Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures
Tuberculosis (TB) is an endemic health-crisis, particularly in sub-Saharan Africa. The rise of multiand extensively-drug resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB, has led to further developments in understanding the physiology of Mtb during infection, as well as searchin...
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
Department of Integrative Biomedical Sciences (IBMS)
2019
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867614460817965056 |
|---|---|
| access_status_str | Open Access |
| author | Kirykowicz, Angela Mary |
| author2 | Woodward, Jeremy David |
| author_browse | Kirykowicz, Angela Mary Woodward, Jeremy David |
| author_facet | Woodward, Jeremy David Kirykowicz, Angela Mary |
| author_sort | Kirykowicz, Angela Mary |
| collection | Thesis |
| description | Tuberculosis (TB) is an endemic health-crisis, particularly in sub-Saharan Africa. The rise of multiand extensively-drug resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB, has led to further developments in understanding the physiology of Mtb during infection, as well as searching for novel drug targets, in order to combat the disease. Our understanding of cells, both eukaryotic and prokaryotic, has changed substantially in the last 50 years, incorporating the role of stable and transient protein-protein interactions which govern cell function and behaviour. Although there are many in vivo and in vitro methods for studying protein-protein interactions, they suffer from the lack of ability to distinguish physiological interactions from interactions that occur which are not physiologically relevant to the cell. Structure-based methods for determining protein interactions have the benefit of screening out false positives whilst simultaneously assessing the possible biological function of the protein complex in question. This study sought to assess different high-throughput methods for capturing stable, water soluble protein complexes from M. smegmatis (Msm), a close relative of Mtb, for structural characterisation by low-resolution transmission electron microscopy (EM). The use of partial biochemical fractionation was assessed, which produced low-resolution structures of glutamine synthetase I, bacterioferritin, and Encapsulin. These structures were unambiguously identified through a combination of fitting of homologous crystal structures into the low-resolution maps, and information obtained by liquid chromatography mass spectrometry (LC-MS/MS) of bands isolated from native- and SDS-PAGE gels. Since Encapsulin is likely to participate in the Msm oxidative stress response and functions to enclose the target proteins DyP-type peroxidase (DyP) and ferritin-family protein (BrfB), optimal conditions for cryo-EM were tested for further efforts to obtain a high-resolution structure. Furthermore, hypotheses were generated for the function of Mtb and Msm Encapsulin based on the Msm Encapsulin structure obtained with the aid of a crystal structure homologue; these related to the mode of cargo binding and pore selectivity. A single-step purification method was also assessed through grid blotting on blue native (BN) PAGE using GroEL as a test protein. The hydrophobicity and charge of the EM copper grid was tested to find the optimal grid property for particle transfer. This established that particles of GroEL could be transferred from BN-PAGE onto an EM copper grid and a successful negative stain reconstruction was obtained. In summary, the pipeline from purifying protein complexes to generating hypotheses based on structure was successfully investigated in Msm, which will aid in the production of novel drug targets for Mtb as well as in the application to other organisms. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/29644 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:52:24.229Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Department of Integrative Biomedical Sciences (IBMS) |
| publisherStr | Department of Integrative Biomedical Sciences (IBMS) |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/29644 High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures Kirykowicz, Angela Mary Woodward, Jeremy David Medical Biochemistry Tuberculosis (TB) is an endemic health-crisis, particularly in sub-Saharan Africa. The rise of multiand extensively-drug resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB, has led to further developments in understanding the physiology of Mtb during infection, as well as searching for novel drug targets, in order to combat the disease. Our understanding of cells, both eukaryotic and prokaryotic, has changed substantially in the last 50 years, incorporating the role of stable and transient protein-protein interactions which govern cell function and behaviour. Although there are many in vivo and in vitro methods for studying protein-protein interactions, they suffer from the lack of ability to distinguish physiological interactions from interactions that occur which are not physiologically relevant to the cell. Structure-based methods for determining protein interactions have the benefit of screening out false positives whilst simultaneously assessing the possible biological function of the protein complex in question. This study sought to assess different high-throughput methods for capturing stable, water soluble protein complexes from M. smegmatis (Msm), a close relative of Mtb, for structural characterisation by low-resolution transmission electron microscopy (EM). The use of partial biochemical fractionation was assessed, which produced low-resolution structures of glutamine synthetase I, bacterioferritin, and Encapsulin. These structures were unambiguously identified through a combination of fitting of homologous crystal structures into the low-resolution maps, and information obtained by liquid chromatography mass spectrometry (LC-MS/MS) of bands isolated from native- and SDS-PAGE gels. Since Encapsulin is likely to participate in the Msm oxidative stress response and functions to enclose the target proteins DyP-type peroxidase (DyP) and ferritin-family protein (BrfB), optimal conditions for cryo-EM were tested for further efforts to obtain a high-resolution structure. Furthermore, hypotheses were generated for the function of Mtb and Msm Encapsulin based on the Msm Encapsulin structure obtained with the aid of a crystal structure homologue; these related to the mode of cargo binding and pore selectivity. A single-step purification method was also assessed through grid blotting on blue native (BN) PAGE using GroEL as a test protein. The hydrophobicity and charge of the EM copper grid was tested to find the optimal grid property for particle transfer. This established that particles of GroEL could be transferred from BN-PAGE onto an EM copper grid and a successful negative stain reconstruction was obtained. In summary, the pipeline from purifying protein complexes to generating hypotheses based on structure was successfully investigated in Msm, which will aid in the production of novel drug targets for Mtb as well as in the application to other organisms. 2019-02-18T11:39:03Z 2019-02-18T11:39:03Z 2018 2019-02-13T11:46:41Z Master Thesis Masters MSc http://hdl.handle.net/11427/29644 eng application/pdf Department of Integrative Biomedical Sciences (IBMS) Faculty of Health Sciences University of Cape Town |
| spellingShingle | Medical Biochemistry Kirykowicz, Angela Mary High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures |
| thesis_degree_str | Master's |
| title | High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures |
| title_full | High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures |
| title_fullStr | High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures |
| title_full_unstemmed | High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures |
| title_short | High-Throughput Determination of Mycobacterium smegmatis Protein Complex Structures |
| title_sort | high throughput determination of mycobacterium smegmatis protein complex structures |
| topic | Medical Biochemistry |
| url | http://hdl.handle.net/11427/29644 |
| work_keys_str_mv | AT kirykowiczangelamary highthroughputdeterminationofmycobacteriumsmegmatisproteincomplexstructures |