Fully Aqueous Self-Assembly of a Gold-Nanoparticle-Based Pathogen Sensor.

Surface plasmon resonance (SPR) is a very sensitive measure of biomolecular interactions but is generally too expensive for routine analysis of clinical samples. Here we demonstrate the simplified formation of virus-detecting gold nanoparticle (AuNP) assemblies on glass using only aqueous buffers at room temperature. The AuNP assembled on silanized glass and displayed a distinctive absorbance peak due to the localized SPR (LSPR) response of the AuNPs. Next, assembly of a protein engineering scaffold was followed using LSPR and a sensitive neutron reflectometry approach, which measured the formation and structure of the biological layer on the spherical AuNP. Finally, the assembly and function of an artificial flu sensor layer consisting of an in vitro-selected single-chain antibody (scFv)-membrane protein fusion was followed using the LSPR response of AuNPs within glass capillaries. In vitro selection avoids the need for separate animal-derived antibodies and allows for the rapid production of low-cost sensor proteins. This work demonstrates a simple approach to forming oriented arrays of protein sensors on nanostructured surfaces that uses (i) an easily assembled AuNP silane layer, (ii) self-assembly of an oriented protein layer on AuNPs, and (iii) simple highly specific artificial receptor proteins.

Exploiting Meltable Protein Hydrogels to Encapsulate and Culture Cells in 3D.

There is a growing realization that 3D cell culture better mimics complex in vivo environments than 2D, lessening aberrant cellular behaviors and ultimately improving the outcomes of experiments. Chemically crosslinked hydrogels which imitate natural extracellular matrix (ECM) are proven cell culture platforms, but the encapsulation of cells within these hydrogel networks requires bioorthogonal crosslinking chemistries which can be cytotoxic, synthetically demanding, and costly. Capsular antigen fragment 1 (Caf1) is a bacterial, polymeric, fimbrial protein which can be genetically engineered to imitate ECM. Furthermore, it can, reversibly, thermally interconvert between its polymeric and monomeric forms even when chemically crosslinked within a hydrogel network. It is demonstrated that this meltable feature of Caf1 hydrogels can be utilized to encapsulate neonatal human dermal fibroblasts at a range of cell densities (2 × 105 -2 × 106  cells mL-1 of hydrogel) avoiding issues with chemical cytotoxicity. These hydrogels supported cell 3D culture for up to 21 d, successfully inducing cellular functions such as proliferation and migration. This work is significant because it further highlights the potential of simple, robust, Caf1-based hydrogels as a cell culture platform.

An oomycete NLP cytolysin forms transient small pores in lipid membranes.

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.

Unraveling the molecular determinants of the anti-phagocytic protein cloak of plague bacteria.

The pathogenic bacterium Yersina pestis is protected from macrophage engulfment by a capsule like antigen, F1, formed of long polymers of the monomer protein, Caf1. However, despite the importance of this pathogen, the mechanism of protection was not understood. Here we demonstrate how F1 protects the bacteria from phagocytosis. First, we show that Escherichia coli expressing F1 showed greatly reduced adherence to macrophages. Furthermore, the few cells that did adhere remained on the macrophage surface and were not engulfed. We then inserted, by mutation, an "RGDS" integrin binding motif into Caf1. This did not change the number of cells adhering to macrophages but increased the fraction of adherent cells that were engulfed. Therefore, F1 protects in two separate ways, reducing cell adhesion, possibly by acting as a polymer brush, and hiding innate receptor binding sites needed for engulfment. F1 is very robust and we show that E. coli expressing weakened mutant polymers are engulfed like the RGDS mutant. This suggests that innate attachment sites on the native cell surface are exposed if F1 is weakened. Single-molecule force spectroscopy (SMFS) experiments revealed that wild-type F1 displays a very high mechanical stability of 400 pN. However, the mechanical resistance of the destabilised mutants, that were fully engulfed, was only 20% weaker. By only marginally exceeding the mechanical force applied to the Caf1 polymer during phagocytosis it may be that the exceptional tensile strength evolved to resist the forces applied at this stage of engulfment.

Investigation of mutations (L41F, F17M, N57E, Y99F_Y134W) effects on the TolAIII-UnaG fluorescence protein's unconjugated bilirubin (UC-BR) binding ability and thermal stability properties.

