1,720,973 research outputs found
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
No full textA 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.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.University of the Witwatersrand, Johannesburg - Postgraduate Merit Award.MM202
HIV-1 subtype C protease: enzyme kinetics, thermodynamics, and X-ray crystal structure
No full textA dissertation submitted in fulfilment of the requirements for the degree Master of Science, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2023.Human immunodeficiency virus (HIV), a precursor for AIDS is still one of the most devastating pandemics in history. In 2021 alone there were 650 000 deaths associated with the virus and the number of people living with the infection was recorded to be 38.4 million globally. Sub-Saharan Africa suffers the most burden of the virus with approximately 8.3 million people living with virus, HIV-1 subtype C is the main driver of the disease in South Africa and accounts for 46% of global infections. Even with these alarming statistics this subtype is not the main focus point for the majority of HIV-1 research which mainly focuses on subtype B though it only accounts for 12% of infections globally. There is no vaccine or cure against HIV; however, great strides have been made in suppressing the virus. Viral suppression drugs have been developed to target different stages of viral replication such as those targeting the three important enzymes (protease, reverse transcriptase and integrase). In this study the focus will be on HIV-1 subtype C protease. This is a homodimeric aspartyl protease with 99 amino acids in each monomer. It plays a crucial role in the replication cycle of HIV-1 by producing mature infectious virions through cleavage of the Gag and Gag-Pol polyproteins. The subtype C protease differs from subtype B protease in that it has eight naturally occurring polymorphisms which are substitution mutations, some occurring in different regions of the protease with some in the fulcrum (T12S, I15V and L19I), others in the hinge region (M36I and R41K), with H69K and L89M found in the loops and I93L in the α helix. In this study, structural and functional characterisation of HIV-1 subtype C protease was carried out. The secondary structure was characterised using far-UV CD, which is a technique that measures the difference in left and right circularly polarised light. The subtype C protease was estimated to be predominantly β-sheeted, with spectra showing a maximum at 195 nm and a minimum between 215-225 nm. Tertiary structure characterisation of protease was performed using fluorescence spectroscopy. The maximum emission at 347 nm close to that of water (350 nm), demonstrated that the tertiary conformation of the HIV-1 protease was conserved, and that the tryptophan residues within the protease are solvent exposed. SE-HPLC was used to characterise the quaternary structure of the protease and the homodimeric size was determined to be approximately 22 kDa. Steady-state enzyme kinetics to assess the catalytic activity of the subtype C protease was performed using a fluorogenic substrate. The activity of the enzyme was confirmed, with the specific activity of 24.22±1.72 µmol. min-1.mg-1 and the binding of the substrate to the HIV-1 protease was demonstrated by the KM value of 79.546±6.491 µM. This correlates to literature indicating that the substrate was weakly bound and that a high substrate concentration will be required to reach the maximum velocity (Vmax), and Vmax was determined to be 0.036±0.003 µmol. min-1. Enzyme kinetics was coupled with displacement isothermal titration calorimetry for determination of thermodynamics parameters using second generation PIs (atazanavir, darunavir and lopinavir). Thermodynamic studies indicated that the HIV-1 protease has a high affinity for LPV (Kd = 1 nM), compared to ATV (Kd = 18.57nM) and DRV (Kd = 42.26 nM) and binding reactions were all spontaneous with ΔG values(ATV = -43.39 kJ/mol, DRV = -41.39 kJ/mol and LPV = -50.51 kJ/mol). The values also indicated that LPV complexed with HIV-1 is more a stable complex. Also, all the binding reactions were exothermic as indicated by the negative ΔH values of ATV = -45.54 kJ/mol, DRV = -55.62 kJ/mol and LPV = -54.71 kJ/mol. The entropy of all the reactions were determined to be unfavourable with the -T∆S of DRV = 14.23 kJ/mol followed by LPV: 4.2 kJ/mol and ATV: 2.15 kJ/mol. Overall this suggested that all the binding reactions were enthalpically driven. Furthermore, the three-dimensional structure of the HIV-1 subtype C protease was elucidated using X-ray crystallography. The three-dimensional structure the HIV-1 CSA (PDB ID: 8CI7) was solved at a 2.4 Å resolution which is better than the 2.7 Å (PDB ID: 3U71) initially solved in our lab. The high-resolution three-dimensional structure of the protease will provide precise information about the arrangement of atoms within the protease molecule, thus enabling the design and development of protease inhibitors that will be specific for the subtype C protease. This study emphasised the significance of investigating subtype C protease in the context of enzyme kinetics, thermodynamics and detailed X-ray crystallography.National Research Foundation (NRF).Global Challenges Research Fund-START.MMM202
Exploring the Structure, Function and Stability of Glutathione Transferases Engineered from Intra- and Inter-class Consensus Sequences: How Forgiving is Nature?
