James, John R., Oliveira, Marta I, Carmo, Alexandre M, Iaboni, Andrea and Davis, Simon J (2006) A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nature Methods, 3 (12). pp. 1001-1006. doi:10.1038/nmeth978 ISSN 1548-7091.
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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.
| Item Type: | Journal Article |
|---|---|
| Divisions: | Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School > Biomedical Sciences Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
| Journal or Publication Title: | Nature Methods |
| Publisher: | Nature Publishing Group |
| ISSN: | 1548-7091 |
| Official Date: | December 2006 |
| Dates: | Date Event December 2006 Published 5 November 2006 Available 17 October 2006 Accepted |
| Volume: | 3 |
| Number: | 12 |
| Page Range: | pp. 1001-1006 |
| DOI: | 10.1038/nmeth978 |
| Status: | Peer Reviewed |
| Publication Status: | Published |
| Access rights to Published version: | Restricted or Subscription Access |
| URI: | https://wrap.warwick.ac.uk/110234/ |
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