Dokument: Towards improved FRET-derived biophysical models of proteins
| Titel: | Towards improved FRET-derived biophysical models of proteins | |||||||
| Weiterer Titel: | Verbesserung von FRET-basierten biophysikalischen Modellen von Proteinen | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=64409 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20231214-134750-1 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Popara, Milana [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus A. M. [Gutachter] Prof. Dr. Gohlke, Holger [Gutachter] | |||||||
| Stichwörter: | fluorescence spectroscopy, FRET, blind study, MD simulations, structural modelling, integrative modelling, ensemble reweighting, maximum entropy method, multi-domain proteins, lipase-specific foldase, U2 auxiliary factor, maltose binding protein | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Förster Resonance Energy Transfer (FRET) measurements are an ever-growing tool in structural biology that have the ability of solving highly sophisticated biological questions, which are otherwise inaccessible or challenging to be addressed by other approaches.
Through technical and methodological developments, FRET measurements have evolved to a multiparameter detecting technique, which is able to infer the structural information with Ångström accuracy. Moreover, in FRET measurements one can detect and quantify conformational dynamics with unrivaled time resolution, spanning twelve orders of magnitudes in time, from sub-ns to thousands of seconds. Despite these advantages, FRET experiments suffer from the intrinsic sparsity of information, which impedes comprehensive structural insights at a satisfying level of detail or resolution. This can be overcome through an alliance with other experimental methods and computational methods that can add insights down to the atomistic level of detail. Driven by these challenges, efforts of this thesis were focused on two main areas. To a large extent, this thesis is aimed at answering the question if precision and accuracy of FRET measurements are at a level that is ready to take on complexity of proteins. In a blind study, we assessed the performance of FRET measurements at a single-molecule level (smFRET). Reproducibility and accuracy of smFRET experiments across instruments, analysis procedures and systems of different complexity were rigorously tested. And much beyond - we answered two questions: (1) Can FRET detect and quantify conformational dynamics on different timescales? (2) What are the minimal structural fluctuations detectable? We were able to extract maximum information from fluorescence burst traces, while pushing the limits of FRET studies and dismissing any doubts about its credibility. At the same time, this work also revealed areas where FRET measurements are still imperfect. In particular, we found that the main cause of discrepancies between lab reported values is the error in correction parameters. To alleviate this issue, in a follow-up project I established a protocol for robust determination of correction parameters. Second major area of research within this thesis was the use of FRET in integrative manner with all-atom MD simulations, with commitment to the principle of maximum entropy. Besides establishing the workflow for FRET/MD integrative modelling, we put on a test bench robustness of posterior reconstructions, and found that while recovering atomistic models is an ill-defined problem, ensemble-integrated representations, such as inter-residue distograms and 3D density maps, can be robustly recovered. While atomistic models are a long-term dream of structural biology, we pose the question if these are indeed useful, when the target are biomolecules which exist as large quasi-continuum of states. With the established FRET/MD modelling workflow, we recovered the first-ever structural models of a steric chaperone PaLif, from the family of lipase-specific foldases, which activate the most important class of enzymes in biotechnology. Besides Lif, using FRET-based tools we decoded plasticity of U2AF2 - yet another system that exceeds the complexity of systems typically studied. All systems studied within this thesis belong to a family of multidomain proteins, which constitute the majority of proteins in both eukaryotes and prokaryotes. The results of this thesis provide a major step ahead for quantitative FRET and FRET-based structure determination. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie | |||||||
| Dokument erstellt am: | 14.12.2023 | |||||||
| Dateien geändert am: | 14.12.2023 | |||||||
| Promotionsantrag am: | 19.07.2023 | |||||||
| Datum der Promotion: | 04.12.2023 |
