Effect of Phospholipid Composition and Phase on Nanodisc Films at the Solid–Liquid Interface as Studied by Neutron Reflectivity

Nanodiscs are disc-like self-assembled structures formed by phospholipids and amphipatic proteins. The proteins wrap like a belt around the hydrophobic part of the lipids, basically producing nanometer-sized patches of lipid bilayers. The bilayer in the nanodisc constitutes a native-like model of the cell membrane and can act as a nanometer-sized container for functional single membrane proteins. In this study, we present a general nanodisc-based system, intended for structural and functional studies of membrane proteins. In this method, the nanodiscs are aligned at a solid surface, providing the ability to determine the average structure of the film along an axis perpendicular to the interface as measured by neutron reflectivity. The nanodisc film was optimized in terms of nanodisc coverage, reduced film roughness, and stability for time-consuming studies. This was achieved by a systematic variation of the lipid phase, charge, and length of lipid tails. Herein, we show that, although all studied nanodiscs align with their lipid bilayer parallel to the interface, gel-phase DMPC nanodiscs form the most suitable film for future membrane protein studies since they yield a dense irreversibly adsorbed film with low roughness and high stability over time. This may be explained by the appropriate matching between the thickness of the hydrophobic lipid core of gel phase DMPC and the height of the belt protein. Moreover, once formed the gel-phase DMPC nanodiscs film can be heated up to melt the lipid bilayer, thus providing a more biologically friendly environment for membrane proteins

Effect of Functionalized Gold Nanoparticles on Floating Lipid Bilayers

The development of novel nano-engineered materials poses important questions regarding the impact of these new materials on living systems. Possible adverse effects must be assessed in order to prevent risks for health and the environment. On the other hand, a thorough understanding of their interaction with biological systems might also result in the creation of novel biomedical applications. We present a study on the interaction of model lipid membranes with gold nanoparticles (AuNP) of different surface modifications. Neutron reflectometry experiments on zwitterionic lipid double bilayers were performed in the presence of AuNP functionalized with cationic and anionic head groups. Structural information was obtained that provided insight into the fate of the AuNPs with regard to the integrity of the model cell membranes. The AuNPs functionalized with cationic head groups penetrate into the hydrophobic moiety of the lipid bilayers and cause membrane disruption at an increased concentration. In contrast, the AuNPs functionalized with anionic head groups do not enter but seem to impede the destruction of the lipid bilayer at an alkaline pH. The information obtained might influence the strategy for a better nanoparticle risk assessment based on a surface charge evaluation and contribute to nano-safety considerations during their design

Nanodisc films for membrane protein studies by neutron reflection : Effect of the protein scaffold choice

Nanodisc films are a promising approach to study the equilibrium conformation of membrane bound proteins in native-like environment. Here we compare nanodisc formation for NADPH-dependent cytochrome P450 oxidoreductase (POR) using two different scaffold proteins, MSP1D1 and MSP1E3D1. Despite the increased stability of POR loaded MSP1E3D1 based nanodiscs in comparison to MSP1D1 based nanodiscs, neutron reflection at the silicon–solution interface showed that POR loaded MSP1E3D1 based nanodisc films had poor surface coverage. This was the case, even when incubation was carried out under conditions that typically gave high coverage for empty nanodiscs. The low surface coverage affects the embedded POR coverage in the nanodisc film and limits the structural information that can be extracted from membrane bound proteins within them. Thus, nanodisc reconstitution on the smaller scaffold proteins is necessary for structural studies of membrane bound proteins in nanodisc films

Additional file 1: of Immunoregulatory soluble CTLA-4 modifies effector T-cell responses in systemic lupus erythematosus

Full analysis of individual patient and donor peptide response datasets used to generate the summary data presented in Fig. 3. (DOCX 495 kb

Modulation of Dipalmitoylphosphatidylcholine Monolayers by Dimethyl Sulfoxide

The action of the penetration-enhancing agent, dimethyl sulfoxide (DMSO), on phospholipid monolayers was investigated at the air–water interface using a combination of experimental techniques and molecular dynamics simulations. Brewster angle microscopy revealed that DPPC monolayers remained laterally homogeneous at subphase concentrations up to a mole fraction of 0.1 DMSO. Neutron reflectometry of the monolayers in combination with isotopic substitution enabled the determination of solvent profiles as a function of distance perpendicular to the interface for the different DMSO subphase concentrations. These experimental results were compared to those obtained from molecular dynamic (MD) simulations of the corresponding monolayer systems. There was excellent agreement found between the MD-derived reflectivity curves and the measured data for all of the H/D contrast variations investigated. The MD provide a detailed description of the distribution of water and DMSO molecules around the phosphatidylcholine headgroup, and how this distribution changes with increasing DMSO concentrations. Significantly, the measurements and simulations that are reported here support the hypothesis that DMSO acts by dehydrating the phosphatidylcholine headgroup, and as such provide the first direct evidence that it does so primarily by displacing water molecules bound to the choline group

The Relationship between Charge Density and Polyelectrolyte Brush Profile Using Simultaneous Neutron Reflectivity and In Situ Attenuated Total Internal Reflection FTIR

We report on a novel experimental study of a pH-responsive polyelectrolyte brush at the silicon/D₂O interface. A poly­[2-(diethylamino)­ethyl methacrylate] brush was grown on a large silicon crystal which acted as both a substrate for a neutron reflectivity solid/liquid experiment but also as an FTIR-ATR spectroscopy crystal. This arrangement has allowed for both neutron reflectivities and FTIR spectroscopic information to be measured in parallel. The chosen polybase brush shows strong IR bands which can be assigned to the N–D⁺ stretch, D₂O, and a carbonyl group. From such FTIR data, we are able to closely monitor the degree of protonation along the polymer chain as well as revealing information concerning the D₂O concentration at the interface. The neutron reflectivity data allows us to determine the physical brush profile normal to the solid/liquid interface along with the corresponding degree of hydration. This combined approach makes it possible to quantify the charge on a polymer brush alongside the morphology adopted by the polymer chains

Is Osmotic Pressure Relevant in the Mechanical Confinement of a Polymer Brush?

The structures of polymer brushes under confinement were measured using a combination of neutron reflectivity and a surface force type apparatus. The samples were either poly­(ethylene oxide), PEO, used to investigate the effect of the grafting density or poly­(acrylic acid), PAA, used to determine the effect of charge on the structure of a polymer brushes under confinement. Without confinement both PEO and PAA brushes are found to be highly swollen with water, >50% v/v, with the expected parabolic brush structure. Compression of the PEO brushes with as little as 0.5 bar of confinement is found experimentally to reduce the brush to a polymer block of uniform density that is significantly dehydrated, <12% v/v. Further subsequent increases in the confinement pressure only marginally decrease the hydration and thickness of the polymer block. The grafting density of the brush does not significantly influence this behavior. PAA polymer brushes with little (pH 3) or an intermediate level of charging (pH 5.5) are also found to be compressed into a single uniform density polymer block with a confinement of 5 bar. However, with a high level of charge (pH 9) the brush structure is believed to be partially retained due to the repulsion between the internal charges. These experimental results are compared against a theoretical model based on numerical self-consistent field (nSCF) theory as well as to osmotic, SFA, and AFM data. While the nSCF model correctly predicts the observed transition from a brush to a block profile, experimentally it occurs at a pressure 2 orders of magnitude lower than the simulations would suggest. The results acquired through simulation are consistent with available osmotic pressure data; however, SFA and AFM measurements are consistent with the neutron reflection experimental data presented here. This significant disagreement between the two data sets indicates that in a confined system the effective osmotic counter pressure to an applied mechanical pressure is much less than the osmotic pressure expected from the local polymer concentration

Nanoscale control of interfacial processes for latent fingerprint enhancement

Latent fingerprints on metal surfaces may be visualized by exploiting the insulating characteristics of the fingerprint deposit as a “mask” to direct electrodeposition of an electroactive polymer to the bare metal between the fingerprint ridges. This approach is complementary to most latent fingerprint enhancement methods, which involve physical or chemical interaction with the fingerprint residue. It has the advantages of sensitivity (a nanoscale residue can block electron transfer) and, using a suitable polymer, optimization of visual contrast. This study extends the concept in two significant respects. First, it explores the feasibility of combining observation based on optical absorption with observation based on fluorescence. Second, it extends the methodology to materials (here, polypyrrole) that may undergo post-deposition substitution chemistry, here binding of a fluorophore whose size and geometry preclude direct polymerization of the functionalised monomer. The scenario involves a lateral spatial image (the whole fingerprint, first level detail) at the centimetre scale, with identification features (minutiae, second level detail) at the 100–200 μm scale and finer features (third level detail) at the 10–50 μm scale. However, the strategy used requires vertical spatial control of the (electro)chemistry at the 10–100 nm scale. We show that this can be accomplished by polymerization of pyrrole functionalised with a good leaving group, ester-bound FMOC, which can be hydrolysed and eluted from the deposited polymer to generate solvent “voids”. Overall the “void” volume and the resulting effect on polymer dynamics facilitate entry and amide bonding of Dylight 649 NHS ester, a large fluorophore. FTIR spectra demonstrate the spatially integrated compositional changes. Both the hydrolysis and fluorophore functionalization were followed using neutron reflectivity to determine vertical spatial composition variations, which control image development in the lateral direction

Interactions of PAMAM Dendrimers with Negatively Charged Model Biomembranes

We have investigated the interactions between cationic poly­(amidoamine) (PAMAM) dendrimers of generation 4 (G4), a potential gene transfection vector, with net-anionic model biomembranes composed of different ratios of zwitterionic phosphocholine (PC) and anionic phospho-l-serine (PS) phospholipids. Two types of model membranes were used: solid-supported bilayers, prepared with lipids carrying palmitoyl-oleoyl (PO) and diphytanoyl (DPh) acyl chains, and free-standing bilayers, formed at the interface between two aqueous droplets in oil (droplet interface bilayers, DIBs) using the DPh-based lipids. G4 dendrimers were found to translocate through POPC:POPS bilayers deposited on silica surfaces. The charge density of the bilayer affects translocation, which is reduced when the ionic strength increases. This shows that the dendrimer–bilayer interactions are largely controlled by their electrostatic attraction. The structure of the solid-supported bilayers remains intact upon translocation of the dendrimer. However, the amount of lipids in the bilayer decreases and dendrimer/lipid aggregates are formed in bulk solution, which can be deposited on the interfacial layers upon dilution of the system with dendrimer-free solvent. Electrophysiology measurements on DIBs confirm that G4 dendrimers cross the lipid membranes containing PS, which then become more permeable to ions. The obtained results have implications for PAMAM dendrimers as delivery vehicles to cells