荧光共振能量转移

荧光共振能量转移(FRET)：当一个荧光分子(又称为供体分子)的荧光光谱与另一个荧光分子(又称为受体分子) 的激发光谱相重叠时, 供体荧光分子的激发能诱发受体分子发出荧光, 同时供体荧光分子自身的荧光强度衰减.FRET 程度与供、受体分子的空间距离紧密相关, 一般为7～10 nm 时即可发生FRET; 随着距离延长, FRET呈显著减弱.

供体和受体之间FRET的效率，可以由E=1/[1+(R/R0)exp6]反映,其中R表示供体和受体之间的距离，R0表示福氏半径，依赖供体发射谱和受体激发谱的重叠程度，以及供体和受体能量转移的偶极子的相对方位。

荧光共振能量转移[是指在两个不同的荧光基团中，如果一个荧光基团（供体 Donor）的发射光谱与另一个基团（受体 Acceptor）的吸收光谱有一定的重叠，当这两个荧光基团间的距离合适时（一般小于100A0），就可观察到荧光能量由供体向受体转移的现象，即以前一种基团的激发波长激发时，可观察到后一个基团发射的荧光。简单地说，就是在供体基团的激发状态下由一对偶极子介导的能量从供体向受体转移的过程，此过程没有光子的参与，所以是非辐射的。给予体分子被激发后，当接受体分子与给予体分子相距一定距离，且给予体和接受体的基态及第一电子激发态两者的振动能级间的能量差相互适应时，处于激发态的给予体将把一部分或全部能量转移给接受体，使接受体被激发，在整个能量转移过程中，不涉及光子的发射和重新吸收。如果接受体荧光量子产率为零，则发生能量转移荧光熄灭；如果接受体也是一种荧光发射体，则呈现出接受体的荧光，并造成次级荧光光谱的红移。

能量供给体－接受体（D–A）对之间发生有效能量转移的条件是苛刻的，主要包括：(1)能量供体的发射光谱与能量受体的吸收光谱必须重叠；(2)能量供体与能量受体的荧光生色团必须以适当的方式排列；(3)能量供体、能量受体之间必须足够接近，这样发生能量转移的几率才会高。此外，对于合适的供体、受体分子在量子产率、消光系数、水溶性、抗干扰能力等方面还有众多的要求。可见，要找到一个合适的D–A对是很不容易的

摘自Wikipedia, the free encyclopediaFörster resonance energy transferFörster resonance energy transfer(abbreviatedFRET), also known asfluorescence resonance energy transfer,resonance energy transfer(RET) orelectronic energy transfer(EET), is a mechanism describing energy transfer between two chromophores.

A donor chromophore, intially in its electronic excited state, may transfer energy to an acceptor chromophore (in close proximity, typically <10nm) through nonradiative dipole-dipole coupling. This mechanism is termed "F&ouml;rster resonance energy transfer" and is named after the German scientist Theodor F&ouml;rster. When both chromophores are fluorescent, the term "fluorescence resonance energy transfer" is often used instead, although the energy is not actually transferred by fluorescence. In order to avoid an erroneous interpretation of the phenomenon that (even when occurring between two fluorescent chromophores) is always a nonradiative transfer of energy, the name "F&ouml;rster resonance energy transfer" is preferred to "fluorescence resonance energy transfer" - although the latter enjoys common usage in scientific literature. FRET is analogous to Near Field Communication, in that the radius of interaction is much smaller than thewavelengthof light emitted. In the near field region, the excited chromophore emits a virtual photon that is instantly absorbed by a receiving chromophore. These virtual photons are undetectable, since their existence violates the conservation of energy and momentum, and hence FRET is known as a radiationless mechanism. From quantum electrodynamical calculations, it is determined that radiationless (FRET) and radiative energy transfer are the short- and long-range asymptotes of a single unified mechanism.

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:

wherekETis the rate of energy transfer,kfthe radiative decay rate and thekiare 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.Edepends on the donor-to-acceptor separation distancerwith an inverse 6th power law due to the dipole-dipole coupling mechanism:

withR0 being the F&ouml;rster distance of this pair of donor and acceptor at which the FRET efficiency is 50%. The F&ouml;rster distance depends on the overlapintegralof the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation:

whereQ0 is the fluorescence quantum yield of the donor in the absence of the acceptor, κ is the dipole orientation factor,nis the refractive index of the medium,NAis Avogadro's_number, andJis the spectral overlap integral calculated as

wherefD is the normalized donor emission spectrum, and εA is the acceptor molar extinction coefficient.κ=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, thenκ=2/3 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging thatκ= 2/3 does not result in a large error in the estimated energy transfer distance due to the sixth power dependence ofR0 onκ. Even whenκis quite different from 2/3 the error can be associated with a shift inR0 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≤ 4.

The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the donor molecule as follows:

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

whereF'D andFD 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 asprotein-protein interactions, protein-DNAinteractions, 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.

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.

ApplicationsFRET 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). The presence of phosgene is detected at 5x10Mwith a typical FRET emission at 464 nm.

MISTAKE: The chromophore on the right must be also coumarine (double bond is missing)

FRET is also used to study lipid rafts in cell membranes.

References