Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1.
In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2. We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases.
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK; Department of Chemistry, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
Louis
L
Perdios
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK.
Tom D
TD
Bunney
Department of Physics, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
Sean C
SC
Warren
Department of Physics, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
Christopher
C
Dunsby
Department of Physics, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
Paul M W
PMW
French
Department of Chemistry, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK.
Edward W
EW
Tate
Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower St, London WC1E 6BT, UK. Electronic address: m.katan@ucl.ac.uk.
Matilda
M
Katan
D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051966:Phospholipase C gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051496:Receptor, Fibroblast Growth Factor, Type 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C496348:FGFR1 protein, human; D051496:Receptor, Fibroblast Growth Factor, Type 1; C522157:PLCG1 protein, human; D051966:Phospholipase C gamma
Fibroblast growth factor receptor; Phospholipase Cγ; Time-resolved FRET