What is FRET?
Fluorescence or Förster resonance energy transfer (FRET) briefly, is a distance-dependent transfer of excited state energy from an initially excited donor to an acceptor, with the donor molecule typically emitting at shorter wavelengths that overlap with the absorption of an acceptor (1-3). FRET occurs when a donor (fluorophore) and an acceptor (another fluorophore or quencher) are within a specified distance, usually within 10-100 Å. The donor-acceptor distance at which the energy transfer is 50% is called the Förster radius (Ro). Within this distance, when a donor transfers its resonance energy to a quencher, a decrease in the donor fluorescence is seen. FRET efficiency falls dramatically as the donor-acceptor distance exceeds the Förster radius. Figure 1 shows a schematic representation of a FRET peptide where the fluorescence of the donor is quenched through resonance energy transfer. Enzyme hydrolysis of the peptide results in spatial separation of the donor and acceptor, which leads to the recovery of the donor’s fluorescence.