Dokument: Mapping protein structure and dynamics by Förster Resonance Energy Transfer in vitro and in live cells
| Titel: | Mapping protein structure and dynamics by Förster Resonance Energy Transfer in vitro and in live cells | |||||||
| Weiterer Titel: | Kartierung von Proteinstruktur und Dynamik mittels Försterresonanzenergietransfer in vitro und in lebenden Zellen | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=40991 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20170130-133636-7 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Peulen, Thomas-Otavio [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Seidel, Claus A. M. [Gutachter] Prof. Dr. Gohlke, Holger [Gutachter] | |||||||
| Stichwörter: | Förster resonance energy transfer, Fluorescence correlation spectroscopy, single molecule detection, protein dynamics, protein structure | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Structural, biochemical and cellular aspects have to be considered to obtain a full picture on the native function of proteins. In this context fluorescence spectroscopy with multi-parameter fluorescence detection (MFD) combined Förster Resonance Energy transfer (FRET) with is an ideal method, as it informs on protein structure and dynamics in living cells. MFD detects spectral and polarization resolved fluorescent photons with picosecond resolution, while FRET measures distances with Ångström accuracy. Therefore, a combination of MFD, FRET and computational methods is ideal to study biochemical-, structural and dynamics. Cellular aspects are considered by a combination of MFD with microscopic imaging (multi-parameter image spectroscopy, MFIS) allowing to solve the multifactorial problem of understanding biomolecular function in their native environment.
FRET-measurements are rarely used to determine quantitative structural models. Therefore, a set fluorescence methods was established and combined with computational simulations to cumulate multiple FRET-measurements into a quantitative structural models and to determine equilibrium constants in in living cells. These methods were tested on a static biomolecular structure. As biomolecules are often dynamic and hence heterogeneous, an analytical method for fluorescence intensity decays was developed to resolve structural heterogeneities of conformational ensembles by FRET. Next, a theory describing multi-dimensional FRET-efficiency histograms of was established to reliably detect conformational dynamics of biomolcules. Finally, methods were developed to determine equilibrium constants in living cells by MFIS. Using the developed methods as toolkit T4 lysozyme was studied as a model system and a transiently populated conformational state was resolved in vitro. Next, the dimerization of guanylate binding proteins (GBPs) was studied by in vitro and live-cell. In vitro a previously postulated binding mode of the human GBP1 (hGBP1) was confirmed and new binding mode was found. Combining the developed analysis methods and the in vitro information GBP oligomerization was quantify in live-cell by equilibrium constants. Finally, the hGBP1 monomer was studied in detail in vitro by ensemble and single-molecule measurements. Here, two conformational states were resolved and described by structural models suggesting a potential dimerization pathway. Multiple FRET fluorescence correlation spectroscopy measurements quantify the transitions among the two monomeric states and mapping conformational dynamics to structure. These presented studies demonstrate how fluorescence and FRET-measurements improve our understanding of molecular machines. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie | |||||||
| Dokument erstellt am: | 30.01.2017 | |||||||
| Dateien geändert am: | 30.01.2017 | |||||||
| Promotionsantrag am: | 21.12.2015 | |||||||
| Datum der Promotion: | 10.03.2016 |
