title: Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).
abstract: We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.
authors.affiliation: Imperial College London, Dept. Physics, Photonics Lab., Blackett building, Prince Consort Road, London, SW7 2AZ, UK.
authors.forename: Anca
authors.initials: A
authors.lastname: Margineanu
authors.affiliation: University College London, Institute of Structural and Molecular Biology, Darwin building, Gower St., London, WC1E 6BT, UK.
authors.forename: Jia Jia
authors.initials: JJ
authors.lastname: Chan
authors.affiliation: Imperial College London, Dept. Physics, Photonics Lab., Blackett building, Prince Consort Road, London, SW7 2AZ, UK.|Imperial College London, Institute of Chemical Biology, London, SW7 2AZ, London, UK.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Kelly
authors.affiliation: Imperial College London, Dept. Physics, Photonics Lab., Blackett building, Prince Consort Road, London, SW7 2AZ, UK.|Imperial College London, Institute of Chemical Biology, London, SW7 2AZ, London, UK.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Warren
authors.affiliation: Université Paris Diderot, Sorbonne Paris Cité, Molécules Thérapeutiques in silico, Inserm UMR-S 973, 35 rue Helene Brion, 75013 Paris, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Flatters
authors.affiliation: Imperial College London, Dept. Physics, Photonics Lab., Blackett building, Prince Consort Road, London, SW7 2AZ, UK.
authors.forename: Sunil
authors.initials: S
authors.lastname: Kumar
authors.affiliation: University College London, Institute of Structural and Molecular Biology, Darwin building, Gower St., London, WC1E 6BT, UK.
authors.forename: Matilda
authors.initials: M
authors.lastname: Katan
authors.affiliation: Imperial College London, Dept. Physics, Photonics Lab., Blackett building, Prince Consort Road, London, SW7 2AZ, UK.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Dunsby
authors.affiliation: Imperial College London, Dept. Physics, Photonics Lab., Blackett building, Prince Consort Road, London, SW7 2AZ, UK.
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007519:Isoantigens / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D054730:Protein Interaction Domains and Motifs; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D000099317:Hippo Kinases
chemical_list: D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007519:Isoantigens; D017346:Protein Serine-Threonine Kinases; D025521:Tumor Suppressor Proteins; C411762:RASSF1 protein, human; C090667:SARAH antigen; C095566:STK4 protein, human; D000099317:Hippo Kinases