Dokument: Accurate structural determination of biomolecules via single-molecule high-precision FRET measurements
| Titel: | Accurate structural determination of biomolecules via single-molecule high-precision FRET measurements | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=52067 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20200123-105752-0 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Doroshenko, Olga [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus A. M. [Gutachter] Prof. Dr. Steger, Gerhard [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Förster resonance energy transfer (FRET) is a rising tool to determine structure and dynamics by structural heterogeneities and underlying kinetics at single molecule (sm) and ensemble level. It is one of few methods that can resolve distances at the nm scale both in vitro and in vivo. The method employs donor (D) and acceptor (A) fluorophores tethered to the macromolecule of interest and measures the radiationless energy transfer from the excited D to A. FRET has a very broad spectrum of applications, as it can cover a wide range of spatial and temporal resolution. It is suitable to study protein folding, recover conformational changes of polypeptides4 up to ribosomal subunits. Also FRET measurements are useful for formulating kinetic schemes and in combination of different approaches can even reveal dynamics across five orders of magnitude in time (Chapter 4).
Yet, it has been challenging to verify and compare experimental results from different labs, as a common protocol for measurements and analysis was missing. Another obstacle is the wide usage of the home-build microscopes and in-house software, implemented individually in many labs. To resolve this issue, my group together with 20 groups from all over the world participated in the blind study to measure FRET distances on the common samples, compare the results and issue the guideline with methodological recommendation (Chapter 2). We demonstrated that FRET Efficiency could be obtained with st.dev. up to +-0.02 with the suggested method. In addition to that I provide the explicit characterization of the dyes properties that are essential for fluorescence spectroscopy with nucleic acids. In my dissertation I combined experimental data obtained from sm-FRET studies with computer simulations to resolve spatial structures: (i) RNA three-way junctions (RNA 3WJs) and (ii) a 12-mer chromatin array. (i) RNA three-way junctions Chapter 3 involved three RNA 3WJ with different sequence at the junction in order to study the effect of this parameter on the structural conformation. To resolve RNA 3WJ 3D structures with high accuracy and precision 42-45 sm-FRET measurements are performed for each structure. The analysis include the studies of the sequence dependent conformations, different way of alignment and formulating a common representative structure for all RNA 3WJ. I found a unique solution for each studied RNA 3WJ. The findings also revealed that all obtained structural models of the RNA 3WJ are close to be planar but exhibit distinct coaxial stacking. Helix a with G-C pair at the junction never participates in the stacking interaction, indicating a specific spatial orientation of the helices that is coded by their sequence at the intersection. Also representing common structure appears to be not symmetrical and with inequivalent helical positions. Thorough analysis of experimental and modeling errors allowed us to determine the precision of 2 Å. (ii) 12-mer chromatin array In Chapter 4 two sm-FRET approaches are utilized: (i) confocal microscopy with Multiparameter Fluorescence Detection (MFD) of freely diffusing molecules and (ii) Total Internal Reflection Fluorescence (TIRF) microscope of the surface-immobilized molecules. Two major conformers of 12-mer chromatin array with a distinct stacking regime are resolved and dynamic-register model with at least eight states is formulated. Moreover, insights on register exchange dynamics are provided, reporting interchange between registers in millisecond time regime and local interaction between neighboring tetranucleosomes on microsecond time scale. This work also demonstrates that the chromatin effector heterochromatin protein 1α (HP1α) affects conformational changes of the chromatin fiber inducing its compactization. The binding mechanism of HP1α and chromatin fibers is formulated and suggests rapid exchange dynamics on hundreds of millisecond to second timescale. Lastly, resolved structural models for RNA 3WJ and 12-mer chromatin array together with their detailed documentation are ready for deposition to PDB-Dev archive. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie | |||||||
| Dokument erstellt am: | 23.01.2020 | |||||||
| Dateien geändert am: | 23.01.2020 | |||||||
| Promotionsantrag am: | 31.07.2019 | |||||||
| Datum der Promotion: | 19.12.2019 |
