Student Number : 0213014A -
MSc dissertation -
School of Molecular and Cell Biology -
Faculty of Science / Cytosolic glutathione transferases (GSTs) are obligate stable homo- and heterodimers
comprising two GST subunits. Interactions across the subunit interface play an
important role in stabilising the subunit tertiary structure and maintain the dimeric
structure required for activity. The crystal structure of a rat Mu class GST consisting
of two type one subunits (rGST M1-1) reveals a lock-and-key motif and a mixedcharge
cluster at the subunit interface. Previous investigations revealed the lock-andkey
motif was not essential for dimerisation. It was therefore postulated that the
mixed-charge cluster at the dimer interface is primarily responsible for subunit
association. Statistical analyses of individual rGST M1-1 chains did not predict the
presence of any charge clusters. This suggests that the mixed-charge cluster forms
only upon dimerisation and reinforces the probability that quaternary structure
stabilisation is a major role of the mixed-charge cluster. Arginine 81 (Arg-81), a
structurally conserved residue in the GST family involved in the mixed-charge
cluster, was mutated to alanine. Phenylalanine 56 (Phe-56), the ‘key’ residue in the
lock-and-key motif, was mutated to serine. These changes were engineered to disrupt
the mixed-charge cluster and the lock-and-key motif situated at the dimer interface of
rGST M1-1. Sizing by gel filtration chromatography of the mutant GST identified
that these engineered amino acids resulted in a stable monomeric protein
(F56S/R81A rGST M1). The F56S/R81A rGST M1 displayed almost no catalytic
activity, suggesting perturbations of the active site or substrate binding sites.
Structural investigations of the monomer by far- and near-UV circular dichroism
revealed a similar secondary structural content to the wild-type. However, the
tryptophan fluorescence properties suggested the tryptophans were situated in more
hydrophilic environments than in the wild-type. ANS binding studies indicated a
large increase in the accessible hydrophobic surface area of the monomer. Ureainduced
equilibrium unfolding of F56S/R81A rGST M1 follows a cooperative twostate
unfolding model. The unfolding data indicates decreased conformational
stability and a large increase in the solvent exposed surface area of the monomer. In
conclusion, the mixed-charge cluster at the dimer interface of rGST M1-1 is essential for monomeric association, which subsequently contributes to catalytic activity of the
dimer and the stabilities of individual rGST M1-1 subunits.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/1656 |
| Date | 14 November 2006 |
| Creators | Walters, John Clive |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 2210111 bytes, application/pdf, application/pdf |
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