Insights into the Physiochemical Properties of the Interaction between the L38↑N↑L+4 HIV-1 Hinge Mutant Subtype C Protease and a Related Gag Cleavage Site
| dc.contributor.author | Adams, Taryn Racheal | |
| dc.contributor.supervisor | Sayed, Yasien | |
| dc.date.accessioned | 2024-10-25T21:34:38Z | |
| dc.date.available | 2024-10-25T21:34:38Z | |
| dc.date.issued | 2023-09 | |
| dc.description | A dissertation submitted in fulfilment of the requirements for the degree Masters of Science, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023. | |
| dc.description.abstract | Frequent mutations in HIV protein drug targets such as the protease (PR), have led to a rise in resistance to clinically available treatments and inquiries into the associated biochemical mechanisms. In this study, the L38↑N↑L+4 PR (mutant) and related gag, were isolated from a PR inhibitor naïve child for further study. Wild-type and mutant PRs were successfully overexpressed and purified using ion exchange chromatography. Intrinsic fluorescence studies probing tryptophan residues located at the hinge revealed variations in tertiary structure. This coincided with significant differences in refolding efficiency (mutant PR recovery ~ 10% and wild-type PR recovery ~ 34%). Furthermore, differences in catalytic efficiency (mutant-specific activity ~ 7.4 µmol.min-1.mg-1, mutant kcat ~ 2.71 sec-1; wild-type specific activity ~ 31.6 µmol.min-1.mg-1, kcat ~ 11.60 sec-1). Thermal shift assays revealed reduced mutant PR structural stability (Tm ~ 67 ℃) compared to the wild-type PR (Tm ~ 64 ℃). Furthermore, reduced stability of inhibitor-mutant PR complexes (Tm ~ 73.5 ℃ Acetyl pepstatin (AP), 90 ℃ Darunavir (DRV), and 88 ℃ Saquinavir (SQV)) compared to complexes involving the wild-type PR (Tm ~ 70 ℃ (AP), 82 ℃ (DRV), and 82 ℃ (SQV)). Overall, the L38↑N↑L+4 PR mutations were found to influence the tertiary structure of the hinge, gag processing, and PI stability within the mutant PR active site. Computational docking studies highlighted the potential role of a gag single nucleotide polymorphism (SNP), located at the 4th amino acid (P4) position of the peptide-2-nucleocapsid (p2/NC) gag cleavage site, for future studies as a compensatory mutation aiding PR polymorphisms. Further studies should focus on the gag-PR functional pair to build a more accurate understanding of HIV drug resistance mechanisms. | |
| dc.description.sponsorship | University of the Witwatersrand, Johannesburg - Postgraduate Merit Award. | |
| dc.description.submitter | MM2024 | |
| dc.faculty | Faculty of Science | |
| dc.identifier | 0000-0001-8973-4235 | |
| dc.identifier.citation | Adams, Taryn Racheal. (2023). Insights into the Physiochemical Properties of the Interaction between the L38↑N↑L+4 HIV-1 Hinge Mutant Subtype C Protease and a Related Gag Cleavage Site. [Master's dissertation, University of the Witwatersrand, Johannesburg]. https://hdl.handle.net/10539/41969 | |
| dc.identifier.uri | https://hdl.handle.net/10539/41969 | |
| dc.language.iso | en | |
| dc.publisher | University of the Witwatersrand, Johannesburg | |
| dc.rights | ©2023 University of the Witwatersrand, Johannesburg. All rights reserved. The copyright in this work vests in the University of the Witwatersrand, Johannesburg. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of University of the Witwatersrand, Johannesburg. | |
| dc.rights.holder | University of the Witwatersrand, Johannesburg | |
| dc.school | School of Molecular and Cell Biology | |
| dc.subject | HIV Protease | |
| dc.subject | Drug Resistance | |
| dc.subject | Hinge Mutations | |
| dc.subject | Insertion Mutations | |
| dc.subject | Gag cleavage site | |
| dc.subject | Substitution Mutations | |
| dc.subject | UCTD | |
| dc.subject.other | SDG-3: Good health and well-being | |
| dc.title | Insights into the Physiochemical Properties of the Interaction between the L38↑N↑L+4 HIV-1 Hinge Mutant Subtype C Protease and a Related Gag Cleavage Site | |
| dc.type | Dissertation |