Journal
METHODS
Volume 45, Issue 3, Pages 214-218Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2008.06.006
Keywords
bioluminescence resonance energy transfer; fluorescence resonance energy transfer; protein complementation assay; protein-protein interaction; G protein signalling complexes; bimolecular fluorescence complementation
Funding
- NIH
- National Institute of Deafness
- National Institute of Neurological Disorders and Stroke
- Canadian Institutes of Health Research
- Heart and Stroke Foundation of Quebec
- CIHR Team in GPCR Allosteric Regulation (CTiGAR)
- Chercheur National of the Fonds de la Recherche en Sante du Quebec
- Fonds de la Recherche en Sante du Quebec
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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 100 angstrom 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. (c) 2008 Elsevier Inc. All rights reserved.
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