Bioluminescence Resonance Energy Transfer Assays for Protein-Protein Interactions in Living Cells

Abstract

We recently developed a bioluminescence resonance energy transfer (BRET) system for assaying protein-protein interactions (1,2), which has been used successfully for studying the interaction of circadian clock proteins isolated from cyanobacteria, and tested in Escherichia coli cells (1), and the dimerization of human β₂-adrenergic receptors in mammalian cells (3). BRET results from the nonradiative energy transfer between a donor and an acceptor. It is related to fluorescence resonance energy transfer (FRET) (4,5), except that, in FRET, there are two fluorophores, one that absorbs exogenous excitation (the donor) and passes the energy to the other fluorophore (the acceptor); in BRET, the donor is a luciferase that generates its own luminescence emission in the presence of a substrate, and can pass the energy to an acceptor fluorophore. For either BRET or FRET to work, the donor’s emission spectrum must overlap the acceptor’s absorption spectrum, their transition dipoles must be in an appropriate orientation, and the donor and acceptor must be in close proximity (usually within 30-80 Å of each other, depending on the degree of spectral overlap) (6). During a BRET assay for molecular interactions, the first criterion is fixed for a given donor-acceptor combination, but the relative orientation and distance between the donor and acceptor will change depending on the strength of the interaction. Although its use as a protein interaction assay is novel, BRET is a natural phenomenon.