title: Determination of tumor necrosis factor receptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRET detected by flow cytometry.
abstract: The availability of protein fluorophores with appropriate spectral properties has made it possible to employ fluorescence resonance energy transfer (FRET) to assess interactions between three proteins microscopically. Flow cytometry offers excellent sensitivity, effective signal separation and the capacity to assess a large number of events, and, therefore, should be an ideal means to explore protein interactions in living cells. Here, we report a flow-cytometric FRET technique that employed both direct energy transfer from CFP-->YFP-->mRFP and donor quenching to assess TRAF2 trimerization in living cells. Initially, a series of fusion proteins incorporating CFP, YFP and mRFP with spacers that did or did not permit FRET were employed to document the magnitude of CFP-->YFP and YFP-->mRFP FRET and to calculate the efficiency of CFP-->YFP-->mRFP two-step FRET. Based upon this, TRAF2 homotrimerization could be detected. This method should have great utility in studying the dynamics of interactions between three specific proteins in vivo.
authors.affiliation: Flow Cytometry Section, Office of Science and Technology, National Institutes of Health Bethesda, MD 20892, USA. Lihe@mail.nih.gov
authors.forename: Liusheng
authors.initials: L
authors.lastname: He
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Wu
authors.forename: James
authors.initials: J
authors.lastname: Simone
authors.forename: Derek
authors.initials: D
authors.lastname: Hewgill
authors.forename: Peter E
authors.initials: PE
authors.lastname: Lipsky
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002460:Cell Line; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000032:analysis; D047992:TNF Receptor-Associated Factor 2 / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D047992:TNF Receptor-Associated Factor 2; D000097573:Red Fluorescent Protein; C485182:Cyan Fluorescent Protein; C054562:yellow fluorescent protein, Bacteria