Background and Objective: ? Previously, all experiments examining protein protein interactions in living cells by FRET have been limited to the use of two fluorescent proteins.? Here, we report the development of a novel FRET technology for studying complexes involving three proteins.? The system utilizes CFP, YFP and mRFP (monomer red fluorescent protein). CFP->YFP FRET1, YFP->mRFP FRET2 and CFP->YFP->mRFP linked FRET signals are monitored separately by flow cytometry. The technology has been validated by constructing plasmids encoding several FRET-positive and -negative controls, including CFP-YFP-mRFP, CFP-T2TD-YFP-mRFP and CFP-YFP-T2TD-mRFP, whereas T2TD (TRAF2 TRAF domain) acts as FRET insulator because of its structure with a distance of 9 nm from the head to the end.? Furthermore, this technology has been used to examine the trimer formation of TRAF2 in living cells.? ? Results: ? 1. The validity of CFP->YFP->mRFP 3-way FRET: For the first time, we validated the practicality of using flow cytometry to determine CFP->YFP->mRFP linked FRET by directly visualizing 2-step-FRET signals and the quenching of CFP->YFP FRET1 signals.? 2. The determination of three protein complexes: with the system, we examined the trimer formation of tumor-necrosis-factor-associated factor 2 in living Hela cells. TRAF2 was tagged separately by CFP, YFP and mRFP. Co-transfection of all three plasmids displayed the 2-step-chained FRET signals, but not in cells co-transfected with any two of them and blank fluorescent protein. ? ? ? Conclusions: we have developed a method to determine interactions among three proteins in living cells using flow cytometry and aCFP-YFP-mRFP FRET system.
