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

Nature Methods volume 3, pages 1001–1006 (2006)Cite this article

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. Please visit methagora to view and post comments on this article

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Figure 1: Effects of acceptor/donor ratio and surface density on BRETeff.
Figure 2: Monomeric proteins expressed at the cell surface give substantial resonance energy transfer in conventional BRET experiments.
Figure 3: Type-1 experiments: varying the acceptor/donor ratio distinguishes between BRET arising from random versus oligomeric interactions.
Figure 4: Type-2 experiments: BRET signals arising from random interactions of molecules are linearly related to surface density at a given acceptor/donor ratio.
Figure 5: Two native class-A GPCRs are monomeric at the cell surface.

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Acknowledgements

We thank A. Wise (GlaxoSmithKline) for the gift of the GABAβR2 template, and E. Evans (Nuffield Dept. Clinical Medicine, Oxford University) and J. McIlhinney (MRC Anatomical Neuropharmacology Unit, Oxford University) for helpful discussion. This work was supported by the Wellcome Trust, the Rhodes Trust and the Programa Operacional Ciência e Inovação 2010, cofunded by the European Regional Development Fund.

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Authors and Affiliations

  1. 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

    John R James, Andrea Iaboni & Simon J Davis

  2. Group of Cell Activation and Gene Expression, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, Porto, 4150-180, Portugal

    Marta I Oliveira & Alexandre M Carmo

  3. Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo do Prof. Abel Salazar, 2, Porto, 4099-003, Portugal

    Marta I Oliveira & Alexandre M Carmo

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  1. John R James
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Contributions

J.R.J., M.I.O. and A.I. executed the experiments; J.R.J., A.M.C. and S.J.D. formulated the experiments and wrote the manuscript.

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Correspondence to Simon J Davis.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Residual analysis of BRET data for all constructs used. (PDF 287 kb)

Supplementary Table 1

Numerical representation of goodness-of-fits. (PDF 27 kb)

Supplementary Methods (PDF 59 kb)

Supplementary Note

Why the dependence of transfer efficiency (BRETeff) on acceptor/donor ratio is systematically different for randomly interacting proteins versus oligomeric proteins, without recourse to theory. (PDF 65 kb)

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James, J., Oliveira, M., Carmo, A. et al. A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat Methods 3, 1001–1006 (2006). https://doi.org/10.1038/nmeth978

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