An increasing number of diseases are associated with protein misfolding, one type of which results in the formation of amyloid fibrils. This research has addressed the hypothesis that all proteins can form amyloid fibrils and investigates what factors protect proteins from forming these macromolecular assemblies. Most analyses of the aggregation propensity of proteins have been limited to the properties of the amino acid sequence, thus fail to consider the roles that higher levels of organisation play in protecting polypeptides from misfolding. The (α/β)8 barrels are a common class of proteins and have never been shown to form amyloid fibrils. This thesis aims to elucidate the characteristics that prevent (α/β)8 barrels from misfolding using Escherichia coli dihydrodipicolinate synthase (DHDPS), a homotetrameric (α/β)8 barrel protein, as a model. It is widely accepted that the precursor of amyloid fibrils is a partially folded species. It is hypothesised in this thesis that the (α/β)8 barrel fold protects the protein against this partial unfolding. This was tested by generating a catalogue of site-directed mutants of DHDPS and screening each of these in a range of pHs and ionic strengths. Amorphous aggregation propensity was assessed by monitoring light scattering at 340 nm and β-sheet specific aggregation was assessed using ThT. Thermal stability was monitored using DSF and CD spectroscopy. Crystallography was used to assess tertiary and quaternary structures and in the cases where crystal structures were not obtained, kinetics was used as a proxy indicator of correct folding and monomeric association. CD spectroscopy was also used to investigate the secondary structure of the DHDPS variants and analytical gel permeation liquid chromatography and AUC were used to confirm quaternary structure.
The stability and aggregation propensity of DHDPS and its variants were assessed under a range of pH and salt conditions. It was established through the characterisation of the wild-type protein that the predominant determinant of stability was, unsurprisingly, pH. This was a trend observed for all the variants described.
Affinity tags were used during the course of this research to facilitate and expedite the production of the protein variants. The introduction of tags containing a polyhistidine motif to DHDPS significantly altered some biophysical properties. Whilst the secondary and quaternary structures were found to be similar to the wild-type enzyme, the catalytic properties were changed. In addition to this, the propensity to aggregate was altered. The full-length polyhistidine tags increased the propensity of DHDPS to form β-sheet-specific aggregate, although this did not result in the formation of amyloid fibrils for most of the variants.
The Zyggregator algorithm was used to predict amino acid substitutions that would increase the aggregation propensity of DHDPS. It identified several amino acids, three of which were chosen for mutation and two of which were expressed in sufficient quantity for further study. DHDPS Q90L and A207V were characterised and the amino acid substitutions did not significantly alter the kinetic parameters of the enzyme. The crystal structure of A207V was solved and confirmed the results of the kinetic analysis demonstrating unchanged tertiary and quaternary structures. Both variants exhibited tertiary and quaternary structures similar to the wild-type enzyme, although Q90L contained more disorder than the wild-type enzyme. The thermal denaturation temperatures and aggregation propensities were also similar to wild-type, although the propensity for both variants to form β-sheet-specific aggregates was reduced. The combinatorial effects of Q90L, A207V and the polyhistidine tags were assessed. This revealed that whilst most biophysical properties were unaffected, the β-sheet-specific aggregation propensity for pET M11 and pET 151/D-TOPO DHDPS Q90L and pET M11 DHDPS A207V, were significantly increased compared to the wild-type enzyme.
The evolutionary forces driving the association of the monomeric and dimeric subunits of DHDPS are undetermined. Investigation of two quaternary structure mutants (DHDPS Y107W and L197Y) revealed that the tetrameric nature of E. coli DHDPS is important for protein activity, stability and the prevention of aggregation. The combinatorial affects of the disrupted quaternary structure and the polyhistidine tags further increased the predisposition of DHDPS to form β-sheet-specific aggregates, resulting in the formation of linear aggregates with some characteristics of amyloid fibrils.
The additive affect of Q90L, Y107W and a polyhistidine tag was assessed and revealed that the major determinant in protein stability and prevention of amorphous and β-sheet specific aggregation is the quaternary structure.
This study demonstrates that the destabilisation of the quaternary structure of DHDPS can result in the formation of amyloid-like aggregates by an (α/β)8 barrel, the first example of an (α/β)8 barrel misfolding in such a way. This finding supports the assertion that all proteins can form amyloid fibrils.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/4427 |
| Date | January 2008 |
| Creators | Walker, Sophie Keziah |
| Publisher | University of Canterbury. School of Biological Sciences |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Sophie Keziah Walker, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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