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

Nat Methods. 2006 Dec;3(12):1001-6. doi: 10.1038/nmeth978. Epub 2006 Nov 5.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Dimerization
  • Fluorescence Resonance Energy Transfer / methods*
  • Luminescent Measurements / methods*
  • Luminescent Proteins / metabolism*
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / metabolism*

Substances

  • Luminescent Proteins
  • Receptors, G-Protein-Coupled