Dr Robert Barker
Lecturer in Chemistry and Forensic Science
Dr Robert Barker is a Lecturer in Chemistry and Forensic Science.
Luchini, A., Nzulumike, A., Lind, T., Nylander, T., Barker, R., Arleth, L., Mortensen, K. and CÃ¡rdenas, M. (2019). Towards biomimics of cell membranes: Structural effect of phosphatidylinositol triphosphate (PIP3) on a lipid bilayer. Colloids and Surfaces B: Biointerfaces [Online] 173:202-209. Available at: https://doi.org/10.1016/j.colsurfb.2018.09.031.Phosphoinositide (PIP) lipids are anionic phospholipids playing a fundamental role for the
activity of several transmembrane and soluble proteins. Among all, phosphoinositol-3',4',5'-
trisphosphate (PIP3) is a secondary signaling messenger that regulates the function of proteins
involved in cell growth and gene transcription. The present study aims to reveal the structure
of PIP-containing lipid membranes, which so far has been little explored. For this purpose,
supported lipid bilayers (SLBs) containing 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myoinositol-
3',4',5'-trisphosphate (DOPIP3) and 1-palmitoyl-2-oleoyl-sn-glycero-3-
phosphocholine (POPC) were used as mimics of biomembranes. Surface sensitive techniques,
i.e. Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Atomic Force
Microscopy (AFM) and Neutron Reflectometry (NR), provided detailed information on the
formation of SLB and the location of DOPIP3 in the lipid membrane. Specifically, QCMD and
AFM were used to identify the best condition for lipid deposition and to estimate the total
bilayer thickness. On the other hand, NR was used to collect experimental structural data on
the DOPIP3 location and orientation within the lipid membrane.
The two bilayer leaflets showed the same DOPIP3 concentration, thus suggesting the formation
of a symmetric bilayer. The headgroup layer thicknesses of the pure POPC and the mixed POPC/DOPIP3 bilayer
suggest that the DOPIP3-headgroups have a preferred orientation , which is not perpendicular
to the membrane surface, but instead it is close to the surrounding lipid headgroups. These
results support the proposed PIP3 tendency to interact with the other lipid headgroups as PC,
so far exclusively suggested by MD simulations.
Rehal, R., Gaffney, P., Hubbard, A., Barker, R. and Harvey, R. (2019). The pH-dependence of lipid-mediated antimicrobial peptide resistance in a model Staphylococcal plasma membrane: a two-for-one mechanism of epithelial defence circumvention. European Journal of Pharmaceutical Sciences [Online] 128:43-53. Available at: https://doi.org/10.1016/j.ejps.2018.11.017.The mechanisms of membrane defence by lysylphosphatidylglycerol (LPG), were investigated using synthetic biomimetic mono- and bilayer models of methicillin resistant S. aureus ST239 TW, based on its lipid composition in both pH 7.4 (28% LPG) and pH 5.5 (51% LPG) cultures. These models incorporated a stable synthetic analogue of LPG (3adLPG) to facilitate long-duration biophysical studies, which were previously limited by the lability native LPG. Both increased 3adLPG content and full headgroup ionization at pH 5.5, increased bilayer order and dampened overall charge, via the formation of neutral ion pairs with anionic lipids. Ion pair formation in air/liquid interface lipid monolayers elicited a significant condensing effect, which correlated with the inhibition of subphase-injected magainin 2 F5W partitioning. In fluid phase lipid vesicles, increasing the proportion of 3adLPG from 28 to 51 mol% completely inhibited the adoption of the membrane-active ?-helical conformation of the peptide, without the need for full headgroup ionization. Neutron reflectivity measurements performed on biomimetic PG/3adLPG fluid floating bilayers, showed a significant ordering effect of mild acidity on a bilayer containing 30 mol% 3adLPG, whilst peptide binding/partitioning was only fully inhibited in a bilayer with 55 mol% 3adLPG at pH 5.5. These findings are discussed with respect to the roles of LPG in resistance to human epithelial defences in S. aureus and the continued evolution of this opportunistic pathogen’s virulence.
