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Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP
Dissertation (MSc)--University of Pretoria, 2020.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2020
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| _version_ | 1867613489248337920 |
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| access_status_str | Open Access |
| author2 | Schubert, Wolf-Dieter |
| author_browse | Schubert, Wolf-Dieter |
| author_facet | Schubert, Wolf-Dieter |
| collection | Thesis |
| dc_rights_str_mv | © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc)--University of Pretoria, 2020. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/75805 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:36:57.501Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/75805 Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP Schubert, Wolf-Dieter kofiboafoh1@gmail.com Boafoh, Kofi Kwantwi UCTD Biochemistry Dissertation (MSc)--University of Pretoria, 2020. Listeriosis is a food-borne disease caused by the gram-positive bacterium Listeria monocytogenes. In extreme cases, listeriosis can lead to sepsis and meningitis. The bacterium uses host actin cytoskeleton for its own motility and to deform the cell membrane and create protrusions into neighbouring cells. Cell-to-cell spread is a hallmark of listeriosis, and it is linked to the virulence protein InlC. InlC binds the sixth SH3 domain of human Tuba (SH3 6), an actin organising protein and in doing so displaces N WASP, the main physiological binding partner of Tuba SH3 6. InlC thereby enables listerial protrusion formation and cell to cell spread. However, knocking out InlC reduces protrusion formation by only 50 %. To help resolve which other listerial or host factors promote protrusion formation and cell-to-cell spread, this study aims to decouple the interdependence of InlC and N WASP binding to Tuba SH3 6. Crystal structures of the Tuba SH3-6 / N-WASP and Tuba SH3 6 / InlC complexes, were analysed and suitable modifications of Tuba SH3 6 were chosen to weaken the interaction of one partner to Tuba SH3 6 without affecting the other. Mutant plasmids of Tuba SH3 6 were generated by site directed mutagenesis and variant proteins produced and purified chromatographically. Binding affinities between Tuba SH3 6 variants and either InlC or N-WASP were quantified by isothermal titration calorimetry, using the interaction of wild-type proteins as control experiments and to establish the congruency with previous studies. Variants of Tuba SH3-6 with reduced affinity for either InlC or N WASP were used in co crystallization experiments with the other partner to confirm the mode of binding. Binding of InlC to wild-type Tuba SH3 6 and InlC yielded a KD value in agreement with published data. However, N WASP titration into wild type Tuba SH3 6, as well as Tuba variants N1535A, Y1570A or W1554A, invariably yielded a dissolution of an N WASP dimer or higher oligomer presumably induced by the high N WASP peptide concentrations required to quantify weak binding. Similar non sigmoidal curves were observed for reactions of InlC with Tuba SH3 6 variants Y1570A or W1554A. This was again interpreted as the probable dissolution of possible concentration induced InlC dimer masking complex formations. Overall, only variant Tuba E1575A had an impact on interaction with its binding partner. The variant E1575A yielded a weaker and stronger interaction with InlC and N WASP, respectively. This variant will be useful in an in vivo study to further investigate the underlying factors involved in cell to cell spread. The wild type InlC crystal structure solved with an improved resolution at 1.85 Å relative to 2.05 Å of a previous study, will be deposited onto the protein data bank. NRF Biochemistry MSc Unrestricted 2020-08-19T08:06:33Z 2020-08-19T08:06:33Z 2020-09 2020 Dissertation Boafoh, KK 2020, Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/75805> http://hdl.handle.net/2263/75805 en © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | UCTD Biochemistry Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP |
| title | Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP |
| title_full | Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP |
| title_fullStr | Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP |
| title_full_unstemmed | Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP |
| title_short | Selectively modifying the interaction of human Tuba SH3-6 domain with listerial InlC and N-WASP |
| title_sort | selectively modifying the interaction of human tuba sh3 6 domain with listerial inlc and n wasp |
| topic | UCTD Biochemistry |
| url | http://hdl.handle.net/2263/75805 |