Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-145).

A dissertation submitted in fulfilment of the requirements for the degree of Master of Science at the University of the Witwatersrand. Johannesburg, October 1996. / Glutathione S-transferase (GST) class Pi has two tryptophan residues which are conserved within domain one. Trp38 plays a functional role in sequestering glutathione at the active site, whereas Trp28 plays a structural role. The effects of the sterically-conservative substitution of Trp28 to Phe were investigated. When the W28F mutant was compared with the wild-type enzyme, mutation of Ttp28 to Phe was not well tolerated and resulted in a dimeric protein with impaired catalytic function and conformational stability. [Abbreviated Abstract. Open document to view full version] / AC2017

Domain interfaces are important to the folding, stability, structure and function of multidomain proteins. In the case of human glutathione S-transferase A1-1 (hGSTA1-1) site-directed mutagenesis studies have previously implicated the interdomain Arg13 residue of the protein in maintaining the proper catalytic function of the GST though its exact role was never determined (Stenberg et al., 1991). In this study it was shown by structural and sequence alignment of many representatives of the GST family and other thioredoxin-fold containing proteins that Arg13 is also highly conserved throughout the Alpha, Mu, Pi, Plasmodium falciparum and Sigma classes, all of which are Y-GSTs, and that it forms an interdomain salt bridge. This study therefore chose to evaluate the contribution of Arg13 towards the structure, stability and function of hGSTA1-1 by mutating the Arg residue to an Ala and performing comparative studies between wild-type and R13A hGSTA1-1. The spectral properties of R13A hGSTA1-1 monitored using far-ultraviolet circular dichroism and fluorescence indicated no significant changes in the secondary structure as compared to the native protein though fluorescence did indicate local tertiary structural changes around Trp21. Additionally, the catalytic activity of the R13A variant was reduced by 70% as compared to that of the wild-type enzyme further indicating local tertiary structural changes at and possibly near the active site which is located near the Trp21 residue. Conformational stability studies were performed by monitoring both thermal- and chemical-induced protein unfolding. The stability of the R13A variant was lower than that of the wild-type protein as revealed by a thermal-induced unfolding study which indicated that the melting point (Tm) of the R13A variant was 6 °C lower than that of the wild-type. Thermal-induced unfolding was shown not to be reversible however and the thermodynamic parameters of unfolding could not be determined. Urea-induced equilibrium unfolding studies on the other hand were reversible and displayed a variant-induced destabilisation of the conformation of the protein with a ΔΔG(H2O) of 16.7 kJ.mol−1 between the mutant and native protein. Additionally urea-induced equilibrium unfolding studies in the presence of ANS indicated that the equilibrium unfolding of both wild-type and R13A hGSTA1-1 was three-state. In summary the Arg13 residue is more important to the function of the protein than it is for its global stability or structure. Also since the Arg13 residue was found to be highly conserved in all the Y-GSTs and that it forms an interdomain interaction, the residue most likely performs a similar role in each of the Y-GSTs as well.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy. / The thermodynamic stability and the properties of the unfolding/refolding pathways of homodimeric human glutathione transferase A1-1 (hGST A1-1) were investigated. The conformational stability, assessed by urea- and temperature-induced denaturation studies, was consistent with a folded dimer/unfolded monomer transition with no stable intermediates. The high energy of stabilisation and the highly co-operative transition implies that the subunit-subunit interactions are necessary to maintain the three-dimensional state of the individual subunits. The stopped-flow-unfolding pathway, monitored using Trp fluorescence, was biphasic with a fast and slow unfolding event. Urea-dependence and thermodynamic activation parameters suggest that the transition state for each phase is well structured and is closely related to the native protein in term., of solvent exposure. The unfolding pathways monitored by energy transfer