Yuen, F., Watson, M., Barker, R., Grillo, I., Heenan, R., Tunnacliffe, A. and Routh, A. (2019). Preferential adsorption to air-water interfaces: a novel cryoprotective mechanism for LEA proteins. Biochemical Journal [Online] 476:1121-1135. Available at: http://dx.doi.org/10.1042/BCJ20180901.Late embryogenesis abundant (LEA) proteins comprise a diverse family whose
members play a key role in abiotic stress tolerance. As intrinsically disordered proteins, LEA
proteins are highly hydrophilic and inherently stress tolerant. They have been shown to
stabilize multiple client proteins under a variety of stresses, but current hypotheses do not
fully explain how such broad range stabilization is achieved. Here, using neutron reflection
and surface tension experiments, we examine in detail the mechanism by which model LEA
proteins, AavLEA1 and ERD10, protect the enzyme citrate synthase from aggregation during
freeze-thaw. We find that a major contributing factor to citrate synthase aggregation is the
formation of air bubbles during the freeze-thaw process. This greatly increases the air-water
interfacial area, which is known to be detrimental to folded protein stability. Both model LEA
proteins preferentially adsorb to this interface and compete with citrate synthase, thereby
reducing surface induced aggregation. This novel surface activity provides a general
mechanism by which diverse members of the LEA protein family might function to provide
aggregation protection that is not specific to the client protein.
Ballantyne, A., Barker, R., Dalgliesh, R., Ferreira, V., Hillman, A., Palin, E., Sapstead, R., Smith, E., Steinke, N. and Ryder, K. (2018). Electrochemical deposition of silver and copper from a deep eutectic solvent studied using time-resolved neutron reflectivity. Journal of Electroanalytical Chemistry [Online] 819:511-523. Available at: https://doi.org/10.1016/j.jelechem.2018.01.032.Here, we describe new developments in the study of electrodeposition processes with time-resolved dynamic neutron reflectivity (NR) methods to achieve insights into the differences between growth of metal films using a range of electrochemical control functions. We show that the temporal resolution has increased from 1 to 2?h per data set (in our previous studies) to approximately 8?min. We have studied the electrochemical deposition of copper and silver as thin-film metals onto a gold electrode substrate from a deep eutectic solvent using potentiodynamic (PD), potentiostatic (PS) and galvanostatic (GS) electrochemical control functions. In particular, we have utilised novel developments in neutron reflectivity methods to acquire real-time data for the growing metal films. Event mode capture of neutron scattering events, as a function of momentum transfer vector, Q, during electrochemical growth has enabled time-resolved measurement of the neutron reflectivity, R(Q), profiles of the growing metal films. Subsequent fitting and iterative optimisation of the R(Q,t) data reveals the thickness, roughness and relative density (spatially resolved solvent content) of the metal film during growth. These data show that the different electrochemical growth methodologies exhibit different trends in thickness, roughness and solvation. Silver films show an increasing roughness trend with time but these trends are largely independent of growth method. In contrast, the roughness of copper films, grown under similar conditions, shows a strong dependency on growth method with PS methods producing smoothest films. These conclusions are confirmed by ex-situ AFM measurements. The fitted NR data show that the Cu and Ag films contain between 5 and 10% volume fraction solvent. Furthermore, we have explored different NR data fitting methodologies in order to process the large numbers of data sets produced. Gratifyingly, the different methodologies and starting conditions yield a very consistent picture of metal film growth.
Browning, K., Lind, T., Maric, S., Barker, R., CÃ¡rdenas, M. and Malmsten, M. (2018). Effect of bilayer charge on lipoprotein lipid exchange. Colloids and Surfaces B: Biointerfaces [Online]. Available at: https://doi.org/10.1016/j.colsurfb.2018.01.043.Lipoproteins play a key role in the onset and development of atherosclerosis, the formation of
lipid plaques at blood vessel walls. The plaque formation, as well as subsequent calcification,
involves not only endothelial cells but also connective tissue, and is closely related to a wide
range of cardiovascular syndromes, that together constitute the number one cause of death in
the Western World. High (HDL) and low (LDL) density lipoproteins are of particular interest
in relation to atherosclerosis, due to their protective and harmful effects, respectively. In an
effort to elucidate the molecular mechanisms underlying this, and to identify factors
determining lipid deposition and exchange at lipid membranes, we here employ neutron
reflection (NR) and quartz crystal microbalance with dissipation (QCM-D) to study the effect
of membrane charge on lipoprotein deposition and lipid exchange.
Dimyristoylphosphatidylcholine (DMPC) bilayers containing varying amounts of negatively
charged dimyristoylphosphatidylserine (DMPS) were used to vary membrane charge. It was
found that the amount of hydrogenous material deposited from either HDL or LDL to the
bilayer depends only weakly on membrane charge density. In contrast, increasing membrane
charge resulted in an increase in the amount of lipids removed from the supported lipid
bilayer, an effect particularly pronounced for LDL. The latter effects are in line with
previously reported observations on atherosclerotic plaque prone regions of long-term
hyperlipidaemia and type 2 diabetic patients, and may also provide some molecular clues into
the relation between oxidative stress and atherosclerosis.
