Förster resonance energy transfer

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Förster resonance energy transfer describes an energy transfer mechanism between two fluorescent molecules. A fluorescent donor is excited at its specific fluorescence excitation wavelength. By a long-range dipole-dipole coupling mechanism, this excited state is then nonradiatively transferred to a second molecule, the acceptor. The donor returns to the electronic ground state. The described energy transfer mechanism is termed "Förster resonance energy transfer" (FRET), named after the German scientist Theodor Förster. When both molecules are fluorescent, the term "fluorescence resonance energy transfer" is often used, although the energy is not actually transferred by fluorescence.

[edit] Theoretical basis

The FRET efficiency is determined by three parameters:

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.

The FRET efficiency $E$ , which is defined as

$E = 1 - {\tau'_{\rm D}}/{\tau_{\rm D}} \!$

where $τ' D$ and $τ D$ are the donor fluorescence lifetimes in the presence and absence of an acceptor, respectively, or as

$E = 1 - {F'_{\rm D}}/{F_{\rm D}} \!$

where $F' D$ and $F D$ are the donor fluorescence intensities with and without an acceptor, respectively. $E$ depends on the donor-to-acceptor separation distance $r$ with an inverse 6th order 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:

${R_0}^6 = 8.8 \times 10^{23} \; \kappa^2 \, n^{-4} \, Q_0 \, J$

where $κ 2$ is the dipole orientation factor, $n$ is the refractive index of the medium, $Q 0$ is the fluorescence quantum yield of the donor in the absence of the acceptor, and $J$ is the spectral overlap integral calculated as

$J = \int f_{\rm D}(\lambda) \, \epsilon_{\rm A}(\lambda) \, \lambda^4 \, d\lambda$

where $f D$ is the normalized donor emission spectrum, and $ε A$ is the acceptor 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 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging that κ² = 2/3 does not result in a large error in the estimated energy transfer distance due to the sixth power dependence of R₀ on κ². Even when κ² is quite different from 2/3 the error can be associated with a shift in R₀ 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 ≤ κ² ≤ 4.

[edit] Applications

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, 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 close 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.

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.

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 photobleaching. To overcome this difficulty, 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 a common tools in the study of reaction kinetics and molecular motors.

A different, but related, mechanism is Dexter Energy 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 60s - 1 hr.

FRET can be quantified in cuvette based experiments or in microscopy images on a pixel by pixel basis. This quantification can be based directly (sesitized 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.

[edit] References

Joseph R. Lakowicz, "Principles of Fluorescence Spectroscopy", Plenum Publishing Corporation, 2nd edition (July 1, 1999)
FRET microscopy tutorial from Olympus

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Categories: Biochemistry | Biophysics | Cell imaging | Molecular biology