FRETFRET (Fluorescence Resonance Energy Transfer) is a technique for measuring interactions between two proteins in vivo. In this technique, two different fluorescent molecules (fluorophores) are genetically fused the two proteins of interest. Regular (non-FRET) fluorescence occurs when a fluorescent molecule (fluorophore) absorbs electromagnetic energy of one wavelength (the excitation frequency) and re-emits that energy at a different wavelength (the emission frequency). Conceptually, one can imagine each fluorophore to have a two-peaked spectrum in which the first peak is the excitation peak, and the second is the emission peak. For the combined FRET effect, the emission peak of the donor must overlap with the excitation peak of the acceptor. In FRET, light energy is added at the excitation frequency for the donor fluorophore, which transfers some of this energy to the acceptor, which then re-emits the light at its own emission wavelength. The net result is that the donor emits less energy than it normally would (since some of the energy it would radiate as light gets transferred to the acceptor instead), while the acceptor emits more light energy at its excitation frequency (because it is getting extra energy input from the donor fluorophore).
Sources of background noise, or cross-talk, occur because (1) the donor radiates slightly (but not optimally) at the acceptor's emission wavelength, and (2) the acceptor is excited somewhat by the donor's excitation wavelength. Both of these will cause a non-FRET signal at the emission wavelength of the acceptor that needs to be controlled for. There are two ways of doing this. One is to express each fluorophore individually in the same conditions in vivo in which FRET will be performed, and measuring this cross-talking. Thus, one would want to measure how much energy the donor radiates at the acceptor emission wavelength, as well as measuring how much the acceptor can be excited by the donor's excitation wavelength. A second, and easier, control to perform is to photobleach the acceptor fluorophore (by overwhelming it with light at its excitation frequency) to "knock out" its activity. This eliminates the energy transfer from donor to acceptor and should cause an increase in the emission from the donor (due to the fact that it is not transferring energy to the acceptor). This increase of donor emission due to photobleaching of the acceptor is known as "dequenching" and allows one to determine how much the donor fluorophore is radiating at the acceptor emission frequency.
The benefit of FRET technology is that it has excellent resolution. The physics of the FRET energy transfer between donor and acceptor (which is non-radiative) is such that the efficiency falls off with the sixth power of the distance between molecules. Thus, FRET only occurs when the two fluorophores are within 20-100Ǻ (0.002-0.01μm) of each other, which means that the fluorophores must be brought together via very close protein-protein interactions. Since biomolecules can be 50-200Ǻ in diameter, the position of the fluorophores within the protein complex is critical. If the fluorophores are over 200Ǻ apart while the proteins to which they are fused interact with each other, no signal will be observed. Often, FRET experiments are done with just the putative interaction domains of the two proteins under examination because of this distance limitation-complete molecules would hold the fluorophores too far apart for FRET to be observed. Editor(s): John Bracht
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