or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding phase (described above). A two-step sequential unfolding mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed. The crystal structures of all cytosolic glutathione transferases show that the alpha helices 5, 6 and 7 pack tightly against each other to form the hydrophobic core of' domain II. Leu164 in class alpha glutathione transferase is a topologically conserved residue in the alpha helix 6. The replacement ofLeu164 with alanine did not impact on the functional or gross structural properties of hGST A1-1. The urea-induced equilibrium and kinetic unfolding pathways were similar to those observed for the wild-type protein. The free energy change of unfolding was equivalent to the energetic cost of deleting three methylene groups. Furthermore, the decreased co-operativity of the unfolding transition is consistent with a decrease in co-operativity of the forces that maintain the native state of hGST A1-1. The biphasic kinetic unfolding pathway indicated that the fast phase was destabilised to a greater extent than the slow unfolding phase. ( Abbreviations abstract) / Andrew Chakane 2019

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy May 2018 / Proteins exist in equilibrium between the native (N) and the denatured (D) states. In order to form the biologically active native state, the amino acid sequence has to fold to form a stable three-dimensional structure. The large scientific community of biochemists and biophysicists has not yet been able to gain a complete understanding of this process. In this study, the unfolding of the homodimeric detoxification enzyme hGST M1a-1a (WT dimer) was investigated. Additionally, an F56S/R81A double-mutant (mutant monomer) was engineered to create a monomeric form of the protein. The mutant monomer was used to gain a better understanding of the unfolding events occurring at the subunit level, in the absence of quaternary interactions. Data from various techniques indicate the mutant monomer to closely resemble the tertiary structure of the subunits in the WT homodimer, making it a suitable model to study the unfolding mechanism of hGST M1a in the absence of quaternary interactions. A four-state equilibrium unfolding mechanism, involving two stable intermediate species, is proposed. HDX-MS studies indicate that disruption of the conserved lock-and-key motif, as well as the structures surrounding the mu loop, results in a destabilisation of domain 1. However, dimer dissociation cannot occur until the mixed charge cluster at the dimer interface has been destabilised. The destabilisation of domain 1 results in destabilisation of α4 and α5 in domain 2, because the domains unfold in a concerted manner. hGST M1a-1a dissociates to form monomeric intermediate (M), with weak interdomain interactions and compromised short-range contacts. The unstable M intermediate self-associates to form an oligomeric intermediate (I). The destabilisation of α6 and α7 in the hydrophobic core of domain 2 drives the formation of the partially structured denatured state. Further investigation will need to be pursued to determine whether hGST M1a-1a unfolds via transient intermediate states; however, the elucidation of the equilibrium unfolding pathway of a complex homodimeric protein is a valuable addition to the ever-growing knowledge base of protein folding. / MT 2018

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 143-154).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 164-182).

The tripeptides glutathione (GSH; γglutamyl-cysteinyl-glycine) and homoglutathione (hGSH; γglutamyl-cysteinyl-β-alanine) are abundant cytosolic tripeptides in legumes. The reactive cysteinyl sulphydryl group enables GSH or hGSH to act as the major cellular redox buffer through the formation of disulphides with other GSH/hGSH molecules. GSH can also form disulphides with cysteinyl groups within proteins, which is termed 5-thiolation, a reversible modification, protecting proteins from irreversible inactivation of thiol residues, as well as being important in regulating protein activity. Following treatment with fungal cell wall elicitors, plant cells produce reactive oxygen species (ROS) which results in cellular oxidative stress. In animal cells ROS generation induces antioxidant defences which include the accumulation of glutathione (GSH) and the formation of mixed disulphides between proteins and GSH. It was hypothesised that following treatment with a fringal elicitor, plant cells also thiolate proteins. It was