Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein–protein interactions in live cells

Abstract

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/β-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.