Unfolding mechanism of human glutathione transferase M1a-1a

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dc.contributor.authorWiid, Kimberly Jade
dc.date.accessioned2018-10-09T08:08:25Z
dc.date.available2018-10-09T08:08:25Z
dc.date.issued2018
dc.descriptionA 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 2018en_ZA
dc.description.abstractProteins 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.en_ZA
dc.description.librarianMT 2018en_ZA
dc.format.extentOnline resource (xiv,133 leaves)
dc.identifier.citationWiid, Kimberley Jade (2018) Unfolding mechanism of human glutathione transferase M1a-1a, University of the Witwatersrand, Johannesburg, <http://hdl.handle.net/10539/25749>
dc.identifier.urihttps://hdl.handle.net/10539/25749
dc.language.isoenen_ZA
dc.subject.lcshGlutathione transferase
dc.titleUnfolding mechanism of human glutathione transferase M1a-1aen_ZA
dc.typeThesisen_ZA
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