Dokument: The investigation of structural dynamics of intrinsically disordered proteins
| Titel: | The investigation of structural dynamics of intrinsically disordered proteins | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=74551 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20261008-114652-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Stief, Tobias [Autor] | |||||||
| Dateien: |
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| Beitragende: | Dr. habil. Lakomek, Nils-Alexander [Gutachter] Prof. Dr. Heise, Henrike [Gutachter] | |||||||
| Stichwörter: | NMR spectroscopy, IDP, Protein dynamics, SNARE proteins, SNAP25, NMR relaxation | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | For information processing in the brain, spatially separated neurons need a mechanism
to exchange information over the synaptic cleft. Essential for this mechanism is the neuronal exocytosis, which involves the fusion of the pre-synaptic plasma membrane with the vesicle membrane for the release of neurotransmitters. These neurotransmitters can cross the synaptic cleft and activate receptors at the postsynaptic neuron. Key proteins of the neuronal exocytosis, such as Synaptobrevin-2, SNAP25a (Synaptosomal-associated protein 25 a) and Syntaxin-1A, exhibit extensive regions of intrinsic disorder. Intrinsic disorder is a widespread feature of the human proteome, with approximately 60% of proteins containing intrinsically disordered regions (IDRs) and about 5% being nearly complete intrinsically disordered (IDPs). In my PhD work, I used solution NMR spectroscopy to study the structural and dynamic properties of IDPs. Widely used structural biology methods, such as X-ray crystallography and electron microscopy generate contrast based on electron density and therefore cannot extract relevant biophysical information about the protein backbone of IDPs and IDRs, as these regions exhibit diffuse electron density due to their high internal flexibility. NMR spectroscopy provides secondary-structure information about the protein, because carbon chemical shifts are sensitive to its backbone structure. 15N relaxation rates reveal the dynamics of the protein backbone on the nanosecond and picosecond time scales, providing a more detailed and comprehensive picture of protein dynamics. In this work, I aimed to develop a robust, reliable, and user-friendly NMR workflow to investigate the structure and the dynamics of IDPs and IDRs. We optimized and tailored 15N relaxation experiments for magnetic fields ranging from 600 MHz to 1200 MHz. Furthermore, we published a step-by-step protocol for setting up and evaluating the experiments, including an instructional video. These 15N relaxation rate experiments, as well as NMR triple-resonance experiments and other biophysical methods such as CD-spectroscopy and X-ray scattering, were applied to the soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) protein SNAP25a, which revealed large regions of intrinsic disorder and an N-terminus with increased α-helical propensity. Three independent approaches were used to extract amplitudes and timescales of protein backbone motion from 15N relaxation rates, yielding to consistent dynamic modes at 50 ps, hundreds of ps, roughly 1 ns, and 5 ns to 20 ns. A 15N Hahn-echo-based R2 experiment was optimized to enable a fast yet reliable assessment of microsecond- and millisecond-scale dynamics in protein backbones. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 08.10.2026 | |||||||
| Dateien geändert am: | 08.10.2026 | |||||||
| Promotionsantrag am: | 29.03.2022 | |||||||
| Datum der Promotion: | 03.07.2026 |
