Anton P. Le Brun

Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg2+ and Ca2+) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.

An ion-channel-containing model membrane: structural determination by magnetic contrast neutron reflectometry

To many biophysical characterisation techniques, biological membranes appear as two-dimensional structures with details of their third dimension hidden within a 5 nm profile. Probing this structure requires methods able to discriminate multiple layers a few Ångströms thick. Given sufficient resolution, neutron methods can provide the required discrimination between different biochemical components, especially when selective deuteration is employed. We have used state-of-the-art neutron reflection methods, with resolution enhancement via magnetic contrast variation to study an oriented model membrane system. The model is based on the Escherichia coli outer membrane protein OmpF fixed to a gold surface via an engineered cysteine residue. Below the gold is buried a magnetic metal layer which, in a magnetic field, displays different scattering strengths to spin-up and spin-down neutrons. This provides two independent datasets from a single biological sample. Simultaneous fitting of the two datasets significantly refines the resulting model. A β-mercaptoethanol (βME) passivating surface, applied to the gold to prevent protein denaturation, is resolved for the first time as an 8.2 ± 0.6 Å thick layer, demonstrating the improved resolution and confirming that this layer remains after OmpF assembly. The thiolipid monolayer (35.3 ± 0.5 Å), assembled around the OmpF is determined and finally a fluid DMPC layer is added (total lipid thickness 58.7 ± 0.9 Å). The dimensions of trimeric OmpF in isolation (53.6 ± 2.5 Å), after assembly of lipid monolayer (57.5 ± 0.9 Å) and lipid bilayer (58.7 ± 0.9 Å), are precisely determined and show little variation.

Low resolution structure and dynamics of a Colicin-Receptor complex determined by neutron scattering

Background: In order to kill E. coli, colicins need to cross the bacterial outer membrane. Results: Neutron scattering data show colicin N at the protein-lipid interface of its receptor OmpF. Conclusion: Colicins can unfold and penetrate membranes via the outside wall of their receptors. Significance: The protein-lipid interface may be the route that colicins take into the cell. Proteins that translocate across cell membranes need to overcome a significant hydrophobic barrier. This is usually accomplished via specialized protein complexes, which provide a polar transmembrane pore. Exceptions to this include bacterial toxins, which insert into and cross the lipid bilayer itself. We are studying the mechanism by which large antibacterial proteins enter Escherichia coli via specific outer membrane proteins. Here we describe the use of neutron scattering to investigate the interaction of colicin N with its outer membrane receptor protein OmpF. The positions of lipids, colicin N, and OmpF were separately resolved within complex structures by the use of selective deuteration. Neutron reflectivity showed, in real time, that OmpF mediates the insertion of colicin N into lipid monolayers. This data were complemented by Brewster Angle Microscopy images, which showed a lateral association of OmpF in the presence of colicin N. Small angle neutron scattering experiments then defined the three-dimensional structure of the colicin N-OmpF complex. This revealed that colicin N unfolds and binds to the OmpF-lipid interface. The implications of this unfolding step for colicin translocation across membranes are discussed.

Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic

The Gram-negative bacterial outer membrane (OM) is a complex and highly asymmetric biological barrier but the small size of bacteria has hindered advances in in vivo examination of membrane dynamics. Thus, model OMs, amenable to physical study, are important sources of data. Here, we present data from asymmetric bilayers which emulate the OM and are formed by a simple two-step approach. The bilayers were deposited on an SiO2 surface by Langmuir–Blodgett deposition of phosphatidylcholine as the inner leaflet and, via Langmuir–Schaefer deposition, an outer leaflet of either Lipid A or Escherichia coli rough lipopolysaccharides (LPS). The membranes were examined using neutron reflectometry (NR) to examine the coverage and mixing of lipids between the bilayer leaflets. NR data showed that in all cases, the initial deposition asymmetry was mostly maintained for more than 16 h. This stability enabled the sizes of the headgroups and bilayer roughness of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and Lipid A, Rc-LPS and Ra-LPS to be clearly resolved. The results show that rough LPS can be manipulated like phospholipids and used to fabricate advanced asymmetric bacterial membrane models using well-known bilayer deposition techniques. Such models will enable OM dynamics and interactions to be studied under in vivo-like conditions.

