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Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.

Original publication

DOI

10.1038/nmeth978

Type

Journal article

Journal

Nat Methods

Publication Date

12/2006

Volume

3

Pages

1001 - 1006

Keywords

Binding Sites, Cell Membrane, Dimerization, Fluorescence Resonance Energy Transfer, Luminescent Measurements, Luminescent Proteins, Protein Binding, Protein Interaction Mapping, Receptors, G-Protein-Coupled