Fluorescence resonance energy transfer (FRET)

Principle

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. This energy transfer mechanism is termed "Förster resonance energy transfer" (FRET) or dipole-dipole resonance energy transfer (called after the German scientist Förster). When both molecules are fluorescent, the term "fluorescence resonance energy transfer" is often used, although the energy is not actually transferred by fluorescence, as illustrated in Fig. 1.

Fig. 1 Principle of FRET. The emitted 480 nm signal is much weaker than the 535 signal emitted by the acceptor YFP. When the distance between the two chromophores is too large, YFP is not excited and CFP emits a strong 480 nm signal.

Primary Conditions for FRET

· Donor and acceptor molecules must be in close proximity (typically 1–10 nm).

· The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor (see Fig. 1).

· Donor and acceptor transition dipole orientations must be approximately parallel.

The efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules. Thus, FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity (1-10 nm). When FRET is used as a contrast mechanism, co-localization of proteins and other molecules can be imaged with spatial resolution beyond the limits of conventional optical microscopy.

Fig. 2 Overlap of the energy spectra.

Donor/acceptor pairs

In most applications, the donor and acceptor dyes are different, in which case FRET can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence. When the donor and acceptor are the same, FRET can be detected by the resulting fluorescence depolarization.

Non-fluorescent acceptors such as dabcyl have the particular advantage of eliminating the potential problem of background fluorescence resulting from direct (i.e., nonsensitized) acceptor excitation. Typical values of the distance between donor and acceptor are 3 to 6 nm, e.g. fluorescein- tetramethylrhodamine 5.5 nm and fluorescein-fluorescein 4.4 nm.

The most used FRET pair for biological use is a cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) pair. Both are color variants of green fluorescent protein (GFP). While labeling with organic fluorescent dyes is laborious, GFP variants can be easily attached to a host protein by genetic engineering.

Application

In fluorescence microscopy (see Optical microscopy, fluorescence), fluorescence confocal laser scanning microscopy (see Optical microscopy: confocal laser scanning), as well as in molecular biology, FRET is a useful tool to quantify molecular dynamics, such as protein-protein interactions, protein-DNA interactions, and protein and nucleic acid structure conformational changes. For monitoring the complex formation between two molecules, one of them is labeled with a donor and the other with an acceptor, and these fluorophore-labeled molecules are mixed. When they are dissociated, the donor emission is detected upon the donor excitation. When the donor and acceptor are in proximity (1-10 nm) due to the interaction of the two molecules, the acceptor emission is predominantly observed because of the intermolecular FRET from the donor to the acceptor.

FRET can further be used for receptor/ligand interactions, immunoassays, probing interactions of single molecules, automated DNA sequencing, and nucleic acid hybridization.

More Info

Förster Radius

The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET) is defined by the Förster radius (R_o). The magnitude of R_o is dependent on the spectral properties of the donor and acceptor dyes. It is equal to:

R₀⁶ = 8.8 x 10^─28κ²n⁴Q₀ J, (1)

where

κ² is the dipole orientation factor (range 0-4);

n is the refractive index quantum yield of the medium;

Q₀ is the fluorescence of the donor in the absence of the acceptor;

J is the spectral overlap integral calculated as:

J = ∫f_D(λ)ε(λ)λ⁴dλ, (2)

where

f_D is the normalized donor emission spectrum, and

ε_A is the acceptor molar extinction coefficient.

κ² =2/3 is often assumed. This value is obtained when both dyes are freely rotating and can be considered to be isotropically oriented during the excited state lifetime. If either dye is fixed or not free to rotate, then κ² =2/3 is not a valid, but the deviation is small.

The efficiency of FRET can be expressed as:

- he intensity difference of the two emitted signals (535 nm minus 480 nm of the example), divided by the emitted donor intensity;

- in terms of the effect of distance, R₀⁶/( r⁶ + R₀⁶);

- in terms of the effect of life time, the difference between life time without acceptor and with acceptor divided by that without acceptor.

Related methods

A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise in the results from direct excitation of the acceptor, or photo-bleaching. To overcome this difficulty, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. This technique uses a bioluminescent luciferase rather than CFP to produce an initial photon emission compatible with YFP.

A different, but related, mechanism is Dexter Electron Transfer (where the energy transfer is triplet-triplet).

An alternative method to detecting protein-protein proximity is Bimolecular fluorescence complementation (BiFC), where two halves of a YFP are fused to a protein. When these two halves meet they form a fluorophore after about 60 s - 1 hr.