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).