Förster resonance energy transfer

Förster resonance energy transfer (abbreviated FRET), also known as Fluoresence resonance energy transfer or resonance energy transfer (RET), describes an energy transfer mechanism between two chromophores.

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 <10nm). This energy transfer mechanism is termed Förster resonance energy transfer , named after the German scientist [Förster T. Zwischenmolekulare Energiewanderung und Fluoreszenz, "Ann. Physik" 1948, "437", 55. DOI|10.1002/andp.19484370105] . When both molecules are fluorescent, the term "fluorescence resonance energy transfer" is often used, although the energy is not actually transferred by fluorescence. [Joseph R. Lakowicz, "Principles of Fluorescence Spectroscopy", Plenum Publishing Corporation, 2nd edition (July 1, 1999)] ^, [ [http://www.olympusfluoview.com/applications/fretintro.html FRET microscopy tutorial from Olympus] ] In order to avoid an erroneous interpretation of the phenomenon that, even when occurring between two fluorescent molecules, is always a nonradiative transfer of energy, the name "Förster resonance energy transfer" may be preferred to "Fluorescence resonance energy transfer," although the latter enjoys common usage in scientific literature.

Theoretical basis

The FRET efficiency ($E$) is the quantum yield of the energy transfer transition, "i.e." the fraction of energy transfer event occurring per donor excitation event:

: $E\; =\; frac\{k\_\{ET\{k\_f+k\_\{ET\}+sum\{k\_i$

where $k\_\{ET\}$ is the rate of energy transfer, $k\_\{f\}$ the radiative decay rate and the $k\_\{i\}$ are the rate constants of any other de-excitation pathway.

The FRET efficiency depends on many parameters that can be grouped as follows:

* The distance between the donor and the acceptor
* The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum.
* The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.

$E$ depends on the donor-to-acceptor separation distance $r$ with an inverse 6th power law due to the dipole-dipole coupling mechanism: : $E=frac\{1\}\{1+(r/R\_0)^6\}!$with $R\_0$ being the Förster distance of this pair of donor and acceptor at which the FRET efficiency is 50%.The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation:: $\{R\_0\}^6\; =\; frac\{9,Q\_0\; ,(ln\; 10)\; kappa^2\; ,\; J\}\{128\; ,\; pi^5\; ,n^4\; ,\; N\_A\}$ where $Q\_0$ is the fluorescence quantum yield of the donor in the absence of the acceptor, $kappa^2$ is the dipole orientation factor, $n$ is the refractive index of the medium, $N\_A$ is Avogadro's_number, and $J$ is the spectral overlap integral calculated as : $J\; =\; int\; f\_\{\; m\; D\}(lambda)\; ,\; epsilon\_\{\; m\; A\}(lambda)\; ,\; lambda^4\; ,\; dlambda$where $f\_\{\; m\; D\}$ is the normalized donor emission spectrum, and $epsilon\_\{\; m\; A\}$ is the acceptor molar extinction coefficient. "κ"² =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 "κ"² =2/3 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging that "κ"² = 2/3 does not result in a large error in the estimated energy transfer distance due to the sixth power dependence of "R"₀ on "κ"². Even when "κ"² is quite different from 2/3 the error can be associated with a shift in "R"₀ and thus determinations of changes in relative distance for a particular system are still valid. Fluorescent proteins do not reorient on a timescale that is faster than their fluorescence lifetime. In this case 0 ≤ "κ"² ≤ 4.

The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the donor molecule as follows:: $E\; =\; 1\; -\; \{\; au\text{'}\_\{\; m\; D/\{\; au\_\{\; m\; D\; !$where $au\text{'}\_\{\; m\; D\}$ and $au\_\{\; m\; D\}$ are the donor fluorescence lifetimes in the presence and absence of an acceptor, respectively, or as : $E\; =\; 1\; -\; \{F\text{'}\_\{\; m\; D/\{F\_\{\; m\; D\; !$where $F\text{'}\_\{\; m\; D\}$ and $F\_\{\; m\; D\}$ are the donor fluorescence intensities with and without an acceptor, respectively.

Methods

In fluorescence microscopy, fluorescence confocal laser scanning microscopy, as well as in molecular biology, FRET is a useful tool to quantify molecular dynamics in biophysics and biochemistry, such as protein-protein interactions, protein-DNA interactions, and protein 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. On the other hand, 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. For monitoring protein conformational changes, the target protein is labeled with a donor and an acceptor at two loci. When a twist or bend of the protein brings the change in the distance or relative orientation of the donor and acceptor, FRET change is observed. If a molecular interaction or a protein conformational change is dependent on ligand binding, this FRET technique is applicable to fluorescent indicators for the ligand detection.

