Förster resonance energy transfer, also known as fluorescence resonance energy transfer (FRET), resonance energy transfer (RET), or electronic energy transfer (EET), is a mechanism describing energy transfer between two colored molecules, also called dyes or fluorophores. This dipole-dipole coupling mechanism is called a Förster transfer. In this process, electrostatic interactions, also called Coulomb interactions, occur between the charge distributions in the two molecules. The excited donor molecule goes from its excited to its ground state. In contrast, the other, the acceptor molecule, inversely goes from its ground state to its own energetically lower excited state. This process can be viewed as a virtual photon exchange, as the electrons remain localized on their individual molecules.
The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making fluorescence resonance energy transfer extremely sensitive to small distances. Hence fluorescence resonance energy transfer allows the determination that two fluorophores are within a certain distance. Typical Förster radii are in the range of 30–50Å. Typically, the donor emits light at a wavelength that overlaps the absorption wavelength of the acceptor molecule. If the donor and acceptor molecule are in close proximity, the energy transfer happens. Depending on the chemical structure of the acceptor molecule or dye, the transferred energy is converted to molecular vibrations; this is called quenching or emitted as light with a longer wavelength resulting in a fluorescent signal. Fluorescence resonance energy transfer-based assays enable well-designed protease assays, assays for the study of structure and conformation of proteins, the spatial distribution and assembly of protein complexes, receptor-ligand interactions, to design immunoassays, to probe interactions of single molecules, design membrane fusion assays, for membrane potential sensing, as fluorogenic protease substrates, and indicators for a small molecule such as cyclic AMP, and many others. Fluorescence resonance energy transfer substrates such as FRET peptides can be easily prepared by chemical synthesis.
The typical structure of a FRET peptide is a “donor-peptide-acceptor.” The distance between the donor and acceptor and the overlap of the absorption spectrum of both the donor and acceptor determine the quality of the FRET peptide. For example, based on a minimal effective recognition sequence, FRET peptide-based protease assays can be developed.
Substrates containing a leaving fluorescent group at the carboxy terminus and a quenching group on the other end of the short peptide allow assaying enzymes specific to the amino acid residues at the cleavage site of the substrate.