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Molecular characterisation of the UgpB substrate binding protein from Mycobacterium tuberculosis
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Fenn, Jonathan (2019) Molecular characterisation of the UgpB substrate binding protein from Mycobacterium tuberculosis. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3714488
Abstract
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB) and remains as one of the leading causes of death worldwide. Mtb encodes a limited number of ATP-binding cassette (ABC) importers for the acquisition of carbohydrates, potentially reflecting the nutrient scarce environment encountered within the host. The focus of this PhD therefore, was to carry out a structural and functional analysis of Mtb UgpB (Rv2833c), the substrate-binding protein of the Mtb UgpABCE transporter.
The Mtb UgpB substrate-binding protein was successfully expressed and purified to homogeneity. Following purification, the function of Mtb UgpB was investigated. A panel of potential substrates were investigated using a thermal shift assay, which found that glycerol-3-phosphocholine (GPC) produced the highest thermal shift. Microscale thermophoresis revealed a clear binding preference for GPC with a Kd of 3.6 M for apo-Mtb UgpB. Further to these studies, alternative glycerophosphodiesters were also found to bind to Mtb UgpB, demonstrating that Mtb UgpB is promiscuous for a wide range of glycerophosphodiester substrates. Structural studies were undertaken in both solid and solution state and revealed, for the first time, the molecular basis of GPC and glycerophosphodiester recognition. Genetically engineered variants of Mtb UgpB were created to investigate the direct role of residues that interact with GPC and further define the molecular basis of substrate recognition and the specificity for glycerophosphodiesters. In addition, further studies were undertaken to produce additional components of the Mtb UgpABCE transporter and the nucleotide binding protein, Mtb UgpC (Rv2832c) was successfully expressed and its ATPase activity investigated.
Combined, these results have provided critical insights into the structural and functional role of Mtb UgpB, revealing the specificity is not limited to GPC, and that Mtb UgpB may have adapted to scavenge alternative glycerophosphodiester metabolites via a single transporter during intracellular infection.
| Item Type: | Thesis (PhD) | ||||
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| Subjects: | Q Science > QP Physiology Q Science > QR Microbiology |
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| Library of Congress Subject Headings (LCSH): | Mycobacterium tuberculosis, Tuberculosis, ATP-binding cassette transporters, Protein binding | ||||
| Official Date: | September 2019 | ||||
| Dates: |
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| Institution: | University of Warwick | ||||
| Theses Department: | School of Life Sciences ; Warwick Medical School | ||||
| Thesis Type: | PhD | ||||
| Publication Status: | Unpublished | ||||
| Supervisor(s)/Advisor: | Fullam, Elizabeth ; Cameron, Alexander | ||||
| Sponsors: | Medical Research Council (Great Britain) | ||||
| Format of File: | |||||
| Extent: | xx, 258 leaves : illustrations | ||||
| Language: | eng |
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