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.

Spatial-Controlled Coating of Pro-Angiogenic Proteins on 3D Porous Hydrogels Guides Endothelial Cell Behavior.

In tissue engineering, the composition and the structural arrangement of molecular components within the extracellular matrix (ECM) determine the physical and biochemical features of a scaffold, which consequently modulate cell behavior and function. The microenvironment of the ECM plays a fundamental role in regulating angiogenesis. Numerous strategies in tissue engineering have attempted to control the spatial cues mimicking in vivo angiogenesis by using simplified systems.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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--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.

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.

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.

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).

Determining the amphipol distribution within membrane-protein fibre samples using small-angle neutron scattering.

Detergent micelles can solubilize membrane proteins, but there is always a need for a pool of free detergent at the critical micellar concentration to maintain the micelle-monomer equilibrium. Amphipol polymeric surfactants (APols) have been developed to replace conventional detergents in membrane-protein studies, but the role of free amphipol is unclear. It has previously been shown that the removal of free APol causes monodisperse outer membrane protein F (OmpF) to form long filaments.

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.

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.

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.

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 outer membrane protein A (OmpA) to create protein-AuNP conjugates.

Liquid crystalline bacterial outer membranes are critical for antibiotic susceptibility.

The outer membrane (OM) of Gram-negative bacteria is a robust, impermeable, asymmetric bilayer of outer lipopolysaccharides (LPSs) and inner phospholipids containing selective pore proteins which confer on it the properties of a molecular sieve. This structure severely limits the variety of antibiotic molecules effective against Gram-negative pathogens and, as antibiotic resistance has increased, so has the need to solve the OM permeability problem. Polymyxin B (PmB) represents those rare antibiotics which act directly on the OM and which offer a distinct starting point for new antibiotic development.

The Two-State Prehensile Tail of the Antibacterial Toxin Colicin N.

Intrinsically disordered regions within proteins are critical elements in many biomolecular interactions and signaling pathways. Antibacterial toxins of the colicin family, which could provide new antibiotic functions against resistant bacteria, contain disordered N-terminal translocation domains (T-domains) that are essential for receptor binding and the penetration of the Escherichia coli outer membrane. Here we investigate the conformational behavior of the T-domain of colicin N (ColN-T) to understand why such domains are widespread in toxins that target Gram-negative bacteria.

Thermal stability and rheological properties of the 'non-stick' Caf1 biomaterial.

The ability to culture cells in three-dimensions has many applications, from drug discovery to wound healing. 3D cell culture methods often require appropriate scaffolds that mimic the cellular environments of different tissue types. The choice of material from which these scaffolds are made is of paramount importance, as its properties will define the manner in which cells interact with the scaffold.