The principles of FRET/BRET and split luciferase-based methods
FRET technology harnesses the natural phenomenon that occurs when two complementary fluorophores are in close proximity and an excited fluorophore non-radiatively transfers energy, through dipole-dipole coupling, to an acceptor fluorophore (Figure 1A). The efficiency of the energy transfer is dictated by three main factors: (1) the emission spectrum of the donor and excitation spectrum of the acceptor fluorophores, which must at least partially overlap; (2) the distance between the two fluorophores, which should be very short (typically less than 10nm); and (3) the relative orientation of the donor and acceptor dipoles in space, which must be favourable (Sekar and Periasamy, 2003). FRET requires external excitation of the donor fluorophore and can be measured using a fluorescence microscope or fluorimeter to determine protein–protein interactions and/or conformational changes (Sekar and Periasamy, 2003).