of interest to determine how protein thiolation changed in response to changes in thiol metabolism known to occur during elicitation, as well as identifying proteins which underwent this modification. Using cell cultures of alfalfa (Medicago saliva L.), a leguminous plant containing both GSH and hGSH, changes in thiol content upon treatment with a fungal cell wall preparation elicitor were determined. By inhibiting protein synthesis and labelling the thiol pools with L-[(^35)S]cysteine, the degree and rate of protein mixed disulphide formation could be monitored in-vivo. To induce the elicitation response, alfalfa cell cultures were treated with a fungal cell-wall elicitor. Following elicitor treatment GSH, but not hGSH, was found to accumulate, with an associated increase in GSH, but not hGSH, forming mixed disulphide with protein. In order to use proteomic tools to identify thiolated proteins, the oxidative stress response in cell cultures of Arabidopsis, a GSH containing species, was then characterised. The level of protein-bound GSH was found to increase following treatment of cell cultures with the oxidant tert-hutyl hydroperoxide and this was associated with changes in cellular thiols. When proteins S-thiolated either in-vivo, or in-vitro, with [(^35)S]-GSH were resolved by SDS-PAGE under non-reducing conditions, a large number of radiolabelled polypeptides were identified in oxidatively stressed preparations. Testing the hypothesis that GSH-dependent enzymes may undergo S-thiolation, proteins which bound GSH were isolated from Arabidopsis using GSH-afFinity chromatography. A number of 30 kDa polypeptides were isolated and found to be S-thiolated under oxidative conditions in-vitro. Several of these were subsequently identified, notably members of the glutathione transferase (GST) superfamily. Representative recombinant GSTs from Arabidopsis, maize and soybean were expressed, Violated in-vitro and the effect on activity determined. Several thiolatable GSTs were identified from Arabidopsis, notably the members of the family of dehydroascorbate reductases (DHAR I, 11, III) and lambda GSTs. Further analysis by elecfrospray mass-spectroscopy confirmed the covalent binding of GSH to DHAR isoenzymes during in-vitro thiolation. It was concluded that S-thiolation of proteins is a commonly observed reversible modification of proteins in plants exposed to oxidative stress with potentially important consequences in cytoprotection and regulation.

The three dimensional native structure of multi domain proteins is only achieved when the adjacent domains recognise each other through the domain-domain interface. The domain-domain interface of the Glutathione S-transferase (GST) family has been studied extensively; however, no studies have been conducted on the role of the linker regions in the domain-domain interactions. Glutaredoxin 2 (Grx2) protein, from the GST family was chosen as model to investigate the possible role of linkers in protein stability by mutational analysis. Bioinformatics data revealed a conserved residue within the linker region (Leu78 in Grx2). A Grx2 mutant was created by replacing the conserved residue (Leu78) within the linker region with an alanine. This mutation (Leu to Ala) was performed in order to assess the role of the conserved residue leucine; whilst maintaining Grx2 function. A previous Grx2 mutant (Grx2 Y58W) was utilised because it incorporates tryptophan into domain 1; therefore it was possible to follow tertiary structural changes in this domain. Grx2 Y58W was compared against the mutant created within the linker Grx2 Y58W/L78A. Far-UV CD spectrum indicated that there was an increase of (~30 %) in ellipticity of Grx2 Y58W/L78A protein whereas; tryptophan fluorescence probes indicated no change in tertiary structure. Conformational stability studies showed a decrease of ΔΔG (H2O) = 3.8 kcal.mol-1 due to the impact of the Y58W/L78A mutation. The m-value which is indicative of the co-operativity between the two domains has decreased slightly by ~0.4 kcal.mol-1 M-1. This reduction in the m-value suggested the formation of intermediate however; it was not evident when using ANS as a probe. This study indicates that replacing a leucine with an alanine in the linker region causes a reduction in domain co-operativity. Therefore, the linker region in addition to separating the two domains plays a role in interdomain co-operativity.