Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein-protein interactions in live cells.
Bioluminescence resonance energy transfer (BRET) is an increasingly popular technique for studying protein-protein interactions in live cells. It is particularly suitable for real-time monitoring of such interactions, however, the timescale over which assays can be carried out is currently relatively short (minutes) due to substrate instability. We present a new derivation of the BRET technology, termed 'extended BRET' (eBRET), which now enables protein-protein interactions to be monitored in real-time for many hours. This capability has significant benefits for investigating cellular function over extended timescales, as we have illustrated using the agonist-induced G-protein coupled receptor/beta-arrestin interaction. The potential for studying the modulation of such interactions by agonists, antagonists, inhibitors, dominant negative mutants and co-expressed accessory proteins is substantial. Furthermore, the advantages of eBRET have important implications for the development of high-throughput BRET screening systems, an ever-expanding area of interest for the pharmaceutical industry.
Pfleger
Kevin D G
KD
7TM Laboratory/Laboratory for Molecular Endocrinology, Western Australian Institute for Medical Research (WAIMR) and Centre for Medical Research, University of Western Australia, Nedlands, Perth, WA 6009, Australia. kpfleger@waimr.uwa.edu.au
Dromey
Jasmin R
JR
Dalrymple
Matthew B
MB
Lim
Esther M L
EM
Thomas
Walter G
WG
Eidne
Karin A
KA
D000818:Animals; D019556:COS Cells; D002470:Cell Survival; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004735:Energy Transfer; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007700:Kinetics; D049410:Luciferases, Renilla / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D011719:Pyrazines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
D007093:Imidazoles; D011506:Proteins; D011719:Pyrazines; D011993:Recombinant Fusion Proteins; C017144:coelenterazine; D049410:Luciferases, Renilla