title: A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer.
abstract: 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.
authors.affiliation: Nuffield Department of Clinical Medicine and Medical Research Council, Human Immunology Unit, Weatherall Institute of Molecular Medicine, The University of Oxford, Oxford Radcliffe Hospital, Oxford, OX3 9DU, UK.
authors.forename: John R
authors.initials: JR
authors.lastname: James
authors.forename: Marta I
authors.initials: MI
authors.lastname: Oliveira
authors.forename: Alexandre M
authors.initials: AM
authors.lastname: Carmo
authors.forename: Andrea
authors.initials: A
authors.lastname: Iaboni
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Davis
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled