Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

Methods. 2008 Jul;45(3):214-8. doi: 10.1016/j.ymeth.2008.06.006. Epub 2008 Jun 27.

Abstract

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100A of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Amino Acid Sequence
  • Bacterial Proteins / analysis
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biological Assay / methods
  • Cell Line, Transformed
  • Cell Line, Tumor
  • Fluorescence Resonance Energy Transfer*
  • Fluorescent Dyes / analysis
  • Fluorescent Dyes / metabolism
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Luciferases / analysis
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescent Proteins / analysis
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Fluorescence, Multiphoton / methods*
  • Molecular Sequence Data
  • Multiprotein Complexes / analysis*
  • Multiprotein Complexes / metabolism
  • Plasmids
  • Protein Interaction Mapping / methods
  • Protein Multimerization
  • Receptors, G-Protein-Coupled / metabolism
  • Recombinant Fusion Proteins / analysis*
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / radiation effects
  • Research Design
  • Spectrometry, Fluorescence / methods
  • Transfection

Substances

  • Bacterial Proteins
  • Fluorescent Dyes
  • Luminescent Proteins
  • Multiprotein Complexes
  • Receptors, G-Protein-Coupled
  • Recombinant Fusion Proteins
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins
  • Luciferases
  • Adenylyl Cyclases