Fluorescence Resonance Energy Transfer (FRET) is the non-radiative transfer of energy from an excited fluorophore (the donor) to a neighbouring fluorophore (the acceptor), thereby causing the acceptor to emit characteristic fluorescence. FRET is highly sensitive to the distance between the donor and acceptor dipoles. An advantage of FRET over biochemical assays is that it is performed optically,allowing interrogation of live cells in a non-destructive and minimally invasive way.
FRET peptides are labelled with two dye molecules. These can be identical, but normally different dyes are used. In all FRET systems, choosing optimal FRET donor and acceptor fluorophores are key to their performance.
FRET peptides have been widely adopted as suitable substrates in enzyme studies, such as functional and kinetic characterization of peptidases, proteases, kinases and phosphatases. Hydrolysis of a peptide bond as the fluorophore and quencher are separated generates fluorescence that allows the measurement of the activity of the enzyme at nanomolar concentrations. They have also found use for screening new proteolytic enzymes and conformational investigation of peptide folding, protein–protein interactions and intracellular ion concentrations.