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Title: | Development and optimization of foundational techniques for the characterization of cytochrome bo3 quinol oxidase from E-coli | Authors: | Paques, Willem | Advisors: | GAO, Ye | Issue Date: | 2025 | Publisher: | tUL | Abstract: | Antimicrobial resistance is a major global threat, requiring new therapeutic targets. Prokaryotic cytochrome bo3 ubiquinol oxidase (cyt bo3) contains a unique transmembrane helix (TM0) involved in substrate/product exchange that represents a druggable site. Structural insights into its conformational dynamics could guide the design of inhibitors that disrupt respiration and kill the host bacterium. Monitoring TM0 charge state dynamics during the redox cycle by native-mass spectrometry (native-MS) may uncover conformational intermediates beyond reported 'open' and 'closed' states. We overexpressed and purified cyt bo3 from Escherichia coli, with SDS-PAGE and Blue-Native-PAGE showing characteristic gel bands, while size exclusion chromatography confirmed sample homogeneity. Next, cyt bo3 ubiquinol oxidation was measured spectrophotometrically, yielding a turnover rate of 153.91 e-/sec/enzyme (±23.76). Additionally, we optimized native-MS methods to detect cyt bo3 on a Q exactive UHMR quadrupole-Orbitrap mass spectrometer. Improved desolvation and ion transmission enabled the detection of a 147,951 Da (±49.01 SD) mass matching the cyt bo3 monomer mass from cryo-electron microscopy structures, and a 330,103 Da (±54.38 SD) mass, a putative dimer, respectively. Ion fragmentation using high energy collision induced dissociation to confirm mass identity was unsuccessful, but future strategies will include UV-photodissociation or electron-based fragmentation. Attempts to study ligand-binding effects on TM0's charging status failed due to native-MS signal loss after decylubiquinol addition. Together, these optimized methods enable reproducible cyt bo3 detection and activity measurements, providing a foundation for future structural studies of TM0 and its potential as an antimicrobial target. | Notes: | Master of Biomedical Sciences-Molecular Mechanisms in Health and Disease | Keywords: | Cytochrome bo3 oxidase;TM0;E coli;spectrophotometric activity assay;Q Exactive UHMR (hybrid) quadrupole-Orbitrap native-mass spectrometry | Document URI: | http://hdl.handle.net/1942/46784 | Category: | T2 | Type: | Theses and Dissertations |
Appears in Collections: | Master theses |
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Thesis+final+-+Willem+Paques.pdf Restricted Access | Published version | 802.35 kB | Adobe PDF | View/Open Request a copy |
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