Dokument: Protein Structure and Dynamics probed by Multiparameter Fluorescence
| Titel: | Protein Structure and Dynamics probed by Multiparameter Fluorescence | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=63448 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20230830-085516-1 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Folz, Julian [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus A. M. [Gutachter] Prof. Dr. Herrmann, Christian [Gutachter] Prof. Dr. Fitter, Jörg [Gutachter] | |||||||
| Stichwörter: | Biophysik, Fluoreszenzspektroskopie | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | The structure and dynamical behavior of biomolecules are key to the understanding of their function and underlying mechanisms. An important experimental method to study structural dynamics over 12 orders of magnitude in time is single-molecule (sm) fluorescence spectroscopy.
The use of more than one fluorophore per molecule opens additional opportunities arising from photon densities, coincidences and dipolar coupling by Förster Resonance Energy Transfer (FRET) to study the stoichiometry, structure, dynamics and conformational transitions of biomolecules. One advantage of the method is that molecules can be studied under ambient conditions using single-molecule concentrations in the pM range. Hence it is possible to follow the mechanism of for instance a single protein that it performs in order to fulfil their biological function. The following thesis is aimed to contribute to discussed topics addressing two different areas, a methodological one and a following application-based one answering open questions related to specific biomolecules. In the first main chapter the accuracy of the method was benchmarked in a worldwide smFRET study on proteins. In the second main chapter I applied smFRET measurements to study various biomolecules performing a wide range of biological functions involved in genetic recombination, the human immune response and how a toxin perforates the membrane of a cell. The results of the worldwide smFRET study yielded good agreement between the different labs when studying the structure and dynamics of biomolecules. In the study two proteins were used with two specific purposes. The first one was used to study the performance of measurement and analysis methods to derive distances from the FRET measurements. The second one was used as a model system for the description of complex conformational dynamics. However, in this study, user bias and non-uniform calibration was observed. In order to resolve these issues, a follow-up study presents a workflow for more robust calibration. Finally, a new idea is introduced which can potentially reduce the effect of dye artifacts occurring from undesired interaction with the surface of the biomolecule it is attached to. For the application of smFRET on biomolecules, I studied various biomolecules that are influenced by different environmental conditions. For the Holliday Junction I was able to track the conformation changes it performs on a sub-ms timescale when adding magnesium ions. To this end, a novel device was used enhancing the obtained fluorescence signal. As a second biologically relevant system, I investigated the influence of a small farnesyl moiety on the human guanylate binding protein 1. For this protein, deeper insights in the pathway from a monomeric to its oligomeric state were gained. The last biomolecule investigated was a large protein complex, the Tc toxin, for which new insights into the syringe-like mechanism were resolved. Here, I combined findings from a range of experimental fluorescence methods and was able to derive a consistent picture of the functional mechanism that involved different stable and transient intermediate states. The obtained results of the global FRET study and the subsequently developed calibration workflow will aid in unifying measurement and analysis methods within the smFRET community, thereby allowing for more consistent and trustworthy smFRET studies on biomolecules. The presented investigation of biomolecules will help to understand basic steps of a fundamental system in genetics, show a protein performing a whole structural pathway to follow its function in the immune response, and how perforating a cell in a syringe like mechanism is performed by a toxin, that has possibly applications as a biopesticide. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 30.08.2023 | |||||||
| Dateien geändert am: | 30.08.2023 | |||||||
| Promotionsantrag am: | 02.11.2022 | |||||||
| Datum der Promotion: | 08.08.2023 |
