A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

• Published in: Nature methods Vol. 3; no. 12; pp. 1001 - 1006
• Main Authors: Davis, Simon J, James, John R, Oliveira, Marta I, Carmo, Alexandre M, Iaboni, Andrea
• Format: Journal Article
• Language: English
• Published: United States Nature Publishing Group 01.12.2006
• Subjects: Binding Sites; Biological assay; Bioluminescence; Cell Membrane - chemistry; Cell Membrane - metabolism; Cellular biology; Dimerization; Energy transfer; Fluorescence Resonance Energy Transfer - methods; Health aspects; Luminescent Measurements - methods; Luminescent Proteins - metabolism; Methods; Molecular structure; Physiological aspects; Protein Binding; Protein Interaction Mapping - methods; Proteins; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - metabolism; Research methodology; Resonance; United Kingdom
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth978

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.