No full textA dissertation submitted in fulfilment of the requirements for the degree Master of Science, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2024.Protein folding is an enigmatic biochemical process that is foundational to the structural and functional requirements of a cell. The problem of protein folding, in a nutshell, concerns itself with the rate of protein folding as well as the conversion of amino acids from a linear sequence to a fully folded structure. This problem is partly answered by the existence of folding pathways. The folding funnel was conceptualised as a depiction of folding pathways, and it is a framework that illustrates that native proteins naturally favour the lowest energy state, encountering kinetic and thermodynamic barriers as they fold. Consensus protein design, based on this understanding, aims to: (1) enhance stability and (2) navigate the pitfalls of folding by modifying the folding funnel of a protein. This approach can also shed light on the significance of evolutionarily conserved residues. In this study, consensus protein mutants were generated for the Alpha and Mu glutathione transferases (GSTs) classes. The consensus proteins were then benchmarked against the parental proteins that were chosen (hGSTA1-1 and hGSTM1-1). The Alpha consensus mutant had 11 consensus mutations, including a notable M50L mutation, which affects the dynamic behaviour of helices α2 and α9, while the Mu consensus mutant had 13 unique mutations. Protein production and purification showed that the Mu consensus mutant had larger and purer yields. Data from far-UV circular dichroism studies and root-mean-squared-fluctuation (RMSF) from molecular dynamics (MD) simulations showed that the secondary structural components of the Alpha and Mu proteins remained largely the same, although the Alpha consensus mutant displayed a far lower molar residue ellipticity reading than its wildtype counterpart, indicating the disruption of secondary structural elements, likely caused by the M50L mutation. The ANS binding results showed that the M50L mutation in the Alpha consensus protein caused an increase in exposure of the surface area of the H-site, while the Mu consensus protein had a decrease in the solvent accessibility of its H-site. Thermal shift assay results indicated the consensus proteins had increased thermal stability. Enzyme kinetics results showed that the functionality of the proteins was severely diminished in the consensus mutants, particularly the Alpha consensus mutant. MD simulation results showed that there was an overall increase in the rigidity and compactness of the consensus mutant proteins, further affirming the improvement of thermal stability, while signalling the loss in functionality. The results produced herein have the potential to facilitate the proliferation of engineered GSTs for biotechnological applications that require proteins with an increased half-life and greater stability.National Research Foundation (NRF)MMM202
In Silico Exploration of Endocannabinoid Receptor–CB1 and CB2–Interactions Comparing Cannabidiol and Cannabidiol Diacetate: A Comprehensive Computational Study
No full textThesis submitted in fulfilment of the requirements for the degree Philosophiae Doctor, to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2024.In the rapidly evolving field of cannabinoid research, acetylated phytocannabinoids such as cannabidiol diacetate (CBDDA) have shown prominence due to its enhanced effects compared to its natural counterpart, cannabidiol (CBD). Despite the growing popularity in the consumption of acetylated phytocannabinoids, in-depth research on its pharmacological impact, especially on CB1 and CB2 receptors, remains scarce. With rising reports of adverse reactions to acetylated phytocannabinoids, a molecular understanding of their interaction with endocannabinoid receptors (CBRs) is imperative. This study aimed to fill this knowledge gap by analysing receptor interactions of CBDDA in comparison with receptor interactions of CBD. The study showed that CBDDA forms stronger interactions with CBRs than CBD. Recognised for its heightened potency, the potential of CBDDA as a biopharmaceutical product was examined. CBR interactions with known endocannabinoids, agonists and inverse agonists validated the computational models used to determine the difference in conformational dynamics upon ligand binding. In this work, bioinformatics, molecular docking, and molecular dynamics (MD) simulations were used to determine the structural differences of CBRs when bound to CBD/CBDDA. Simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and water environment successfully mimicked physiological conditions. Subsequent high-throughput virtual screening (HTVS) was conducted using CBDDA as a reference where ligands 142730975 and 21568811 were identified as the top scoring hits for CB1 and CB2 receptors, respectively. The identification of these ligands via HTVS highlights the therapeutic potential of targeting CBRs and the biopharmaceutical potential of CBDDA. This study elucidates the specific interactions of CBD and CBDDA with CB1 and CB2 receptors, laying a foundation for assessing the safety and efficacy of acetylated phytocannabinoids. Overall, the differential interaction of CBDDA compared to CBD with CBRs suggests that acetylation changes the conformational dynamics of CBRs thereby potentially affecting signalling. The identification of ligands 142730975 and 21568811 as strong interactors with the receptors may provide valuable leads for the development of new cannabinoid-based therapies.Wits University Postgraduate Merit Award ScholarshipMMM202
Management of Research Data in ETD Systems
Full text linkPurpose
To establish the research data management practices in institutions which have ETD programmes. These may include the systems in place (software and hardware), and the integration of data and text.
Design/methodology/approach
Along with a review of the literature the authors carried out a brief survey of existing practices in the management of the raw data on which a thesis/dissertation is based. A questionnaire was sent to the members of the Networked Digital Library of Theses and Dissertations (NDLTD) and a number of other institutions.
Findings
Sixty percent of the respondents indicated that their institutions have research data management centres/services. Only in one institution is there a relationship between the ETD programme and the data management centre/service. In this instance, ETDs were previously housed on the Academic Computing Services servers. Most of the institutions have no policy on the stewardship of raw data of a thesis/dissertation, and raw data is only stored if provided by the author along with the full text. Other forms of data pose challenges to institutions, and they are investigating options for including other data such as artwork, datasets and recordings, that form part of the submission.
