FtsZ, the primary bacterial cytoskeleton protein, drives cytokinesis in the vast majority of archae and eubacteria by forming single-stranded filaments that coat the cell membrane and scaffold the peptidoglycan synthesis machinery. While the biochemistry and kinetics of FtsZ filaments are well studied, the structure details of the filaments remain elusive. Across species, FtsZ monomers are highly homologous, with all but two published monomer crystal structures assuming the same "Relaxed" conformation. However, a second "Tense" conformation has been identified in Staphylococcus aureus FtsZ monomers that is presumed to correspond to the monomer form present in active filaments. As of this writing, Tense monomers have only been directly observed in S. aureus, and while it is widely thought that they correspond to the active monomer form, this has not been confirmed.
This dissertation presents a series of structural studies of Escherichia coli FtsZ filaments, primarily using the magic angle spinning solid-state NMR (MAS NMR) technique dynamic nuclear polarization (DNP). DNP uses cryogenic temperatures and high-powered microwave radiation to dramatically increase the NMR signal, making signal-to-noise limited systems, such as the ones presented here, much more efficient. I employ a differential isotopic labelling scheme to selective observe nuclei present at the inter-monomer interface using ZF-TEDOR to recouple the heteronuclear dipolar coupling. When combined with homology modelling and chemical shift prediction, this strategy allows for the generation of distance restraints across the interface and direct model comparison in the absence of full resonance assignments.
The size of the EcFtsZ monomer (~300 structured residues), in comparison to the size of the intermonomer interface (30--80 residues depending on the model) makes it very difficult to generate enough signal-to-noise to do multi-dimensional NMR studies and obtain unambiguously assigned spectra. However, by combining 1D NMR with various sparse 13C labelling schemes, I was able to observe inter-monomer 13C--15N contacts and measure a set of 12 distances between 3.0--6.0 Å. Using this set of restraints, along with chemical shift predictions for three potential interface models---one corresponding to the Tense monomer state and two corresponding to different Relaxed states---I performed several different structural analyses on the ZF-TEDOR data, including residue counting, identifying peaks best described by a single model, and chemical shift difference analysis. This study provides multiple sets of evidence that active EcFtsZ filaments are primarily composed of Tense monomers. This is the first such direct structural evidence of the presence of Tense monomers in FtsZ filaments, and the first direct observation of Tense monomers in EcFtsZ.
Additionally, I present a previously published study where we characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in DNP experiments, specifically probing the stability in whole-cell pellets and lysates. DNP experiments use paramagnetic species to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-psmm-5t69 |
| Date | January 2021 |
| Creators | McCoy, Kelsey |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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