3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The conformational stability of a detoxification enzyme widely used as a fusion-protein affinity tag.(1997) Kaplan, Warren HA glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is widely used as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an ad titional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible for-ration of significant amounts of 160 -kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, ultraviolet melting, differential scanning micro calorimetry , and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the umolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration with a ~GO(H20) = 26 ±1.7 kcal/mol. The conformational stability was unchanged in the presence of the leading antischistosomal drug Praziquantel, which bound the protein with a Kd = 9 ±1.8 p,M. The strong relationship observed between the m-v,llue and the size of the protein indicates that the amount of protem. surface exposed to solvent upon unfolding is the major structural de.erminant for the dependence of the protein's free energy of unfolding on urea concentration. 'Ihermograms obtained by differential scanning calorimetry also fitted to a two-state irreversible unfolding transition, both in the presence and absence of Praziquantel, with values of ~Cp = 1779 cal mol-IK-I , ~HcaI = 227 kcal/mol, AHVH ::::::233 kcal/mol (r :::::~:HVHIAlIcal = 1.02) and AS = 354 cal mol''K". The low ~Cp and ~S, when compared with the theoretically determined values, implied that the thermal denaturation of Sj26GST did not result in complete unfolding of the protein,Item Regulation of glutathione transferase P1-1 by S-nitrosation(2014-06-12) Balchin, DavidS-Nitrosation is a post-translational modification of protein cysteine residues, which occurs in response to cellular oxidative stress. Although it is increasingly being linked to physiologically important processes, the molecular basis for protein regulation by this modification remains poorly understood. Biophysical methods were used to elucidate the mechanism and molecular consequences of S-nitrosation of glutathione transferase (GST) P1-1, a ubiquitous homodimeric detoxification enzyme and important target for cancer therapeutics. Transient kinetic techniques, isothermal titration calorimetry and protein engineering were used to develop a minimal mechanism for S-nitrosation of GSTP1-1, the first for any protein. Cys47 of GSTP1-1 is S-nitrosated according to a conformational selection mechanism, with the chemical step limited by a pre-equilibrium between the open and closed conformations of a dynamic helix at the active site. Cys101, in contrast, is Snitrosated in a single step but is subject to negative cooperativity due to steric hindrance at the dimer interface. S-Nitrosation at Cys47 and Cys101 was found to reduce the detoxification activity of GSTP1-1 by 94%. Circular dichroism spectroscopy, acrylamide quenching and amide hydrogen-deuterium exchange mass spectrometry experiments revealed that the loss of activity is due to the introduction of local disorder at the active site. Furthermore, the modification destabilises domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation, and provide general insight into the mechanism of S-nitrosation and its effect protein stability and dynamics.Item The N-subdomain of the thioredoxin fold of glutathione transferase is stabilised by topologically conserved leucine residue(2013-04-30) Khoza, Thandeka NtokozoThe 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.Item The role of a conserved interdomain salt bridge on the structure, function and stability of the Y-GSTs(2013-01-29) Robertson, Gary JayDomain 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.