Hillman, R., Barker, R., Dalgliesh, R., Ferreira, V., Palin, E., Sapstead, R., Smith, E., Steinke, N., Ryder, K. and Ballantyne, A. (2018). Real-time in situ dynamic sub-surface imaging of multi-component electrodeposited films using event mode neutron reflectivity. Faraday Discussions [Online]. Available at: http://dx.doi.org/10.1039/C8FD00084K.Exquisite control of the electrodeposition of metal films and coatings is critical to a number
of high technology and manufacturing industries, delivering functionality as diverse as anticorrosion
and anti-wear coatings, electronic device interconnects and energy storage. The
frequent involvement of more than one metal motivates the capability to control, maintain
and monitor spatial disposition of the component metals, whether as multilayers, alloys or
composites. Here we investigate the deposition, evolution and dissolution of single and twocomponent
metal layers involving Ag, Cu, and Sn on Au substrates immersed in the deep
eutectic solvent (DES) Ethaline. During galvanostatically controlled stripping of the metals
from two-component systems the potential signature in simultaneous thickness
electrochemical potential (STEP) measurements provides identification of the dissolving
metal; coulometric assay of deposition efficiency is an additional outcome. When combined
with quartz crystal microbalance (QCM) frequency responses, the mass change:charge ratio
provides oxidation state data; this is significant for Cu in the high chloride environment
provided by Ethaline. The spatial distribution (solvent penetration and external roughness)
of multiple components in bilayer systems is provided by specular neutron reflectivity (NR).
Significantly, the use of recently established event mode capability shortens the
observational timescale of the NR measurements by an order of magnitude, permitting
dynamic in situ observations on practically useful timescales. Ag,Cu bilayers of both spatial
configurations give identical STEP signatures indicating that, despite the extremely low layer
porosity, thermodynamic constraints (rather than spatial accessibility) dictate reactivity;
thus, surprisingly, Cu dissolves first in both instances. Sn penetrates the Au electrode on the
timescale of deposition; this can be prevented by interposing a layer of either Ag or Cu.
Urbano, L., Clifton, L., Ku, H., Kendall-Troughton, H., Vandera, K., MatarÃ¨seB., Abelha, T., Li, P., Desai, T., Dreiss, C., Barker, R., Green, M., Dailey, L. and Harvey, R. (2018). Influence of Surfactant Structure on Photoluminescent ?-Conjugated Polymer Nanoparticles: Interfacial Properties and Protein Binding. Langmuir [Online] 34:6125-6137. Available at: https://doi.org/10.1021/acs.langmuir.8b00561.?-conjugated polymer nanoparticles (CPNs) are under investigation as photoluminescent agents for diagnostics and bioimaging. To determine whether the choice of surfactant can improve CPN properties and prevent protein adsorption, five non-ionic polyethylene glycol alkyl ether surfactants were used to produce CPNs from three representative ?-conjugated polymers. The surfactant structure did not influence size or yield, which were dependent on the nature of the conjugated polymer. Hydrophobic interaction chromatography, contact angle, quartz crystal microbalance and neutron reflectivity studies were used to assess the affinity of the surfactant to the conjugated polymer surface, and indicated that all surfactants were displaced by the addition of a model serum protein. In summary, CPN preparation methods which rely on surface coating of a conjugated polymer core with amphiphilic surfactants may produce systems with good yields and colloidal stability in vitro, but may be susceptible to significant surface alterations in physiological fluids.
Raasakka, A., Ruskamo, S., Kowal, J., Barker, R., Baumann, A., Martel, A., Tuusa, J., Myllykoski, M., BÃ¼rckJ., Ulrich, A., Stahlberg, H. and Kursula, P. (2017). Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line. Scientific reports [Online] 7:4974. Available at: https://doi.org/10.1038/s41598-017-05364-3.Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between
two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple
sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBPdriven formation of the MDL remains elusive at the biomolecular level. We employed complementary
biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a
single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for
MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions.
