Fluorescence Energy Transfer
 
In principle, the fluorescent light emitted by a fluorophore (the energy donor) can be reabsorbed by another chromophore (the energy acceptor) in the same molecule or somewhere else in the solution, provided that the absorption spectrum of the acceptor overlaps with the fluorescence emission spectrum of the donor. Such an energy transfer by emission and reabsorption of light is extremely inefficient. A much more efficient energy transfer between donor and acceptor can occur in a nonradiative process, which involves the coupling of their transition dipoles. This process is called resonance energy transfer. It depends on:
1. the extent of overlap between the fluorescence band of the donor and the absorption band of the acceptor,
2. the distance between the two chromophores, and
3. the relative orientation of their transition dipoles.
Resonance energy transfer is strongly sensitive to distance and varies with the inverse sixth power of the distance R between donor and acceptor. This is very useful to measure distances in macromolecules and changes in distances as a result of conformational changes in proteins and nucleic acids. An important number in energy transfer is the characteristic transfer distance R₀. R₀ is the distance between donor and acceptor at which fluorescence emission from the donor and energy transfer to the acceptor are equally probable. Förster developed a theory to correlate the efficiency of energy transfer with R and R₀. The characteristic distance R₀ can be calculated (primarily from the spectral overlap between donor and acceptor), and consequently the distance R between donor and acceptor can be determined from the measured efficiency of fluorescence energy transfer. Values for R₀ often lie in the range between 1 and 5 nm. Thus, distances up to about 8 nm can be measured by energy transfer.
 In proteins, energy is transferred primarily from tyrosine to tryptophan residues. Tryptophan residues can also transfer their energy to natural acceptors, such as NADH, riboflavin, or heme. For measuring distances in proteins or nucleic acids, often both donor and acceptor are introduced by site-directed labeling. A naphthyl group is often used as the donor and a dansyl group as the acceptor in such experiments. A detailed list of useful donor/acceptor pairs is found in (1).
References
1. M. R. Eftink (1991) "Fluorescence techniques for studying protein structure". In Methods of Biochemical Analysis (C. H. Suelter, ed.), Vol. 35, Wiley, New York, pp. 127–205.