Forster resonance energy transfer pairs

Fluorescence or Forster resonance energy transfer (FRET) is widely accepted as being one of the most useful methods to observe biochemical events in vitro and in living cells. Generally, there are two forms of FRET sensors those based on a pair of genetically encoded fluorophores, usually employing fluorescent proteins from jellyfish or corals, or those based on small molecules that make use of small organic fluorophores. 

In the case of Forster resonance energy transfer (FRET) [13], i.e. energy transfer by the dipole-dipole mechanism, and for randomly oriented donor-acceptor pairs, the depolarization after one transfer step (ensemble average) is almost complete [27]. For this reason, fluorescence anisotropy is a good indicator of energy transfer between identical fluorophores, hence of relative distances. Existence of efficient FRET may therefore reflect an association process. 

Deniz A A, Dahan M, Grunwell J R, Ha T, Faulhaber A E, Chemla D S, Weiss S and Schultz P G 1999 Single-pair fluorescence resonance energy transfer on freely diffusing molecules observation of Forster distance dependence and subpopulations Proc. Natl Acad. Sc/. USA 96 3670-5... 

Forster (1968) points out that R0 is independent of donor radiative lifetime it only depends on the quantum efficiency of its emission. Thus, transfer from the donor triplet state is not forbidden. The slow rate of transfer is partially offset by its long lifetime. The importance of Eq. (4.4) is that it allows calculation in terms of experimentally measured quantities. For a large class of donor-acceptor pairs in inert solvents, Forster reports Rg values in the range 50-100 A. On the other hand, for scintillators such as PPO (diphenyl-2,5-oxazole), pT (p-terphenyl), and DPH (diphenyl hexatriene) in the solvents benzene, toluene, and p-xylene, Voltz et al. (1966) have reported Rg values in the range 15-20 A. Whatever the value of R0 is, it is clear that a moderate red shift of the acceptor spectrum with respect to that of the donor is favorable for resonant energy transfer. 

Deniz, A. A., Dahan, M., Gmnwell, J. R., Ha, T., Faulhaber, A. E., Chemla, D. S., Weiss, S., and Schultz, P. G. (1999). Single-pair fluorescence resonance energy transfer on freely diffusing molecules Observation of Forster distance dependence and subpopulations. Proc. Natl. Acad. Sci. USA 96, 3670—3675. 

Fluorescence Resonance Energy Transfer (FRET), Table 1 Approximate Forster distances for three common FRET pairs... 

Fluorescence resonance energy transfer (FRET), a phenomenon first described by Forster in 1948 [27], involves dipole-dipole energy transfer from the emitting fluorophore moiety (donor) to the absorbing moiety (acceptor). The rate of energy transfer (kx) for any specific donor (D) and acceptor (A) pair is given by... 

According to Forster s theory [13], the efficiency of the resonance energy transfer ( ret) is inversely proportional to the sixth-power of the distance (r) between donor and acceptor (2). In (2), Rq is the so-called Forster distance at which the transfer efficiency is 50%. Forster RET is therefore a very sensitive process and can transduce small conformational changes into large intensity modulations. Rq is characteristic for a particular donor-acceptor pair and depends on the overlap integral J between >ret emission and Aret absorption, the PLQY of >ret in its unperturbed state < d and the mutual orientation of the two partners expressed as geometry factor which equals 2/3 for random orientation of the partners (3). For a more detailed discussion of the mechanistics of RET, the reader is referred to the literature [12]. 

In 1948 Forster proposed a theory for the resonance energy transfer. He postulated that the rate of transfer depends on the inverse sixth power of the distance between the donor and the acceptor. This predicted distance dependence was verified later by experimental studies of fluorescent donor-acceptor pairs separated by a known distance in defined systems. 

Luminescence resonance energy transfer (LRET) proceeds by radiationless dipole-dipole coupled energy transfer from an excited luminophore through space to another lumino-phore [30]. There are several criteria that must be met for LRET. First, the emission band of the excited luminophore donor must overlap the absorbance band of the acceptor luminophore. The distance between the donor and acceptor molecules can be measured by using Equation 8.2 [31]. In Equation 8.2, the efficiency of energy transfer is E, tda is the time-resolved luminescence lifetime of the donor-acceptor pair, tq is the lifetime of the donor, Rq is the distance for 50% energy transfer to occur (or Forster distance), and r is the calculated distance between the donor and acceptor. 

All of the examples of singlet energy transfer we have considered take place via the long-range resonance mechanism. When the oscillator strength of the acceptor is very small (for example, n ir transitions) so that the Forster critical distance Ro approaches or is less than the collision diameter of the donor-acceptor pair, then all evidence indicates that the transfer takes place at a diffusion-controlled rate. Consequently, the transfer mechanism should involve exchange as well as Coulomb interaction. Good examples of this type of transfer have been provided by Dubois and co-workers. " ... 

---