Inducible chomosomal AmpC β-lactamase (AmpC) is present in many Gram-negative opportunistic human pathogens. Expressed in response to β-lactam antibiotics, AmpC is an enzyme that can deactivate an extended spectrum of β-lactam antibiotics and thereby promote bacterial survival. Inducible chromosomal ampC is associated with ampR, a gene that encodes a LysR-type transcriptional regulator that suppresses ampC expression in the absence of β-lactam exposure. Together, ampR and ampC form a divergent operon with overlapping promoters to which the AmpR protein binds and regulates the transcription of both genes. AmpR induces ampC expression by interacting with 1,6-anhydro-N-acetylmuramyl peptide, an intermediate of peptidoglycan recycling that is generated by a glycoside hydrolase encoded by nagZ. Given the role of NagZ and AmpR in the AmpC induction pathway, the structure and function of these proteins were investigated to understand the molecular basis for how they participate in AmpC production. The crystal structure of NagZ from Vibrio cholerae was determined in complex with the glycoside hydrolase inhibitor PUGNAc (O-(2-Deoxy-2-N-2-ethylbutyryl-D-glucopyranosylidene)amino-N-phenylcarbamate) to 1.8 Å resolution. Since PUGNAc also inhibits functionally related human enzymes, the structure of the enzyme was also determined in complex with the NagZ selective PUGNAc derivatives N-butyryl-PUGNAc (2.3 Å resolution) and N-valeryl-PUGNAc (2.4 Å resolution). These structural studies revealed the molecular basis for how 2-N-acyl derivatives of PUGNAc selectively inhibit the bacterial enzyme NagZ. The effector binding domain of AmpR from Citrobacter Spp. was determined to 1.83 Å resolution and lead to the identification of a putative effector molecule binding site. In vivo functional analysis of site directed mutants of AmpR containing amino acid substitutions at the base of the putative binding pocket verified its role in AmpR function. A protocol was subsequently devised to purify milligram quantities of soluble full-length AmpR. Biochemical and biophysical analysis, including non-denaturing mass spectrometry and small angle X-ray scattering, revealed that the purified full-length protein is tetrameric and specifically binds ampC promoter DNA. In summary, this research provides the basis for the development of small-molecules that could specifically block the activity of these proteins to suppress AmpC β-lactamase production during β-lactam therapy.

EmrE from Escherichia coli is a member of the small multidrug resistance protein family that oligomerizes to export hydrophobic cationic antimicrobials by utilizing the proton motive force. We studied the helix-helix interactions of the four transmembrane (TM) segments of EmrE to determine how this protein might assemble into its oligomeric forms. Using a combination of biochemical and biophysical techniques, we assessed the oligomerization propensities of Lys-tagged EmrE TM peptides in membrane-mimetic environments. Our results established that each of the TMs of EmrE display detergent-sensitive self-association, but in particular, TM2 had the greatest dimerization capability that was not completely abolished even by scrambling the native sequence. Mutations made to TM2 in full-length EmrE also revealed that efflux-defective mutations are located on one face of the helix. These findings reveal another potential oligomerization site for EmrE - and perhaps SMRs - and may provide a target for development of novel efflux-inhibitors.

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.

The unfolding and refolding of a number of oligomeric enzymes have been studied. These were: fumarase from pig heart, the NAD+ -dependent isocitrate dehydrogenase from yeast, the citrate synthases from pig hean, Acinetobacter anitratum and Thermoplasma acidophilum and the chaperone protein GroEL from Escherichia coli. In each case the unfolding by guanidinium chloride (GdnHCI) was monitored by enzyme activity (to detect possible perturbations at the active site), protein fluorescence (to detect changes in tertiary structure) and far U.v. circular dichroism (to detect changes in protein secondary structure). In general the losses in secondary and tertiary structure were found to run broadly in parallel, whereas the enzyme activity was lost at much lower concentrations of GdnHCl. This sensitivity to mild, denaturing conditions may reflect the greater flexibility of the active site compared with the molecule as a whole. Interestingly) the bacterial citrate synthases were activated in the presence of low concentrations of GdnHCl. Following denaturation) refolding was initiated by lowering the concentration of GdnHCI by dilution or dialysis. Only the dimeric citrate synthases (from pig heart and Thermoplasma acidophilum) could be reactivated to a moderate extent using the dilution procedure; less than 5% reactivation was observed for the other enzymes. In the cases of fumarase, NAD+ -dependent isocitrate dehydrogenase and the dimeric citrate synthases the degrees of reactivation following dialysis were significantly greater (approximately 50-75% of the native enzymes) than those obtained following the dilution procedure. Factors such as protein concentration and the inclusion of dithiothreitol in the dialysis or dilution buffer were found to influence significantly the extent of reactivation. The greater yield of reactivation of unfolded protein using the dialysis procedure probably reflects the ability of the enzyme to make the correct structural adjustments between intermediates when the concentration of GdnHCI is lowered gradually. In the case of Thermoplasma acidophilum the recovery of citrate synthase activity was much greater at 20 ·C than at 55 ·C (the optimal temperature for growth of this organism). This has implications for the folding process in vivo under the extreme growth conditions of thermophiles and possibly other extremophiles. The hexameric citrate synthase from Acinetobacter anitratum and the tetradecameric chaperonin, GroEL could not be reactivated following denaturation. Far u.v. circular dichroism measurements on GroEL indicated that the native secondary structure of this protein was regained to a large extent. In vivo a number of the proteins studied (fumarase and citrate synthase from pig hean and yeast NAD+ -dependent isocitrate dehydrogenase)are translocated into mitochondria as precursors in a non-native state prior to processing, folding and assembly. The lack of complete refolding of the proteins studied in this work points to the existence of specialised mechanisms in vivo to promote efficient folding. Chaperone proteins have been implicated in the assistance of protein folding in vivo. Intriguingly. the studies on the inefficient refolding of the chaperonin GroEL support the proposal that this protein may fold in vivo by way of a "self chaperoning" mechanism.

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