Influence of adhesion-promoting glycolipids on the structure and stability of solid-supported lipid double-bilayers.

Glycolipids have a considerable influence on the interaction between adjacent biomembranes and can promote membrane adhesion trough favorable sugar-sugar "bonds" even at low glycolipid fractions. Here, in order to obtain structural insights into this phenomenon, we utilize neutron reflectometry in combination with a floating lipid bilayer architecture that brings two glycolipid-loaded lipid bilayers to close proximity. We find that selected glycolipids with di-, or oligosaccharide headgroups affect the inter-bilayer water layer thickness and appear to contribute to the stability of the double-bilayer architecture by promoting adhesion of adjacent bilayers even against induced electrostatic repulsion. However, we do not observe any redistribution of glycolipids that would maximize the density of sugar-sugar contacts. Our results point towards possible strategies for the investigation of interactions between cell surfaces involving specific protein-protein, lipid-lipid, or protein-lipid binding.

Interaction of a Histidine-Rich Antimicrobial Saliva Peptide with Model Cell Membranes: The Role of Histidines.

Histatin 5 is a histidine-rich, intrinsically disordered, multifunctional saliva protein known to act as a first line of defense against oral candidiasis caused by Candida albicans. An earlier study showed that, upon interaction with a common model bilayer, a protein cushion spontaneously forms underneath the bilayer. Our hypothesis is that this effect is of electrostatic origin and that the observed behavior is due to proton charge fluctuations of the histidines, promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Here we are investigating the role of the histidines in more detail by defining a library of variants of the peptide, where the former have been replaced by the pH-insensitive amino acid glutamine. By using experimental techniques such as circular dichroism, small angle X-ray scattering, quartz crystal microbalance with dissipation monitoring, and neutron reflectometry, it was determined that changing the number of histidines in the peptide sequence did not affect the structure of the peptide dissolved in solution. However, it was shown to affect the penetration depth of the peptide into the bilayer, where all variants except the one with zero histidines were found below the bilayer. A decrease in the number of histidine from the original seven to zero decreases the ability of the peptide to penetrate the bilayer, and the peptide is then also found residing within the bilayer. We hypothesize that this is due to the ability of the histidines to charge titrate, which charges up the peptide, and enables it to penetrate and translocate through the lipid bilayer.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Structure of Self-Initiated Photopolymerized Films: A Comparison of Models.

Self-initiated photografting and photopolymerization (SI-PGP) uses UV illumination to graft polymers to surfaces without additional photoinitiators using the monomers as initiators, "inimers". A wider use of this method is obstructed by a lack of understanding of the resulting, presumably heterogeneous, polymer structure and of the parallel degradation under continuous UV illumination. We have used neutron reflectometry to investigate the structure of hydrated SI-PGP-prepared poly(HEMA-co-PEG10MA) (poly(2-hydroxyethyl methacrylate-co-(ethylene glycol)10 methacrylate)) films and compared parabolic, sigmoidal, and Gaussian models for the polymer volume fraction distributions. Results from fitting these models to the data suggest that either model can be used to approximate the volume fraction profile to similar accuracy. In addition, a second layer of deuterated poly(methacrylic acid) (poly(dMAA)) was grafted over the existing poly(HEMA-co-PEG10MA) layer, and the resulting double-grafted films were also studied by neutron reflectometry to shed light on the UV-polymerization process and the inevitable UV-induced degradation which competes with the grafting.

New Insights into the Interaction of Class II Dihydroorotate Dehydrogenases with Ubiquinone in Lipid Bilayers as a Function of Lipid Composition.

The fourth enzymatic reaction in the de novo pyrimidine biosynthesis, the oxidation of dihydroorotate to orotate, is catalyzed by dihydroorotate dehydrogenase (DHODH). Enzymes belonging to the DHODH Class II are membrane-bound proteins that use ubiquinones as their electron acceptors. We have designed this study to understand the interaction of an N-terminally truncated human DHODH (HsΔ29DHODH) and the DHODH from Escherichia coli (EcDHODH) with ubiquinone (Q10) in supported lipid membranes using neutron reflectometry (NR). NR has allowed us to determine in situ, under solution conditions, how the enzymes bind to lipid membranes and to unambiguously resolve the location of Q10. Q10 is exclusively located at the center of all of the lipid bilayers investigated, and upon binding, both of the DHODHs penetrate into the hydrophobic region of the outer lipid leaflet towards the Q10. We therefore show that the interaction between the soluble enzymes and the membrane-embedded Q10 is mediated by enzyme penetration. We can also show that EcDHODH binds more efficiently to the surface of simple bilayers consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine, and tetraoleoyl cardiolipin than HsΔ29DHODH, but does not penetrate into the lipids to the same degree. Our results also highlight the importance of Q10, as well as lipid composition, on enzyme binding.

