Rhomboids are intramembrane serine proteases that cleave transmembrane (TM) protein substrates within the phospholipid bilayer. Since the discovery of the first rhomboid protease, many homologous rhomboids have been identified in all kingdoms illustrating their biological significance. Rhomboids are key players in a variety of biological processes such as, cell signalling, protein degradation, mitochondria health, apoptosis, and pathogenicity. While the mechanism of substrate entry into the rhomboid active site is still not clear, it is thought to involve dynamics around the putative substrate gate, of which appears to be comprised of the fifth transmembrane a-helix. A powerful tool that can be used to investigate conformational dynamics around the substrate gate is solution-state nuclear magnetic resonance (NMR). However, due to the size restriction of solution-state NMR, only detergent micelles have been able to produce well- resolved 1H-15N HSQC spectra of rhomboids. However, the lipid membrane environment has a significant impact on rhomboid structure and function. The use of membrane-scaffolding proteins (MSPs) in the formation of nanodiscs has the potential to allow the study of rhomboid dynamics in lipid bilayers by solution-state NMR. Therefore, this thesis investigates the plausibility of incorporating rhomboid into nanodiscs that would be compatible with solution NMR with a focus on the E. coli rhomboid, ecGlpG. The formation of empty (no ecGlpG) and ecGlpG-encapsulated nanodiscs was attempted using two MSP variants. While some successful nanodisc formation was possible, MSP degradation and low yields were seen for all nanodisc samples. Further optimization or alternate nanodisc systems will be required to incorporate ecGlpG into more membrane-like environments in a state that is compatible with solution-state NMR.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/45566 |
| Date | 20 October 2023 |
| Creators | Semotiuk, Brittany |
| Contributors | Goto, Natalie K. |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Attribution-NonCommercial-NoDerivatives 4.0 International, http://creativecommons.org/licenses/by-nc-nd/4.0/ |
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