Martin, C., Barker, R., Watkins, E., Dubreuil, F., Cranston, E., Heux, L. and Jean, B. (2017). Structural Variations in Hybrid All-Nanoparticle Gibbsite Nanoplatelet/Cellulose Nanocrystal Multilayered Films. Langmuir [Online] 33:7896-7907. Available at: https://doi.org/10.1021/acs.langmuir.7b02352.Cellulose nanocrystals (CNCs) are promising bio-sourced building blocks for the production of high performance materials. In the last ten years, CNCs have been used in conjunction with polymers for the design of multilayered thin films via the layer-by-layer assembly technique. Herein, polymer chains have been replaced with positively charged inorganic gibbsite nanoplatelets (GN) to form hybrid “nanoparticle-only” composite films. A combination of atomic force microscopy and neutron reflectivity experiments was exploited to investigate the growth and structure of the films. Data show that the growth and density of GN/CNC films can be tuned over a wide range during preparation by varying the ionic strength in the CNC suspension and the film drying protocol. Specifically, thin and dense multilayered films or very thick, more porous mixed slabs, as well as intermediate internal structures could be obtained in a predictable manner. The influence of key physicochemical parameters on the multilayer film build up was elucidated and the film architecture was linked to the dominating interaction forces between components. The degree of structural control over these hybrid nanoparticle-only films is much higher than that reported for CNC/polymer films, which offers new properties and potential applications as separation membranes or flame retardant coatings.
Auriemma, F., De Rosa, C., Malafronte, A., Di Girolamo, R., Santillo, C., Gerelli, Y., Fragneto, G., Barker, R., Pavone, V., Maglio, O. and Lombardi, A. (2017). Nano-in-Nano Approach for Enzyme Immobilization Based on Block Copolymers. ACS Applied Materials & Interfaces [Online] 9:29318-29327. Available at: http://dx.doi.org/10.1021/acsami.7b08959.We set up a facile approach for fabrication of supports with tailored nanoporosity for immobilization of enzymes. To this aim block copolymers (BCPs) self-assembly has been used to prepare nanostructured thin films with well-defined architecture containing pores of tailorable size delimited by walls with tailorable degree of hydrophilicity. In particular, we employed a mixture of polystyrene-block-poly(l-lactide) (PS-PLLA) and polystyrene-block-poly(ethylene oxide) (PS-PEO) diblock copolymers to generate thin films with a lamellar morphology consisting of PS lamellar domains alternating with mixed PEO/PLLA blocks lamellar domains. Selective basic hydrolysis of the PLLA blocks generates thin films, patterned with nanometric channels containing hydrophilic PEO chains pending from PS walls. The shape and size of the channels and the degree of hydrophilicity of the pores depend on the relative length of the blocks, the molecular mass of the BCPs, and the composition of the mixture. The strength of our approach is demonstrated in the case of physical adsorption of the hemoprotein peroxidase from horseradish (HRP) using 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with H2O2 as substrate. The large surface area, the tailored pore sizes, and the functionalization with hydrophilic PEO blocks make the designed nanostructured materials suitable supports for the nanoconfinement of HRP biomolecules endowed with high catalytic performance, no mass-transfer limitations, and long-term stability.
Foglia, F., Karan, S., Nania, M., Jiang, Z., Porter, A., Barker, R., Livingston, A. and Cabral, J. (2017). Neutron Reflectivity and Performance of Polyamide Nanofilms for Water Desalination. Advanced Functional Materials [Online] 27:1701738. Available at: https://doi.org/10.1002/adfm.201701738.The structure and hydration of polyamide (PA) membranes are investigated with a combination of neutron and X-ray reflectivity, and their performance is benchmarked in reverse osmosis water desalination. PA membranes are synthesized by the interfacial polymerization of m-phenylenediamine (MPD) and trimesoyl chloride (TMC), varying systematically reaction time, concentration, and stoichiometry, to yield large-area exceptionally planar films of ?10 nm thickness. Reflectivity is employed to precisely determine membrane thickness and roughness, as well as the (TMC/MPD) concentration profile, and response to hydration in the vapor phase. PA film thickness is found to increase linearly with reaction time, albeit with a nonzero intercept, and the composition cross-sectional profile is found to be uniform, at the conditions investigated. Vapor hydration with H2O and D2O from 0 to 100% relative humidity results in considerable swelling (up to 20%), but also yields uniform cross-sectional profiles. The resulting film thickness is found to be predominantly set by the MPD concentration, while TMC regulates water uptake. A favorable correlation is found between higher swelling and water uptake with permeance. The data provide quantitative insight into the film formation mechanisms and correlate reaction conditions, cross-sectional nanostructure, and performance of the PA active layer in RO membranes for desalination.
Raasakka, A., Ruskamo, S., Barker, R., Krokengen, O., Vatne, G., Kristiansen, C., Hallin, E., Skoda, M., Bergmann, U., Wacklin-Knecht, H., Jones, N., Hoffmann, S. and Kursula, P. (2019). Neuropathy-Related Mutations Alter the Membrane Binding Properties of the human 1 Myelin Protein P0 Cytoplasmic Tail. PLOS ONE [Online]. Available at: https://doi.org/10.1371/journal.pone.0216833.Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-4 Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 8 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane.