title: Investigating protein-protein interactions in live cells using bioluminescence resonance energy transfer.
abstract: Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.
authors.affiliation: Language and Genetics Department, Max Planck Institute for Psycholinguistics.
authors.forename: Pelagia
authors.initials: P
authors.lastname: Deriziotis
authors.affiliation: Language and Genetics Department, Max Planck Institute for Psycholinguistics.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Graham
authors.affiliation: Language and Genetics Department, Max Planck Institute for Psycholinguistics.
authors.forename: Sara B
authors.initials: SB
authors.lastname: Estruch
authors.affiliation: Language and Genetics Department, Max Planck Institute for Psycholinguistics; Donders Institute for Brain, Cognition and Behaviour; simon.fisher@mpi.nl.
authors.forename: Simon E
authors.initials: SE
authors.lastname: Fisher
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D060066:Protein Interaction Maps; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D048790:Renilla / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases