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.

Engineered self-assembling monolayers for label free detection of influenza nucleoprotein.

Integrating nanotechnology into useable devices requires a combination of bottom up and top down methodology. Often the techniques to measure and control these different components are entirely different, so methods that can analyse the nanoscale component in situ are of increasing importance. Here we describe a strategy that employs a self-assembling monolayer of engineered protein chimeras to display an array of oriented antibodies (IgG) on a microelectronic device for the label free detection of influenza nucleoprotein. The structural and functional properties of the bio-interface were characterised by a range of physical techniques including surface plasmon resonance, quartz-crystal microbalance and neutron reflectometry. This combination of methods reveals a 13.5 nm thick engineered-monolayer that (i) self-assembles on gold surfaces, (ii) captures IgG with high affinity in a defined orientation and (iii) specifically recognises the influenza A nucleoprotein. Furthermore we also show that this non-covalent self-assembled structure can render the dissociation of bound IgG irreversible by chemical crosslinking in situ without affecting the IgG function. The methods can thus describe in detail the transition from soluble engineered molecules with nanometre dimensions to an array that demonstrates the principles of a working influenza sensor.

Recombinant anthrax protective antigen: Observation of aggregation phenomena by TEM reveals specific effects of sterols.

Negatively stained transmission electron microscope images are presented that depict the aggregation of recombinant anthrax protective antigen (rPA83 monomer and the PA63 prepore oligomer) under varying in vitro biochemical conditions. Heat treatment (50°C) of rPA83 produced clumped fibrils, but following heating the PA63 prepore formed disordered aggregates. Freeze-thaw treatment of the PA63 prepore generated linear flexuous aggregates of the heptameric oligomers. Aqueous suspensions of cholesterol microcrystals were shown to bind small rPA83 aggregates at the edges of the planar bilayers. With PA63 a more discrete binding of the prepores to the crystalline cholesterol bilayer edges occurs. Sodium deoxycholate (NaDOC) treatment of rPA83 produced quasi helical fibrillar aggregate, similar but not identical to that produced by heat treatment. Remarkably, NaDOC treatment of the PA63 prepores induced transformation into pores, with a characteristic extended ß-barrel. The PA63 pores aggregated as dimers, that aggregated further as angular chains and closed structures in higher NaDOC concentrations. The significance of the sterol interaction is discussed in relation to its likely importance for PA action in vivo.

Reversible non-stick behaviour of a bacterial protein polymer provides a tuneable molecular mimic for cell and tissue engineering.

Yersina pestis, the bubonic plague bacterium, is coated with a polymeric protein hydrogel for protection from host defences. The protein, which is robust and non-stick, resembles structures found in many eukaryotic extracellular-matrix proteins. Cells grown on the natural polymer cannot adhere and grow poorly; however, when cell-adhesion motifs are inserted into the protein, the cells proliferate.

High coverage fluid-phase floating lipid bilayers supported by ω-thiolipid self-assembled monolayers.

Large area lipid bilayers, on solid surfaces, are useful in physical studies of biological membranes. It is advantageous to minimize the interactions of these bilayers with the substrate and this can be achieved via the formation of a floating supported bilayer (FSB) upon either a surface bound phospholipid bilayer or monolayer. The FSB's independence is enabled by the continuous water layer (greater than 15 Å) that remains between the two. However, previous FSBs have had limited stability and low density. Here, we demonstrate by surface plasmon resonance and neutron reflectivity, the formation of a complete self-assembled monolayer (SAM) on gold surfaces by a synthetic phosphatidylcholine bearing a thiol group at the end of one fatty acyl chain. Furthermore, a very dense FSB (more than 96%) of saturated phosphatidylcholine can be formed on this SAM by sequential Langmuir-Blodgett and Langmuir-Schaefer procedures. Neutron reflectivity used both isotopic and magnetic contrast to enhance the accuracy of the data fits. This system offers the means to study transmembrane proteins, membrane potential effects (using the gold as an electrode) and even model bacterial outer membranes. Using unsaturated phosphatidylcholines, which have previously failed to form stable FSBs, we achieved a coverage of 73%.

In vitro fibrillogenesis of collagen type I in varying ionic and pH conditions.

