NMR experiments to characterize cellular and molecular mechanisms : from metabolomics to protein biogenesis.
PhD Thesis, University of Basel,
Faculty of Science.
Available under License CC BY-NC-ND (Attribution-NonCommercial-NoDerivatives).
Official URL: http://edoc.unibas.ch/diss/DissB_11515
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique that allows non-invasive studies of biomolecules at atomic resolution. It provides information on structure and dynamics of biomolecules and is also broadly used in small molecule characterisation. This thesis explores new possibilities of NMR spectroscopy to characterise molecular and cellular systems, the!chaperone–protein interactions in the periplasm of E. coli and the metabolism of eukaryotic cells upon external modifications. In the first part of this thesis, basic concepts of NMR spectroscopy are described, as well as the specific NMR experiments used in the experimental part. The second part of the thesis describes the application of NMR spectroscopy to characterise chaperone–protein complexes. Site-specific intermolecular short-range contacts were detected in a membrane–protein–chaperone complex. This was achieved by an orthogonal isotope–labeling scheme that permits the unambiguous detection of intermolecular NOEs between the well–folded Skp chaperone and the unfolded outer membrane protein A substrate ensemble. The residues involved in these contacts are part of the chaperone–substrate interface. Furthermore, the interaction between the periplasmic chaperone SurA and the BamA–POTRA domains was characterised by NMR spectroscopy. This interaction is supposed to induce the delivery of unfolded outer membrane protein substrates to the BAM complex for their insertion into the outer membrane of E. coli. The combination of sequence–specific assignment using triple–resonance experiments and chemical shift mapping upon interaction revealed the mechanism of SurA interaction with POTRA. A destabilization of SurA and the release of a hydrophobic surface on POTRA1 upon interaction presumably lead to the handover of the OMP precursor to the Bam complex.
The third part of the thesis describes studies of cellular metabolism by NMR spectroscopy by footprinting method and in living cells. 1D NMR experiments, combined with metabolite quantification methods characterise the metabolic changes in cells infected by S. flexneri and provide new insights into the infection mechanism of this highly virulent bacterium. Furthermore, the potential of dissolution dynamic nuclear polarisation (DNP) NMR spectroscopy in the characterisation of real time metabolic processes in living macrophages was successfully explored showing that dissolution–DNP NMR spectroscopy can be applied to a broad range of cell systems, and can become routinely applied for metabolic studies in the cell.
|Committee Members:||Stahlberg, Henning|
|Faculties and Departments:||05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Hiller)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||128 S.|
|Last Modified:||30 Jun 2016 10:59|
|Deposited On:||07 Dec 2015 13:49|
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