The UnaG protein is a ligand (unconjugated bilirubin) dependent fluorescence protein isolated from Unagi freshwater eel larvae and expressed as fusion in heterologous expression systems. Bilirubin is a tetrapyrrole molecule mainly produced from heme catabolism by the destruction of erythrocytes in the body. Bilirubin can cause kernicterus, a serious condition associated with permanent neurological damage in neonates with the passage of brain tissue. Different methods have been developed for plasma bilirubin analysis and quantification. The use of UnaG fluorescence protein triggered by bilirubin has become a new approach in bilirubin studies. In this study, we aimed to investigate the biophysical characterization of ligand interactions with the proteins obtained as a result of mutations (UnaGY99F_Y134W, UnaGN57E, UnaGL41F, and UnaGF17M) on the amino acid sequence of TolAIII-UnaG protein. After the purity levels of the expressed proteins have been analyzed by SDS-PAGE, secondary structures and thermal melting temperatures of the proteins have been examined by circular dichroism spectroscopy. Then determination of excitation and emission points by fluorescence spectroscopy, titration studies have been performed with bilirubin, and dissociation constant was calculated. According to the biophysical characterization studies, UnaGL41F has the highest affinity and stability among the mutants.

Exploiting neutron scattering contrast variation in biological membrane studies.

Biological membranes composed of lipids and proteins are central for the function of all cells and individual components, such as proteins, that are readily studied by a range of structural approaches, including x-ray crystallography and cryo-electron microscopy. However, the study of complex molecular mixtures within the biological membrane structure and dynamics requires techniques that can study nanometer thick molecular bilayers in an aqueous environment at ambient temperature and pressure. Neutron methods, including scattering and spectroscopic approaches, are useful since they can measure structure and dynamics while also being able to penetrate sample holders and cuvettes. The structural approaches, such as small angle neutron scattering and neutron reflectometry, detect scattering caused by the difference in neutron contrast (scattering length) between different molecular components such as lipids or proteins. Usually, the bigger the contrast, the clearer the structural data, and this review uses examples from our research to illustrate how contrast can be increased to allow the structures of individual membrane components to be resolved. Most often this relies upon the use of deuterium in place of hydrogen, but we also discuss the use of magnetic contrast and other elements with useful scattering length values.

Probing the oligomeric re-assembling of bacterial fimbriae in vitro: a small-angle X-ray scattering and analytical ultracentrifugation study.

Capsular antigen fragment 1 (Caf1) is an oligomeric protein consisting of 15 kDa monomeric subunits that are non-covalently linked through exceptionally strong and kinetically inert interactions into a linear polymer chain. It has been shown that after its thermal depolymerisation into unfolded monomeric subunits, Caf1 is able to efficiently repolymerise in vitro to reform its polymeric structure. However, little is known about the nature of the repolymerisation process. An improved understanding of this process will lead to the development of methods to better control the lengths of the repolymerised species, and ultimately, to better design of the properties of Caf1-based materials. Here we utilize small-angle X-ray scattering to estimate the size of Caf1 polymers during the first 24 h of the re-polymerisation process. Analytical ultracentrifugation measurements were also used to investigate the process post-24 h, where the rate of repolymerisation becomes considerably slower. Results show that in vitro polymerisation proceeds in a linear manner with no evidence observed for the formation of a lateral polymer network or uncontrolled aggregates. The rate of Caf1 in vitro repolymerisation was found to be concentration-dependent. Importantly, the rate of polymer growth was found to be relatively fast over the first few hours, before continuing at a dramatically slower rate. This observation is not consistent with the previously proposed step-growth mechanism of in vitro polymerisation of Caf1, where a linear increase in polymer length would be expected with time. We speculate how our observations may support the idea that the polymerisation process may be occurring at the ends of the chains with monomers adding sequentially. Our findings will contribute towards the development of new biomaterials for 3D cell culture and bio-printing.

Hydrogels of engineered bacterial fimbriae can finely tune 2D human cell culture.

Demand continues to grow for biomimetic materials able to create well-defined environments for modulating the behaviour of living cells in culture. Here, we describe hydrogels based upon the polymeric bacterial fimbriae protein capsular antigen fragment 1 (Caf1) that presents tunable biological properties for enhanced tissue cell culture applications. We demonstrate how Caf1 hydrogels can regulate cellular functions such as spreading, proliferation and matrix deposition of human dermal fibroblast cells (hDFBs). Caf1 hydrogels exploring a range of mechanical properties were prepared using copolymers featuring controlled compositions of inert wild-type Caf1 subunits and a mutant subunit displaying the RGDS peptide motif. The hydrogels showed excellent cytocompatibility with hDFBs and the ability to modulate both cell morphology and matrix deposition. Interestingly, Caf1 hydrogels displaying faster stress relaxation were demonstrated to show the highest metabolic activities of growing cells in comparison with other Caf1 hydrogel formulations. The stiffest Caf1 hydrogel impacted cellular morphology, inducing alignment of the cells. This work is significant as it clearly indicates that Caf1-based hydrogels offer tuneable biochemical and mechanical substrates conditions suitable for cell culture applications.

Studying the surfaces of bacteria using neutron scattering: finding new openings for antibiotics.

The use of neutrons as a scattering probe to investigate biological membranes has steadily grown in the past three decades, shedding light on the structure and behaviour of this ubiquitous and fundamental biological barrier. Meanwhile, the rise of antibiotic resistance has catalysed a renewed interest in understanding the mechanisms underlying the dynamics of antibiotics interaction with the bacterial cell envelope. It is widely recognised that the key reason behind the remarkable success of Gram-negative pathogens in developing antibiotic resistance lies in the effectiveness of their outer membrane (OM) in defending the cell from antibacterial compounds. Critical to its function, the highly asymmetric lipid distribution between the inner and outer bilayer leaflets of the OM, adds an extra level of complexity to the study of this crucial defence barrier. Here we review the opportunities offered by neutron scattering techniques, in particular reflectometry, to provide structural information on the interactions of antimicrobials with in vitro models of the OM. The differential sensitivity of neutrons towards hydrogen and deuterium makes them a unique probe to study the structure and behaviour of asymmetric membranes. Molecular-level understanding of the interactions between antimicrobials and the Gram-negative OM provides valuable insights that can aid drug development and broaden our knowledge of this critically important biological barrier.

Outer membrane protein size and LPS O-antigen define protective antibody targeting to the Salmonella surface.

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.

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.

Self-Assembled Fluid Phase Floating Membranes with Tunable Water Interlayers.

We present a reliable method for the fabrication of fluid phase, unsaturated lipid bilayers by self-assembly onto charged Self-Assembled Monolayer (SAM) surfaces with tunable membrane to surface aqueous interlayers. Initially, the formation of water interlayers between membranes and charged surfaces was characterized using a comparative series of bilayers deposited onto charged, self-assembled monolayers by sequential layer deposition. Using neutron reflectometry, a bilayer to surface water interlayer of ∼8 Å was found between the zwitterionic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membrane and an anionic carboxyl terminated grafted SAM with the formation of this layer attributed to bilayer repulsion by hydration water on the SAM surface. Furthermore, we found we could significantly reduce the technical complexity of sample fabrication through self-assembly of planar membranes onto the SAM coated surfaces. Vesicle fusion onto carboxyl-terminated monolayers yielded high coverage (>95%) bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) which floated on a 7-11 Å solution interlayer between the membrane and the surface. The surface to membrane distance was then tuned via the addition of 200 mM NaCl to the bulk solution immersing a POPC floating membrane, which caused the water interlayer to swell reversibly to ∼33 Å. This study reveals that biomimetic membrane models can be readily self-assembled from solution onto functionalized surfaces without the use of polymer supports or tethers. Once assembled, surface to membrane distance can be tailored to the experimental requirements using physiological concentrations of electrolytes. These planar bilayers only very weakly interact with the substrate and are ideally suited for use as biomimetic models for accurate in vitro biochemical and biophysical studies, as well as for technological applications, such as biosensors.

Engineered mosaic protein polymers; a simple route to multifunctional biomaterials.

BACKGROUND: Engineered living materials (ELMs) are an exciting new frontier, where living organisms create highly functional materials. In particular, protein ELMs have the advantage that their properties can be manipulated via simple molecular biology. Caf1 is a protein ELM that is especially attractive as a biomaterial on account of its unique combination of properties: bacterial cells export it as a massive, modular, non-covalent polymer which is resistant to thermal and chemical degradation and free from animal material. Moreover, it is biologically inert, allowing the bioactivity of each 15 kDa monomeric Caf1 subunit to be specifically engineered by mutagenesis and co-expressed in the same Escherichia coli cell to produce a mixture of bioactive Caf1 subunits. RESULTS: Here, we show by gel electrophoresis and transmission electron microscopy that the bacterial cells combine these subunits into true mosaic heteropolymers. By combining two separate bioactive motifs in a single mosaic polymer we demonstrate its utility by stimulating the early stages of bone formation by primary human bone marrow stromal cells. Finally, using a synthetic biology approach, we engineer a mosaic of three components, demonstrating that Caf1 complexity depends solely upon the variety of monomers available. CONCLUSIONS: These results demonstrate the utility of engineered Caf1 mosaic polymers as a simple route towards the production of multifunctional biomaterials that will be useful in biomedical applications such as 3D tissue culture and wound healing. Additionally, in situ Caf1 producing cells could create complex bacterial communities for biotechnology.

