Site-specific modification strategies for unravelling energetics and dynamics of type I interferon receptor complex

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Titel: Site-specific modification strategies for unravelling energetics and dynamics of type I interferon receptor complex
Autor(en): Podoplelova, Yulia
Erstgutachter: Prof. Dr. Jacob Piehler
Zweitgutachter: Prof. Dr. Richard Wagner
Zusammenfassung: Signal propagation across the membrane by cytokine receptor signalling involves a complex interplay of receptor-ligand interactions, allostery and conformational changes. Type I interferons (IFNs) exert their biological activities through binding to a shared receptor consisting of the type II cytokine receptor subunits IFNAR1 and IFNAR2. The aim of this thesis was to establish biochemical and biophysical approaches for exploring in vitro interactions and conformational changes accompanying the formation of type I IFN receptor complex. For these purposes, in this work a versatile combination of covalent vs. non-covalent reversible site-specific protein modification chemistries was exploited for their surface immobilization, incorporation of fluorescence probes or crosslinkers. The generic bioorthogonal strategy of PPTase-catalysed covalent modification of ybbR short peptide tag fused to a protein of interest enabled highly efficient site-directed fluorescence labelling of wild type IFNα2 and mutants, IFNAR1 and IFNAR2 receptors as well as their functional immobilization onto surfaces. These modified proteins were confirmed to be active by studying their interactions in ligand-receptor surface binding assays in real time by total internal reflection fluorescence spectroscopy and reflectance interference. A rapid quantitative surface assay for probing binding kinetics of proteins captured directly via His-tags from cell supernatants was established and employed for screening of IFNAR1 mutants in order to identify the residues responsible for differential recognition of various IFN subtypes. Thus the fine-tuned IFN binding chemistries through few ligand-specific interaction points as the basis for receptor plasticity was identified. Site-specific covalent immobilization allowed exploring cooperativity in ligand recognition by the receptor subunits. The observed small allosteric effect is apparently not related to the potential contact of membrane-proximal receptor domains but probably mediated through conformational cross-communication of binding sites on the ligand. Substantial conformational changes of IFNAR1 upon ligand binding were exploited as fluorescence readout to monitor the assembly of ternary complexes on artificial membranes. This enabled exploring the life times of ternary complexes with IFNα2 combined mutants targeting binding to IFNAR1 and IFNAR2 and corroborated to the suggestion that the differential physiological activity of IFN subtypes is related to the total ternary complex affinity and not to ligand affinity towards individual receptor subunits. Finally, in vitro stabilization of dual-colour labelled weakly interacting IFNα2/IFNAR1/IFNAR2 complex by means of an entropic clamp was implemented, enabling to analyze ternary complexes by fluorescence cross-correlation spectroscopy Förster resonance energy transfer on the single molecule level. These novel tools will prove valuable for unravelling the subtle interplay of interactions and conformational changes in cytokine receptor complexes.
Schlagworte: Cytokine; Receptor
Erscheinungsdatum: 19-Apr-2013
Enthalten in den Sammlungen:FB05 - E-Dissertationen

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