the acceptor probe in the circled area. The loss of acceptor fluorescence in (d) correlates with an increase in donor fluorescence in (c).

Figure 7.

Bleed‐through of donor fluorescence (shaded) detected at the acceptor emission wavelength. This must be taken into account when performing FRET calculations.

Figure 8.

Unintentional excitation of acceptor probes when exciting at the donor wavelength. This can give overestimations of the degree of direct FRET.



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Further Reading

Damelin M and Silver PA (2000) Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex. Molecular Cell 5: 133–140.

Habuchi S, Cotlet M, Hofkens J et al. (2002) Resonance energy transfer in a calcium concentration‐dependent Cameleon protein. Biophysical Journal 83: 3499–3506.

Karpova TS, Baumann CT, He L et al. (2003) Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. Journal of Microscopy 209: 56–70.

Kenworthy AK (2001) Imaging protein–protein interactions using fluorescence resonance energy transfer. Methods 24: 289–296.

van Kuppeveld FJ, Melchers WJ, Willems PH and Gadella TWJ (2002) Homomultimerization of the coxsackievirus 2B protein in living cells visualized by fluorescence resonance energy transfer microscopy. Journal of Virology 76: 9446–9456.

Larijani B, Allen‐Baume V, Morgan CP, Li M and Cockcroft S (2003) EGF regulation of PITP dynamics is blocked by inhibitors of phospholipase C and of the Ras‐MAP kinase pathway. Current Biology 13: 78–84.

Sorkin A, McClure M, Huang F and Carter R (2000) Interaction of EGF receptor and grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy. Current Biology 10: 1395–1398.

Tramier M, Gautier I, Piolot T et al. (2002) Picosecond‐hetero‐FRET microscopy to probe protein–protein interactions in live cells. Biophysical Journal 83: 3570–3577.

Zimmermann T, Rietdorf J, Girod A, Georget V and Pepperkok R (2002) Spectral imaging and linear un‐mixing enables improved FRET efficiency with a novel GFP2‐YFP FRET pair. FEBS Letters 531: 245–249.

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Chhabra, Deepak, and dos Remedios, Cristobal G(Sep 2005) Fluorescence Resonance Energy Transfer. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0004170]