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2012 / The thioredoxin-like (Trx-like) fold is preserved in various protein families with diverse functions despite their low sequence identity. Glutathione transferases (GSTs) are characterised by a conserved N-terminal domain with a thioredoxin–like βαβαββα secondary structure topology and an all alpha-helical domain. GSTs are the principal phase II enzymes involved in protecting cellular macromolecules from a wide variety of reactive electrophilic compounds. It catalyses the conjugation of reduced glutathione (GSH) to an electrophilic substrate to form a hydrophilic and non-toxic compound. The binding site for GSH (G-site) is located in the N-terminal domain of GSTs. The sequence identity within members of the Trx-like superfamily is low; however, the members of this family fold into a conserved βαβαββα topology. It, therefore, seems reasonable that there are topologically conserved residues within this fold whose main role is to drive folding and/or maintain the structural integrity of the Trx-like fold. Structural alignments of the N-subdomain (βαβ motif) of the GST family shows that Leu7 in β1 and Leu23 in α1 are topologically conserved residues. The Leu7 side chain is involved in the packing of α1β1α2 and α3, whilst Leu23 is mainly involved in van der Waals interactions with residues in α1 and the loop region connecting α1 and β2. Taking into account the types of interaction that both Leu7 and Leu23 are involved in, as well their location in close proximity to the G-site, it was postulated that both these residues may play a role in the structure, function and stability of the GST family of proteins. Leu7 and Leu23 are not directly involved in the binding of GSH but they could be important in maintaining the G-site in a functional conformation via correct packing of the Nsubdomain. The homodimeric human class Alpha of GST (hGSTA1-1) was used as the representative of the GST family to test this hypothesis. The bulky side chains of Leu7 and Leu23 were replaced with a less bulky alanine residue to prevent altering the hydrophobicity of the βαβ motif. The effect of the mutation on the structure, function and stability of hGSTA1-1 was, therefore, studied in comparison with the wild-type using spectroscopic tools, X-ray crystallography, functional assays and conformational stability studies. The impact of the mutations on the structure of the enzyme was determined using spectroscopic tools and X-ray crystallography. The X-ray structures of the L7A and L23A mutants were resolved at 1.79 Å and 2.2 Å, respectively. Analysis of both X-ray structures shows that the mutation did not significantly perturb the global structure of the protein, which correlates with far-UV CD and intrinsic fluorescence spectroscopic data. In addition, structural alignments using the C-alpha gave root mean square deviation (r.m.s.d) values of 0.63 Å (L7A) and 0.67 Å (L23A) between the wild-type and mutant structures. However, both the L7A and L23A structures showed the presence of a cavity within the local environment of each mutation. The functional properties of the mutants were also similar to those of the wild-type as determined by specific activity and 8-anilino-1-naphthalene sulfonate (ANS)-binding, indicating that Leu7 and Leu23 are not involved in the function of hGSTA1- 1. The conformational stability of L7A and L23A proteins was probed using thermal-induced unfolding, pulse proteolysis and urea-induced equilibrium unfolding studies. The thermal stability of L7A and L23A hGSTA1-1 was reduced in comparison to the wild-type protein. This was consistent with proteolytic susceptibility of L7A and L23A proteins which indicates that both mutants are more prone to thermolysin digestion when compared to wild-type hGSTA1-1. This also correlates with urea-induced equilibrium studies. The ΔG(H2O) value (23.88 kcal.mol-1) for the wild-type protein was reduced to 12.6 and 10.49 kcal.mol-1 in L7A and L23A hGSTA1-l, respectively. Furthermore, the m-values obtained for the L7A and L23A proteins were 1.46 and 1.06 kcal.mol-1.M-1 urea, respectively; these were much lower than that obtained for the wild-type protein (4.06 kcal.mol-1.M-1 urea). The low m-values obtained for the mutant proteins indicated that the cooperativity of hGSTA1-1 unfolding was significantly diminished in both mutations. The results obtained in this study indicate that the topologically conserved Leu7 and Leu23 in the N-subdomain of hGSTA1-1 play a crucial role in maintaining the structural stability of the thioredoxin-like domain and are not involved in the function of the enzyme.