Practical implications The paper will be a source of information to institutions considering embarking on the management of research data, especially that research data on which a thesis/dissertation is based.
Originality/value
The work draws attention to the need to collect research data of long term value. Such data may for example be utilised for the transfer of knowledge and skills by using postgraduate students to train less experienced students, using the archived/stored data without duplicating costly experiments, or for multiple projects based on the same or expanded datasets
Biochemical and thermodynamic characterisation of ligand-binding to class alpha glutathione transferase A1-1
No full textPhD, Faculty of Science (Molecular and Cell Biology), University of the Witwatersrand, 2001.Phenylalanine 51 (F51) in the human class alpha GST forms part of a hydrophobic lockand-
key intersubunit motif at the dimer interface. Protein engineering techniques were
used to replace the phenylalanine key with serine. The results indicated that the mutant
protein is dimeric with a native-like core structure indicating that F51 at the dimer
interface is not essential for dimerisation to occur. Replacing F51 with serine impacts on
the catalytic and ligandin function suggesting that tertiary structural changes have
occurred at/near the active and non-substrate ligand binding sites. The F51S mutant also
displays an enhanced exposure of hydrophobic surface as well as ligandin function. The
F51S mutant displays a diminished conformational stability when compared to the wildtype
protein. The lock-and-key intersubunit motif, therefore, although not essential for
dimerisation to occur does stabilise the quaternary structure at the dimer interface.
A unique structural feature of the class alpha GSTs is the C-terminal helix (residues 207-
221). In this study, the role of F221 was assessed by deleting it from the C-terminal helix
9 of hGSTA1-1. The results showed that the deletion of F221 does not affect the
secondary, tertiary and quaternary structure of the protein as observed using far-UV CD
measurements, enzyme activity and conformational stability as probes, respectively. The
wild-type protein binds ~ 1.7-fold more ANS than the F221del protein. Binding affinity
studies indicated that although both proteins bind ANS with the same affinity, the wildtype
protein binds ANS with a higher capacity than the F221del protein. ANS binding to
the wild-type and F221del proteins in the presence of urea (0 - 5.5 M urea) indicated that
F221 is required for stabilising helix 9 at the C-terminal of hGSTA1-1. Therefore, F221
is not required for catalysis nor does it impact on the conformational stability of the
protein. F221 does, however, affect the ligandin function and is required for the stability
of helix 9 at the C-terminus of hGSTA1-1.
ITC was used to dissect the binding energetics of glutathione (GSH) and glutathione
sulfonate (GSO3
-) to the wild-type and Y8F hGSTA1-1 proteins. The contribution of the
tyrosyl hydroxyl group to the binding of GSH and GSO3
- indicated that the Y8F mutant
binds GSH tighter than the wild-type protein and the wild-type protein, in turn, binds
GSO3
- tighter than the Y8F mutant protein. The Y8F mutant displays a larger negative
vi
DCp than the wild-type protein when complexed with either GSH or GSO3
-. This
indicates the burial of a larger solvent-exposed hydrophobic surface area for the Y8F
mutant than the wild-type protein. The burial of a large solvent-exposed hydrophobic
surface area is related to the immobilisation of helix 9 onto domain I in the presence of
active site ligands. The observation that the Y8F mutant displays burial of larger solventexposed
hydrophobic surface area suggests that the tyrosyl hydroxyl group controls the
dynamics of helix 9 at the C-terminal of hGSTA1-1. The DDG values also suggest that
the tyrosyl hydroxyl group stabilises the thiolate anion at the active site in the wild-type
protein.
The binding energetics of non-substrate ligands (ANS and BSP) to the wild-type human
class alpha GSTA1-1 were evaluated. The stoichiometry of the interaction between the
wild-type protein and ANS indicated that one molecule of ANS binds per protein
monomer. The binding interactions between ANS and the wild-type protein are
enthalpically favourable indicating the possibility of hydrogen bond formation. ANS
binding to the wild-type protein also resulted in the reduction of non-polar surface area
exposed to solvent. It is proposed that the ANS binding site is the region adjacent to
domain I that becomes buried when helix 9 is immobilised.
The binding of BSP to the wild-type protein involves a high and low affinity set of
binding sites. The high affinity binding site binds one molecule of BSP per protein
monomer whereas the low affinity site is capable of accommodating a minimum of ~ four
BSP molecules. The binding energetics to the high affinity site is both enthalpically and
entropically favourable with each term contributing favourably to the favourable Gibbs
free energy of binding. Binding to the lower affinity site is not very favourable
enthalpically and the major driving force behind the favourable Gibbs free energy of
association is the entropic factor. This interaction, therefore, appears to be entropically
driven
How the Library can Best Serve the Academic Research Community at Wits
No full textPPT PresentationDr. Yasien Sayed -
School of Molecular and Cell Biology - How the Library can Best Serve the Academic Research Community at Wit
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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