Nature Methods
- 3, 1001 - 1006 (2006)
Published online: 5 November 2006; | doi:10.1038/nmeth978
A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transferJohn R James1, Marta I Oliveira2, 3, Alexandre M Carmo2, 3, Andrea Iaboni1 & Simon J Davis11
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. 2
Group of Cell Activation and Gene Expression, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180, Porto, Portugal. 3
Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo do Prof. Abel Salazar, 2, 4099-003, Porto, Portugal.
Correspondence should be addressed to Simon J Davis simon.davis@ndm.ox.ac.uk 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.
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