Student Number : 9306227A -
PhD thesis -
School of Molecular and Cell Biology -
Faculty of Science / It is well documented that N-capping motifs are stabilising local motifs for
-helices. N-capping motifs have been identified within hGST A1-1 at the
N-terminal ends of -helix 9 and helix 6. The conservational role of these
two motifs in protein stability, folding and function was investigated. -Helix
9 is a unique structural feature to class Alpha GSTs that is important for its
catalytic functioning. This amphipathic helix is highly dynamic, where upon
ligand binding at the active-site, the delocalised C-terminal region becomes
immobilised to form a structured helix forming a “lid” over the active-site.
The specific role of the Asp N-cap motif toward the stability and dynamics of
helix 9 was determined by substituting the Asp-209 for a Gly. ANS binding
and urea-induced activity studies showed that by removing the N-cap motif
of helix 9 in hGST A1-1, the helix 9 is destabilised rendering a less hydrophobic
binding site compared to that in the wild-type. The helical content
of the peptide, corresponding to helix 9 in the C-terminal region of hGST
A1-1 (208 -222), decreased significantly upon the removal of the N-cap motif.
The explanation for the conservation of the Asp N-cap residue can be found
in its stabilising role of the C-terminal region of class Alpha GSTs. This stabilising
role was however less apparent in context of the protein compared to
that in the peptide. Majority of the atomic contacts owing to the stability
of helix 9 appear to be governed by non-local tertiary interactions rather
than local interactions, such as the N-cap motif. These tertiary interactions
are likely to include short and long range contacts between residues on the
surface of the protein that are already known to contribute towards the stability
of the C-terminal region. In this study, the ligand displacement-studies
and the molecular docking results strongly suggest that 8-aniline-1-napthalene sulfonate binds at the H-site in hGST A1-1.
The N-capping motif of helix 6 identified in class Alpha GSTs is located
within the core of domain 2. This motif is a common feature found amongst
almost all GST-like proteins and is thought to be the folding nucleation site
(Stenberg et al. J. Biol. Chem. 275 (2000), 10421-10428). The N-cap (Ser-
154) and N3 (Asp-157) residues were each substituted with an Ala in hGST
A1-1 to investigate the role of this motif in the folding of hGST A1-1. Both
substitutions resulted in thermal sensitive mutants compared to that of the
wild-type. The N3 substitution (D157A) was however too disruptive, where
the yields of this mutant were insufficient for any further studies to be carried
out. For the N-cap mutant (S154A), the unfolding kinetic studies revealed a
significantly destabilised core in domain 2 compared to that of the wild-type.
The kinetic folding studies monitored by fluorescence spectroscopy, revealed
that the N-cap motif contributes to the efficient folding and dimerisation of
the subunits, and to a far lesser extent towards the final tight packing and
reorganisation of tertiary interactions in hGST A1-1. Since no changes in the
burst-phase of S154A was evident compared to that of the wild-type, it seems
unlikely that this motif is a folding nucleation site in hGST A1-1. These results
do not exclude the possibility that this motif contributes to the rapid formation
secondary structure during the burst-phase of folding. Due to the highly
conserved region surrounding helix 6 , the role of this motif contributing
to the stability of hGST A1-1 could be a general feature for GSTs and GSTlike
proteins. In this study, further insight into the mechanism of folding for
hGST A1-1 was gained. The hydrophobic core packing surrounding helix 6
occurred as a late folding event, that is during the final packing and reorganisation
of tertiary interactions of the protein. The N-cap motif is an important
structural feature for the fast folding of domain 2. This N-cap motif is a unique
structural feature important for the efficient folding of the monomers, which
is exclusive to its role in stabilising helix 6 in hGST A1-1.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/1804 |
| Date | 16 November 2006 |
| Creators | Little, Tessa |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 17757356 bytes, application/pdf, application/pdf |
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