Structural Investigations of Protein-Lipid Complexes Using Neutron Scattering.

Neutron scattering has significant benefits for examining the structure of protein-lipid complexes. Cold (slow) neutrons are nondamaging and predominantly interact with the atomic nucleus, meaning that neutron beams can penetrate deeply into samples, which allows for flexibility in the design of samples studied. Most importantly, there is a strong difference in neutron scattering length (i.e., scattering power) between protium ([Formula: see text], 99.98% natural abundance) and deuterium ([Formula: see text] or D, 0.015%). Through the mixing of H2O and D2O in the samples and in some cases the deuterium labeling of the biomolecules, components within a complex can be hidden or enhanced in the scattering signal. This enables both the overall structure and the relative distribution of components within a complex to be resolved. Lipid-protein complexes are most commonly studied using neutron reflectometry (NR) and small angle neutron scattering (SANS). In this review the methodologies to produce and examine a variety of model biological membrane systems using SANS and NR are detailed. These systems include supported lipid bilayers derived from vesicle dispersions or Langmuir-Blodgett deposition, tethered bilayer systems, membrane protein-lipid complexes and polymer wrapped lipid nanodiscs. The three key stages of any SANS/NR study on model membrane systems-sample preparation, data collection, and analysis-are described together with some background on the techniques themselves.

Induction of the immunoprotective coat of Yersinia pestis at body temperature is mediated by the Caf1R transcription factor.

BACKGROUND: Thermal regulation of gene expression occurs in many microorganisms, and is mediated via several typical mechanisms. Yersinia pestis is the causative agent of the plague and spreads by zoonotic transfer from fleas to mammalian blood with a concomitant rapid temperature change, from ambient to 37 °C, which induces the expression of capsular antigen (Caf1) that inhibits phagocytosis. Caf1 is formed into long polymeric fimbriae by a periplasmic chaperone (Caf1M) and outer membrane usher (Caf1A). All three are encoded on an operon regulated by an AraC-type transcription factor Caf1R. The aim of this study was to determine the role of Caf1R in the thermal control of caf1 operon gene expression. RESULTS: PCR analysis of cDNA demonstrated that the genes of the operon are transcribed as a single polycistronic mRNA. Bioinformatic analysis, supported by deletion mutagenesis, then revealed a region containing the promoter of this polycistronic transcript that was critical for Caf1 protein expression. Caf1R was found to be essential for Caf1 protein production. Finally, RT-PCR analysis and western blot experiments showed large, Caf1R dependent increases in caf1 operon transcripts upon a shift in temperature from 25 °C to 35 °C. CONCLUSIONS: The results show that thermal control of Caf1 polymer production is established at the transcriptional level, in a Caf1R dependent manner. This gives us new insights into how a virulent pathogen evades destruction by the immune system by detecting and responding to environmental changes.

Human Miro Proteins Act as NTP Hydrolases through a Novel, Non-Canonical Catalytic Mechanism.

Mitochondria are highly dynamic organelles that play a central role in multiple cellular processes, including energy metabolism, calcium homeostasis and apoptosis. Miro proteins (Miros) are "atypical" Ras superfamily GTPases that display unique domain architecture and subcellular localisation regulating mitochondrial transport, autophagy and calcium sensing. Here, we present systematic catalytic domain characterisation and structural analyses of human Miros. Despite lacking key conserved catalytic residues (equivalent to Ras Y32, T35, G60 and Q61), the Miro N-terminal GTPase domains display GTPase activity. Surprisingly, the C-terminal GTPase domains previously assumed to be "relic" domains were also active. Moreover, Miros show substrate promiscuity and function as NTPases. Molecular docking and structural analyses of Miros revealed unusual features in the Switch I and II regions, facilitating promiscuous substrate binding and suggesting the usage of a novel hydrolytic mechanism. The key substitution in position 13 in the Miros leads us to suggest the existence of an "internal arginine finger", allowing an unusual catalytic mechanism that does not require GAP protein. Together, the data presented here indicate novel catalytic functions of human Miro atypical GTPases through altered catalytic mechanisms.