FRET studies are scalable: the extent of energy transfer is often quantified from the milliliter scale of cuvette-based experiments to the femtoliter scale of microscopy-based experiments. This quantification can be based directly (sensitized emission method) on detecting two emission channels under two different excitation conditions (primarily donor and primarily acceptor). However, for robustness reasons, FRET quantification is most often based on measuring changes in fluorescence intensity or fluorescence lifetime upon changing the experimental conditions (e.g. a microscope image of donor emission is taken with the acceptor being present. The acceptor is then bleached, such that it is incapable of accepting energy transfer and another donor emission image is acquired. A pixel-based quantification using the second equation in the theory section above is then possible.) An alternative way of temporarily deactivating the acceptor is based on its fluorescence saturation.Exploiting polarisation characteristics of light, a FRET quantification is also possible with only a single camera exposure.

CFP-YFP pairs

The most popular 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 requires troublesome processes of purification, chemical modification, and intracellular injection of a host protein, GFP variants can be easily attached to a host protein by genetic engineering. By virtue of GFP variants, the use of FRET techniques for biological research is becoming more and more popular.

BRET

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 to photobleaching. To avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. This technique uses a bioluminescent luciferase (typically the luciferase from "Renilla reniformis") rather than CFP to produce an initial photon emission compatible with YFP.

FRET and BRET are also the common tools in the study of biochemical reaction kinetics and molecular motors.

Photobleaching FRET

FRET efficiencies can also be inferred from the photobleaching rates of the donor in the presence and absence of an acceptor. This method can be performed on most fluorescence microscopes; one simply shines the excitation light (of a frequency that will excite the donor but not the acceptor significantly) on specimens with and without the acceptor fluorophore and monitors the donor fluorescence (typically separated from acceptor fluorescence using a bandpass filter) over time. The timescale is that of photobleaching, which is seconds to minutes, with fluorescence in each curve being given by

$(background)+(constant)*e^\{-(time)/\{\; au\_\{\; m\; pb\}$

where $\{\; au\_\{\; m\; pb$ is the photobleaching decay time constant and depends on whether the acceptor is present or not. Since photobleaching consists in the permanent inactivation of excited fluorophores, resonance energy transfer from an excited donor to an acceptor fluorophore prevents the photobleaching of that donor fluorophore, and thus high FRET efficiency leads to a longer photobleaching decay time constant:

$E\; =\; 1\; -\; \{\; au\_\{\; m\; pb/\{\; au\text{'}\_\{\; m\; pb\; !$

where $\{\; au\text{'}\_\{\; m\; pb$ and $\{\; au\_\{\; m\; pb$ are the photobleaching decay time constants of the donor in the presence and in the absence of the acceptor, respectively. (Notice that the fraction is the reciprocal of that used for lifetime measurements).

This technique was introduced by Jovin in 1989. [Jovin, T.M. and Arndt-Jovin, D.J. FRET microscopy: Digital imaging of fluorescence resonance energy transfer. Application in cellbiology. In Cell Structure and Function by Microspectrofluometry, E. Kohen, J. G. Hirschberg and J. S. Ploem. London: Academic Press, 1989. pp. 99-117.] Its use of an entire curve of points to extract the time constants can give it accuracy advantages over the other methods. Also, the fact that time measurements are over seconds rather than nanoseconds makes it easier than fluorescence lifetime measurements, and because photobleaching decay rates do not generally depend on donor concentration (unless acceptor saturation is an issue), the careful control of concentrations needed for intensity measurements is not needed. It is, however, important to keep the illumination the same for the with- and without-acceptor measurements, as photobleaching increases markedly with more intense incident light.

Other methods

A different, but related, mechanism is Dexter Electron Transfer.

An alternative method to detecting protein-protein proximity is BiFC where two halves of a YFP are fused to a protein (Hu, Kerppola et al. 2002). When these two halves meet they form a fluorophore after about 60 s - 1 hr.

Applications

FRET has been applied in an experimental method for the detection of phosgene. In it, phosgene or rather triphosgene as a safe substitute serves as a linker between an acceptor and a donor coumarine (forming urea groups). ["A FRET approach to phosgene detection" Hexiang Zhang and Dmitry M. Rudkevich Chem. Commun., 2007, 1238 - 1239, DOI|10.1039/b614725a] The presence of phosgene is detected at 5x10^-5M with a typical FRET emission at 464 nm.

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FRET is also used to study lipid rafts in cell membranes.Fact|date=February 2008

External links

[http://www.calctool.org/CALC/chem/photochemistry/fret Browser-based calculator to find the critical distance and FRET efficiency with known spectral overlap]

References