Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684350
Title: Monitoring protein structure and dynamics by optical proteomics : applications of site-specific labelling of FGFR in vitro
Author: Perdios, Louis
ISNI:       0000 0004 5920 944X
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Abstract:
Imaging of protein tyrosine kinase activity requires negligibly intrusive, molecularly precise optical probes to provide spatiotemporal insights on conformational dynamics appertained to receptor dimerization and activation, which govern the engagement of downstream signalling networks in cancer and other pathologies. We present two sets of constructs with genetically encoded, site-specifically incorporated, bioorthogonal reporters that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of an oncogenic tyrosine kinase, fibroblast growth factor receptor 1 (FGFR1). In this study, the conformational changes in the kinase domain of FGFR1 (FGFR1K) were monitored by imaging technologies that afford analysis at great spatial and temporal resolution. First, a novel set of recombinant constructs was engineered allowing the incorporation of BCNK, a bicyclononyne-containing, lysine-like unnatural amino acid (UAA) into the activation loop or the kinase insert region of FGFR1K. BCNK incorporation was accomplished by genetic code expansion, in which an evolved tRNA/synthetase pair from Methanosarcina species directed the site-specific installation of the UAA in response to an amber codon by orthogonal translation. We report here for the first time the efficient labelling of FGFR1K via fluorogenic bioorthogonal Diels-Alder cycloadditions using a tetramethylrhodamine dienophile, Tet1-TAMRA-X. We developed a single molecule Förster resonance energy transfer (smFRET) assay that uses an alternating laser excitation (ALEX) scheme in association with a surface immobilization strategy to examine changes in the conformational states of FGFR1K. FRET efficiency distributions and interdye distances provide evidence of large-amplitude domain motions in the tyrosine kinase domain and can be correlated to autophosphorylation-dependent changes in conformational states of FGFR1. The second construct, GyrB.FGFR1K3Y.TC, comprises a coumermycin-induced artificial dimerizer (GyrB), FGFR1K exhibiting wild-type like intrinsic kinase activity and a TC motif that enables fluorescent labelling with the biarsenicals FlAsH-EDT2 and REAsH-EDT2. We conceived bimolecular systems for in vitro studies by implementing time-resolved fluorescence lifetime imaging technologies. Our approach allowed examination of the interaction between GyrB.FGFR1K3Y.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase C-γ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise Fluorescent Protein (mTFP). Furthermore, we were able to probe FGFR1 dimerization by artificially inducing dimerization of GyrB using a chemical agent. We report here phosphorylation-dependent FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1K3Y.TC as well as artificial dimerization of GyrB.FGFR1K3Y.TC.
Supervisor: Tate, Ed ; Katan, Matilda ; French, Paul ; Dunsby, Chris Sponsor: Engineering and Physical Sciences Research Council ; Biotechnology and Biological Sciences Research Council ; AstraZeneca (Firm)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.684350  DOI: Not available
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