Distribution of mechanical stress in the Escherichia coli cell envelope.

The cell envelope in Gram-negative bacteria comprises two distinct membranes with a cell wall between them. There has been a growing interest in understanding the mechanical adaptation of this cell envelope to the osmotic pressure (or turgor pressure), which is generated by the difference in the concentration of solutes between the cytoplasm and the external environment. However, it remains unexplored how the cell wall, the inner membrane (IM), and the outer membrane (OM) effectively protect the cell from this pressure by bearing the resulting surface tension, thus preventing the formation of inner membrane bulges, abnormal cell morphology, spheroplasts and cell lysis. In this study, we have used molecular dynamics (MD) simulations combined with experiments to resolve how and to what extent models of the IM, OM, and cell wall respond to changes in surface tension. We calculated the area compressibility modulus of all three components in simulations from tension-area isotherms. Experiments on monolayers mimicking individual leaflets of the IM and OM were also used to characterize their compressibility. While the membranes become softer as they expand, the cell wall exhibits significant strain stiffening at moderate to high tensions. We integrate these results into a model of the cell envelope in which the OM and cell wall share the tension at low turgor pressure (0.3 atm) but the tension in the cell wall dominates at high values (>1 atm).

Tuneable hydrogels of Caf1 protein fibers.

Capsular antigen fraction 1 (Caf1) is a robust polymeric protein forming a protective layer around the bacterium Yersinia pestis. Occurring as ≈1 µm polymeric fibers, it shares its immunoglobulin-like fold with the majority of mammalian extracellular proteins such as fibronectin and this structural similarity suggests that this unusual polymer could form useful mimics of the extracellular matrix. Driven by the pressing need for reliable animal-free 3D cell culture environments, we showed previously that recombinant Caf1 produced in Escherichia coli can be engineered to include bioactive peptides, which influence cell behavior. Here, we demonstrate that through chemical crosslinking with a small palette of PEG-based crosslinkers, Caf1-based hydrogels can be prepared displaying a wide range of mechanical and morphological properties that were studied by rheology, compressive testing, SDS-PAGE and scanning electron microscopy. By varying the Caf1 protein concentration, viscoelasticity and stiffness (~11-2300 Pa) are reproducibly tunable to match natural and commercial 3D gels. Hydrogel porosity and swelling ratios were found to be defined by crosslinker architecture and concentration. Finally the hydrogels, which are 95-99% water, were shown to retain the high stability of the native Caf1 protein in a range of aqueous conditions, including extended immersion in cell culture media. The unusual Caf1 polymer thus offers the possibility of presenting bioactive protein subunits in a precisely tuneable hydrogel for use in cell culture and drug delivery applications.

Modular Protein Engineering Approach to the Functionalization of Gold Nanoparticles for Use in Clinical Diagnostics.

Functional protein-gold nanoparticle (AuNP) conjugates have a wide variety of applications including biosensing and drug delivery. Correct protein orientation, which is important to maintain functionality on the nanoparticle surface, can be difficult to achieve in practice, and dedicated protein scaffolds have been used on planar gold surfaces to drive the self-assembly of oriented protein arrays. Here we use the transmembrane domain of Escherichia coli outer membrane protein A (OmpATM) to create protein-AuNP conjugates. The addition of a single cysteine residue into a periplasmic loop, to create cysOmpATM, drives oriented assembly and increased equilibrium binding. As the protein surface concentration increases, the sulfur-gold bond in cysOmpATM creates a more densely populated AuNP surface than the poorly organized wtOmpATM layer. The functionalization of AuNP improved both their stability and homogeneity. This was further exploited using multidomain protein chimeras, based on cysOmpATM, which were shown to form ordered protein arrays with their functional domains displayed away from the AuNP surface. A fusion with protein G was shown to specifically bind antibodies via their Fc region. Next, an in vitro selected single chain antibody (scFv)-cysOmpATM fusion protein, bound to AuNP, detected influenza A nucleoprotein, a widely used antigen in diagnostic assays. Finally, using the same scFv-cysOmpATM-AuNP conjugates, a prototype lateral flow assay for influenza demonstrated the utility of fully recombinant self-assembling sensor layers. By simultaneously removing the need for both animal antibodies and a separate immobilization procedure, this technology could greatly simplify the development of a range of in vitro diagnostics.