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

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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.