Structural analysis of recombinant Caf1 and PA in alhydrogel-adsorbed and free forms

In many cases conventional vaccines, based on live attenuated micro-organisms or killed whole cell preparations, are now being superseded by a new generation of subunit vaccines, which contain one or more highly purified recombinant protein antigens that offer a substantial improvement in safety, protection and manufacturing costs. Since purified proteins are often poor immunogens they need to be formulated with an adjuvant, a substance that non-specifically augments the immune response, to form the vaccine. Despite intensive research into novel adjuvants, still the most common clinical adjuvants are aluminium based hydrated gels. These aluminium gels readily adsorb protein antigens from aqueous solutions, forming insoluble protein-adjuvant micro-complexes. Yet despite their extensive use in vaccine formulations, very little is known of how these adjuvants affect the structure and stability of proteins. Thus there is urgent need for new biophysical analysis methods to characterise this final drug product. The work presented in this thesis aims to address this issue. Two model subunit vaccines were evaluated; namely recombinant capsular antigen fraction 1 (rCaf1) and recombinant protective antigen (rPA) used in new recombinant subunit vaccines against plague and anthrax, respectively. The interaction of rPA and rCaf1 with the Alhydrogel adjuvant was investigated by limited proteolysis, fluoresce nce, chemical modification, differential scanning and isothermal titration micro-calorimetry, electron microscopy and image processing techniques. The results reveal that protein structure is largely retained while cooperativity of folding is reduced. These small perturbations in protein structure can be modulated by addition of phosphate, in agreement with previous data from potency studies. The methods used here reveal new insights into how proteins interact with adjuvants and extend the capability for the characterisation of subunit vaccines. Furthermore, a combination of negative stain transmission electron microscopy and linear dichroism determined that the quaternary structure of Caf1 is a long flexible polymer up to 1.5 um long. It was found that donor strand exchange at the cohesive ends of shorter Caf1 polymers produces circular molecules. Site directed mutagenesis was used to study the unusual circular dichroism spectrum of Caf1. The contribution of aromatic residues in the far- and near ultra-violet spectra was assessed by circular dichroism and the protein fold was examined by Raman optical activity. This revealed unexpected contributions of aromatic residues to both the far- and near-ultraviolet circular dichroism spectra.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A

Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P's structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second