Fluorescence resonance energy transfer (FRET) is a nonradiative process by which the excitation energy can be passed from a fluorescent donor molecule (D) to an acceptor chromophore (A) over long distances, typically 10 – 100 Å. Because the FRET sensitivity range is comparable to the typical dimensions of biological macromolecules, FRET is extensively utilized to study interactions and structural dynamics of proteins, ribozymes and DNAs.
A typical FRET experiment
to investigate conformational changes of a protein is explained on
Fig.1.
The
protein is labeled with 2 fluorescent dyes so that in one of the protein
conformations these chromophores are very close to each other (the left
panel). Upon the laser excitation of the donor, the excitation energy
passes to the acceptor which then emits. The right panel catches the
protein in a different state. Now the average donor-acceptor distance is
increased; the energy transfer is less effective and that is reflected
in the enhancement of the donor fluorescence and anticorrelated drop of
fluorescence intensity from the acceptor.
The efficiency of energy transfer, E, is calculated from the photon
counts from the donor, ID, and the acceptor, IA, as
Here, is a correction factor that accounts for the different quantum yields and detection efficiencies of the donor and the acceptor. The FRET efficiency depends strongly, i.e. to the sixth power, on the distance between the two dye molecules, R, (Fig. 2):
Here, R0 is the characteristic Förster distance at which FRET efficiency
is 0.5, i.e. exactly half of the photons are transferred from the donor
to the acceptor. Förster distance can be calculated directly from the
spectral properties of the dyes and typically lies between 50 and 80 Å.
Fig. 2
Fig. 3 shows an example of the signal recorded from a
single protein molecule fluctuating between different structural
conformations (R0 for the dyes used in this experiment was 53 Å).
Fig.3