Structural Characterization of a Model Gram-Negative Bacterial Surface Using Lipopolysaccharides from Rough Strains of Escherichia coli

Lipopolysaccharides (LPS) make up approximately 75% of the Gram-negative bacterial outer membrane (OM) surface, but because of the complexity of the molecule, there are very few model OMs that include LPS. The LPS molecule consists of lipid A, which anchors the LPS within the OM, a core polysaccharide region, and a variable O-antigen polysaccharide chain. In this work we used RcLPS (consisting of lipid A plus the first seven sugars of the core polysaccharide) from a rough strain of Escherichia coli to form stable monolayers of LPS at the air–liquid interface. The vertical structure RcLPS monolayers were characterized using neutron and X-ray reflectometry, while the lateral structure was investigated using grazing incidence X-ray diffraction and Brewster angle microscopy. It was found that RcLPS monolayers at surface pressures of 20 mN m–1 and above are resolved as hydrocarbon tails, an inner headgroup, and an outer headgroup of polysaccharide with increasing solvation from tails to outer headgroups. The lateral organization of the hydrocarbon lipid chains displays an oblique hexagonal unit cell at all surface pressures, with only the chain tilt angle changing with surface pressure. This is in contrast to lipid A, which displays hexagonal or, above 20 mN m–1, distorted hexagonal packing. This work provides the first complete structural analysis of a realistic E. coli OM surface model.

Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection

Protein arrays are used in a wide range of applications. The array described here binds IgG antibodies, produced in rabbit, to gold surfaces via a scaffold protein. The scaffold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron Reflection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deuterium exchange in the biological layer. Polarised Neutron Reflection allows for the modelling of many and complex layers with good fits. The total thickness of the biological layer immobilised on the gold surface is found to be 187 Å and the layer can thus far be separated into its lipid, protein and solvent parts.

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH2) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.

Investigating the interactions of the 18kDa translocator protein and its ligand PK11195 in planar lipid bilayers.

The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug-membrane interactions may contribute to the apparent affinity of TSPO ligands.

Neutron Reflectometry Studies Define Prion Protein N-terminal Peptide Membrane Binding

The prion protein (PrP), widely recognized to misfold into the causative agent of the transmissible spongiform encephalopathies, has previously been shown to bind to lipid membranes with binding influenced by both membrane composition and pH. Aside from the misfolding events associated with prion pathogenesis, PrP can undergo various posttranslational modifications, including internal cleavage events. Alpha- and beta-cleavage of PrP produces two N-terminal fragments, N1 and N2, respectively, which interact specifically with negatively charged phospholipids at low pH. Our previous work probing N1 and N2 interactions with supported bilayers raised the possibility that the peptides could insert deeply with minimal disruption. In the current study we aimed to refine the binding parameters of these peptides with lipid bilayers. To this end, we used neutron reflectometry to define the structural details of this interaction in combination with quartz crystal microbalance interrogation. Neutron reflectometry confirmed that peptides equivalent to N1 and N2 insert into the interstitial space between the phospholipid headgroups but do not penetrate into the acyl tail region. In accord with our previous studies, interaction was stronger for the N1 fragment than for the N2, with more peptide bound per lipid. Neutron reflectometry analysis also detected lengthening of the lipid acyl tails, with a concurrent decrease in lipid area. This was most evident for the N1 peptide and suggests an induction of increased lipid order in the absence of phase transition. These observations stand in clear contrast to the findings of analogous studies of Ab and ?-synuclein and thereby support the possibility of a functional role for such N-terminal fragment-membrane interactions.

Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology

This study explains the importance of the phosphate moiety and H3O+ in controlling the ionic flux through phospholipid membranes. We show that despite an increase in the H3O+ concentration when the pH is decreased, the level of ionic conduction through phospholipid bilayers is reduced. By modifying the lipid structure, we show the dominant determinant of membrane conduction is the hydrogen bonding between the phosphate oxygens on adjacent phospholipids. The modulation of conduction with pH is proposed to arise from the varying H3O+ concentrations altering the molecular area per lipid and modifying the geometry of conductive defects already present in the membrane. Given the geometrical constraints that control the lipid phase structure of membranes, these area changes predict that organisms evolving in environments with different pHs will select for different phospholipid chain lengths, as is found for organisms near highly acidic volcanic vents (short chains) or in highly alkaline salt lakes (long chains). The stabilizing effect of the hydration shells around phosphate groups also accounts for the prevalence of phospholipids across biology. Measurement of ion permeation through lipid bilayers was made tractable using sparsely tethered bilayer lipid membranes with swept frequency electrical impedance spectroscopy and ramped dc amperometry. Additional evidence of the effect of a change in pH on lipid packing density is obtained from neutron reflectometry data of tethered membranes containing perdeuterated lipids.