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
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/26456 |
| Date | January 1998 |
| Creators | Wallace, Louise Annette |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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