Browsing by Author "Lakey, JH"
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- ItemAsymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic(The Royal Society, 2013-10-16) Clifton, LA; Skoda, MWA; Daulton, E; Hughes, AV; Le Brun, AP; Lakey, JH; Holt, SAThe 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. © 2013, The Royal Society.
- ItemHigh coverage fluid-phase floating lipid bilayers supported by ω-thiolipid self-assembled monolayers(The Royal Society Publishing, 2014-09-06) Hughes, AV; Holt, SA; Daulton, E; Soliakov, A; Charlton, TR; Roser, SJ; Lakey, JHLarge 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%. © 2014 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited.
- ItemIn situ study of the impact of acidic and neutral deposition pH on alkane phosphate film formation and stability on TiO2(Royal Society Chemistry, 2013-02-28) Holt, SA; Le Brun, AP; Nelson, A; Lakey, JHTiO2 has been used as a model system for the surface of medical implant devices based upon titanium alloys. Self Assembled Monolayers (SAM) can be applied via a specific phosphate interaction to modify the surface properties of the TiO2 substrate. Here it is demonstrated via in situ Quartz Crystal Microbalance and Neutron Reflectometry experiments that the deposition of dodecyl phosphate onto TiO2 proceeds more rapidly at pH 4.5 than at pH 7.0. Conversely, the film stability was enhanced for films deposited at pH 7.0, demonstrating that while the initial association with the surface is driven by electrostatics it does not determine the SAM density. While the adsorbed amount appeared to be relatively constant after a few minutes incubation time it was found that washing with buffer removed about 50% of the adsorbed material after these short incubation times. With incubation time of the order of hours the proportion of the film washed off the surface decreased demonstrating that the specific phosphate-TiO2 interaction was a slow process. The slower initial surface interaction at pH 7.0 therefore allowed greater re-arrangement of the dodecyl phosphate resulting in more complete and robust monolayers than at pH 4.5. This was demonstrated by washing the film with buffer of increasing pH of up to 9.5. For SAM longevity on titanium alloys it is clear that slow deposition at pH 7 produces more robust films than rapid deposition at lower pH values.© 2013, Royal Society of Chemistry.
- ItemSelf-assembly of protein monolayers engineered for improved monoclonal immunoglobulin G binding(MDPI Publishing, 2011-08-01) Le Brun, AP; Shah, DSH; Athey, D; Holt, SA; Lakey, JHBacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG) to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated. © The Authors - This is an open access article distributed under the Creative Commons Attribution License
- ItemStructural characterization of a model gram-negative bacterial surface using lipopolysaccharides from rough strains of escherichia coli(American Chemical Society, 2013-06-01) Le Brun, AP; Clifton, LA; Halbert, CE; Lin, B; Meron, M; Holden, PJ; Lakey, JH; Holt, SALipopolysaccharides (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. © 2013, American Chemical Society.
- ItemThe structural orientation of antibody layers bound to engineered biosensor surfaces(Elsevier, 2011-04-01) Le Brun, AP; Holt, SA; Shah, DSH; Majkrzak, CF; Lakey, JHThis paper describes a membrane protein array that binds immunoglobulin G at its constant regions whilst leaving the variable regions free to bind antigen. The scaffold of the array is the transmembrane domain of outer membrane protein A (tOmpA) from Escherichia colt engineered to assemble as an oriented monolayer on gold surfaces via a single cysteine residue. Other protein domains can be fused to the N and C termini of the scaffold. In this study we use circularly permuted ctOmpA fused to two Z domains of Staphylococcus aureus protein A (ZZctOmpA) to create the immunoglobulin G-binding array. The solution structure of the engineered proteins was assessed by circular dichroism spectroscopy. Assembly of the array, attachment of antibodies and antigen binding were measured using surface plasmon resonance and neutron reflection. Compared to mouse IgG2, polyclonal IgG from rabbit bound very strongly to ZZctOmpA and the dissociation of the immunoglobulin was slow enough to allow neutron reflection studies of the assembled layer with antigen. Using both magnetic and isotopic contrasts a complete layer by layer model was defined which revealed that the 223 A high layer contains antibodies in an upright orientation. (C) 2011 Elsevier Ltd. All rights reserved.
- ItemStructurally characterising biomolecular recognition at the solid-liquid interface: an example using immunoglobulin G bound to membrane protein arrays on gold(Neutron Scattering Society of America, 2010-06-29) Le Brun, AP; Holt, SA; Lakey, JH; Shah, D; Majkrzak, CFProteins can be immobilised on surfaces to make arrays with potential uses in tissue engineering, proteomics and point of use diagnostic devices. Outer membrane proteins (OMP) from Escherichia coli have a beta-barrel structure, making ideal protein engineering scaffolds for building arrays. The proteins can be immobilised onto flat gold surfaces by introducing a cysteine residue into their periplasmic turns. The thiol group of the cysteine will form a strong gold-thiolate bond immobilising the OMP to the surface in a specific and correct orientation. The membrane layer is completed by the immobilisatin of a lipid with a thiol head group to the gold surface. Here we use the transmembrane section of the monomeric protein OmpA (TmOmpA). The Z domain of Staphylococcus aureau protein A has been engineered into the N - terminal of a circularly permuted TmOmpA to create the protein ZZctOmpA. The Z domain can bind immunoglobulin G (IgG) at its constant region leaving the variable regions free to bind antigen. The structure of this model protein array was probed using magnetic contrast neutron reflection (MCNR). MCNR uses polarised neutrons which reflect differently from a magnetic metal layer according to their two spin states (spin up and spin down). The magnetic layer deposited under the gold surface and provides additional scattering length density contrast to very complex layer systems without needing to make any changes to the biological layer. The collection of two complimentary but independent data sets allows for more accurate modelling of the resulting high resolution data. The data presented will show the assembly steps for creating the array detailing the surfaces used, the ZZctOmpA and lipid components as well as information on the orientation of bound antibody and antigen.