Collagen is the most abundant protein in the human body, and has primary roles in the formation of tendons, cartilage and bone, it provides mechanical strength to skin and indeed almost every organ and muscle is associated with a layer of collagen. It is thus a key component of the extracellular matrix. Here we have studied the in vitro fibrillogenesis of acetic acid-soluble collagen type I under physiological and varying non-physiological conditions by TEM from negatively stained specimens. At pH 2.5 the collagen heterotrimer remains soluble at increasing buffer concentrations and in the presence of increasing NaCl concentrations. At pH 4.5 molecular aggregates form at low NaCl concentrations, but at higher NaCl concentrations fibrils with a diffuse ~11 nm banding are formed. At pH 7.0, initial molecular aggregates form at low NaCl concentrations that progressively form characteristic ~67 nm D-banded collagen fibrils at intermediate NaCl concentrations that cluster to form thicker multi-fibril D-banded fibres in higher NaCl concentrations. By contrast, increasing concentrations of sodium phosphate at pH 7.0 leads to the formation of flexuous, unbanded fibrils at higher concentrations from the initial, loosely aggregated form of collagen. At higher pHs, the formation of D-banded fibrils is less efficient, particularly at pH 9.0. Thus at neutral pH, the presence of chloride anions, rather than sodium cations, is required for the production of D-banded collagen fibrils; higher than normal physiological chloride concentrations in the form of NaCl or Tris·HCl at neutral pH, but not phosphate buffer, can also lead to the efficient in vitro formation of D-banded collagen fibrils.

Increasing the potency of an alhydrogel-formulated anthrax vaccine by minimizing antigen-adjuvant interactions.

Aluminum salts are the most widely used vaccine adjuvants, and phosphate is known to modulate antigen-adjuvant interactions. Here we report an unexpected role for phosphate buffer in an anthrax vaccine (SparVax) containing recombinant protective antigen (rPA) and aluminum oxyhydroxide (AlOH) adjuvant (Alhydrogel). Phosphate ions bind to AlOH to produce an aluminum phosphate surface with a reduced rPA adsorption coefficient and binding capacity. However, these effects continued to increase as the free phosphate concentration increased, and the binding of rPA changed from endothermic to exothermic. Crucially, phosphate restored the thermostability of bound rPA so that it resembled the soluble form, even though it remained tightly bound to the surface. Batches of vaccine with either 0.25 mM (subsaturated) or 4 mM (saturated) phosphate were tested in a disease model at batch release, which showed that the latter was significantly more potent. Both formulations retained their potency for 3 years. The strongest aluminum adjuvant effects are thus likely to be via weakly attached or easily released native-state antigen proteins.

Anthrax sub-unit vaccine: the structural consequences of binding rPA83 to Alhydrogel®.

An anthrax sub-unit vaccine, comprising recombinant Protective Antigen (rPA83) and aluminium hydroxide adjuvant (Alhydrogel®) is currently being developed. Here, a series of biophysical techniques have been applied to free and adjuvant bound antigen. Limited proteolysis and fluorescence identified no changes in rPA83 tertiary structure following binding to Alhydrogel and the bound rPA83 retained two structurally important calcium ions. For adsorbed rPA83, differential scanning calorimetry revealed a small reduction in unfolding temperature but a large decrease in unfolding enthalpy whilst urea unfolding demonstrated unchanged stability but a loss of co-operativity. Overall, these results demonstrate that interactions between rPA83 and Alhydrogel have a minimal effect on the folded protein structure and suggest that antigen destabilisation is not a primary mechanism of Alhydrogel adjuvancy. This study also shows that informative structural characterisation is possible for adjuvant bound sub-unit vaccines.

Alhydrogel® adjuvant, ultrasonic dispersion and protein binding: a TEM and analytical study.

Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (∼ 10-100 nm), that are significantly smaller than those present the untreated Alhydrogel (∼ 2-12 µm). However, even prolonged ultrasonication did not produce a homogenous suspension of single aluminium hydroxide micro-fibres. The TEM images of unstained and negatively stained air-dried Alhydrogel are very similar to those obtained by cryo-electron microscopy. Visualization of protein on the surface of the finely dispersed Alhydrogel by TEM is facilitated by prior ultrasonication. Several examples are given, including some of medical relevance, using proteins of widely ranging molecular mass and oligomerization state. Even with the smaller mass proteins, their presence on the Alhydrogel surface can be readily defined by TEM. It has been found that low quantities of protein tend to cross-link and aggregate the small Alhydogel clusters, in a more pronounced manner than high protein concentrations. This indicates that complete saturation of the available Alhydrogel surface with protein may be achieved, with minimal cross-linkage, and future exploitation of this treatment of Alhydrogel is likely to be of immediate value for more efficient vaccine production.

The structure of Yersinia pestis Caf1 polymer in free and adjuvant bound states.

Caf1 of the plague bacterium, Yersinia pestis is a polymeric virulence factor and vaccine component, formed from monomers by a donor strand exchange (DSE) mechanism. Here, EM images of Caf1 reveal flexible polymers up to 1.5 microm long (4MDa). The bead-like structures along the polymer are 5.8 + or - 1 nm long and correspond to single Caf1 proteins. Short polymers often form circles, presumably by DSE. We also provide the first images of proteins bound to alhydrogel adjuvant. Caf1, hemocyanin and anthrax PA are all resolved clearly and Caf1 exhibits adjuvant bound stretches with long intervening loops draped from the edges.