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.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2017. / Glutathione transferase P1-1 (GSTP1-1) is an enzyme belonging to the glutathione transferases superfamily of enzymes responsible for xenobiotic detoxification metabolism in the cells. It has been shown recently that GSTP1-1 performs a distinct function from its family members in that it acts as a carrier of the glutathione in the reactivation and glutathionylation of oxidised peroxiredoxin 6 (Prdx6). Prdx6 is a peroxidase belonging to the peroxiredoxin superfamily. The family functions to reduce organic peroxides which are sources of oxidative stress. Prdx6, however, differs from its family members as it is a bi-functional enzyme and it only contains one cysteine in its catalytic centre. The interaction of GSTP1-1 with Prdx6 has proven to be vital for the functioning of the Prdx6. The recombinant Prdx6 and GSTP1-1 proteins have been over-expressed and purified to homogeneity. The secondary structure of the proteins was studied using circular dichroism which has shown that GSTP1-1 is predominantly alpha helical and Prdx6 is mainly alpha helical with aspects of a beta sheet. The tertiary structural analysis has been carried out using tryptophan fluorescence which revealed that in both proteins the tryptophans are partially exposed to solvent. Furthermore, the quaternary structure was analysed using size exclusion-HPLC which indicated that the proteins are homodimeric in solution (both ~50 kDa). This study will present the findings on the overall characterisation and the implications of the findings on the interaction of these proteins. / LG2018

Graduation date: 1992

Quarter Horse mares (n=28, 465-612 kg BW, 6-19 yrs of age) were used to investigate the effect of organic selenium (Se) supplementation (Selenosource, Diamond V Mills, Inc. Cedar Rapids, IA (SeM)) and DE manipulation on plasma, muscle, and colostrum Se concentrations, plasma glutathione peroxidase (Gsh-Px) activity, foaling parameters, and physical characteristics in mares and their foals. Mares were arranged in a 2x2 factorial with two levels of nutrition, pasture (100% NRC DE) or pasture plus grain (120% NRC DE) (fed at 0.75% BW (0.63 ppm Se)) and two levels of Se supplementation (0 or 0.3 mg/kg DM) equaling four treatment groups: pasture (P), pasture grain (PG), pasture grain Se (PGS), or pasture Se (PS). Mares were blocked by expected foaling date and randomly assigned to dietary treatment within block. Body condition score (BCS), BW, and rump fat (RF) were observed every 14 d beginning at d 0. Mare and foal plasma and muscle sampling began on d 0 (birth in foals). Plasma continued every 14 d and muscle every 28 d until parturition (d 56 in foals). Upon parturition, foaling parameters consisting of times: water break to birth, birth to placenta expulsion, foal standing, and nursing were recorded. Colostrum quality was determined via refractometer and colostrometer analysis, and placenta weight, foal birth weight, whither and hip height and body length were recorded. Maternal SeM supplementation influenced (P<0.05) mare and foal plasma, muscle and colostrum Se concentrations. Increased maternal DE influenced (P<0.05) mare and foal plasma and foal muscle Se, mare BW, BCS, and RF. However, mare muscle Se was unaffected (P>0.05) by DE. Mare and foal plasma Gsh-Px, foal physical characteristics, and foaling parameters were unaffected by treatment (P>0.05). Greater (P<0.02) colostrum refractometer values (Brix%) for P, PS mares were noted and PGS, P mares had shorter gestational lengths (nutrition x SeM interaction (P<0.05)). These data indicate that maternal DE manipulation and SeM supplementation influences mare and foal Se status, mare BW and colostrum quality (Brix%), but not plasma Gsh-Px activity. Additionally, nutrition and SeM supplementation may affect gestational length. However, despite treatments there was no difference in foaling parameters or foal physical characteristics.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 141-175). Available also in a digital version from Dissertation Abstracts.

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This study was done to investigate the effect of ischemia-reperfusion injury on cytosolic and mitochondrial glutathione levels in the rat kidney. Glutathione is the main cellular defense against free radicals that are thought to cause ischemia-reperfusion injury. Right kidneys from anesthetized female Lewis rats (9-12 months old) were exposed to 60 minutes of ischemia followed by 0, 30, or 120 minutes of reperfusion. The kidneys were perfused with isotonic saline, harvested, homogenized, and separated into cytosolic and mitochondrial fractions by differential centrifugation. Reduced (GSH) and oxidized (GSSG) glutathione levels were measured spectrophotometrically. There were significant decreases in both the GSH levels and the % GSH/Total Glutathione in the cytosol and mitochondria of kidneys exposed to ischemia-reperfusion injury when compared to control kidneys. The glutathione levels in either the cytosol or mitochondria did not recover even after 120 minutes of reperfusion. This study demonstrates that 60 minutes of ischemia followed by 0, 30, or 120 minutes of reperfusion decreases both cytosolic and mitochondrial levels of glutathione in the rat kidney. / Department of Physiology and Health Science

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.