title: Probing protein conformations by in situ non-covalent fluorescence labeling.
abstract: The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for site-specific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1-EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.
authors.affiliation: Institute of Biochemistry and Cluster of Excellence Macromolecular Complexes (CEF), Johann Wolfgang Goethe-University, Frankfurt/Main, Germany.
authors.forename: Jennifer Julia
authors.initials: JJ
authors.lastname: Strunk
authors.forename: Ingo
authors.initials: I
authors.lastname: Gregor
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Becker
authors.forename: Peter
authors.initials: P
authors.lastname: Lamken
authors.forename: Suman
authors.initials: S
authors.lastname: Lata
authors.forename: Annett
authors.initials: A
authors.lastname: Reichel
authors.forename: Jörg
authors.initials: J
authors.lastname: Enderlein
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006639:Histidine; D006801:Humans; D015336:Molecular Probe Techniques; D018583:Pliability; D011487:Protein Conformation; D011506:Proteins; D053633:Receptor, Interferon alpha-beta
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D053633:Receptor, Interferon alpha-beta; D006639:Histidine