Nanostructural Characterization of Cardiolipin-Containing Tethered Lipid Bilayers Adsorbed on Gold and Silicon Substrates for Protein Incorporation.

A key to the development of lipid membrane-based devices is a fundamental understanding of how the molecular structure of the lipid bilayer membrane is influenced by the type of lipids used to build the membrane. This is particularly important when membrane proteins are included in these devices since the precise lipid environment affects the ability to incorporate membrane proteins and their functionality. Here, we used neutron reflectometry to investigate the structure of tethered bilayer lipid membranes and to characterize the incorporation of the NhaA sodium proton exchanger in the bilayer. The lipid membranes were composed of two lipids, dioleoyl phosphatidylcholine and cardiolipin, and were adsorbed on gold and silicon substrates using two different tethering architectures based on functionalized oligoethylene glycol molecules of different lengths. In all of the investigated samples, the addition of cardiolipin caused distinct structural rearrangement including crowding of ethylene glycol groups of the tethering molecules in the inner head region and a thinning of the lipid tail region. The incorporation of NhaA in the tethered bilayers following two different protocols is quantified, and the way protein incorporation modulates the structural properties of these membranes is detailed.

Encapsulation of Graphene in the Hydrophobic Core of a Lipid Bilayer.

Theoretical simulations have predicted that a lipid bilayer forms a stable superstructure when a sheet of graphene is inserted in its hydrophobic core. We experimentally produced for the first time a lipid-graphene-lipid assembly by combining the Langmuir-Blodgett and the Langmuir-Schaefer methods. Graphene is sandwiched and remains flat within the hydrophobic core of the lipid bilayer. Using infrared spectroscopy, ellipsometry, and neutron reflectometry, we characterized the superstructure at every fabrication step. The hybrid superstructure is mechanically stable and graphene does not disturb the natural lipid bilayer structure.

Interaction with Human Serum Proteins Reveals Biocompatibility of Phosphocholine-Functionalized SPIONs and Formation of Albumin-Decorated Nanoparticles.

Nanoparticles (NPs) are increasingly exploited as diagnostic and therapeutic devices in medicine. Among them, superparamagnetic nanoparticles (SPIONs) represent very promising tools for magnetic resonance imaging, local heaters for hyperthermia, and nanoplatforms for multimodal imaging and theranostics. However, the use of NPs, including SPIONs, in medicine presents several issues: first, the encounter with the biological world and proteins in particular. Indeed, nanoparticles can suffer from protein adsorption, which can affect NP functionality and biocompatibility. In this respect, we have investigated the interaction of small SPIONs covered by an amphiphilic double layer of oleic acid/oleylamine and 1-octadecanoyl-sn-glycero-3-phosphocholine with two abundant human plasma proteins, human serum albumin (HSA) and human transferrin. By means of spectroscopic and scattering techniques, we analyzed the effect of SPIONs on protein structure and the binding affinities, and only found strong binding in the case of HSA. In no case did SPIONs alter the protein structure significantly. We structurally characterized HSA/SPIONs complexes by means of light and neutron scattering, highlighting the formation of a monolayer of protein molecules on the NP surface. Their interaction with lipid bilayers mimicking biological membranes was investigated by means of neutron reflectivity. We show that HSA/SPIONs do not affect lipid bilayer features and could be further exploited as a nanoplatform for future applications. Overall, our findings point toward a high biocompatibility of phosphocholine-decorated SPIONs and support their use in nanomedicine.

Anesthetics significantly increase the amount of intramembrane water in lipid membranes.

The potency of anesthesia was directly linked to the partitioning of the drug molecules in cell membranes by Meyer and Overton. Many molecules interact with lipid bilayers and lead to structural and functional changes. It remains an open question which change in membrane properties is responsible for a potential anesthetic effect or if anesthetics act by binding to direct targets. We studied the effect of ethanol, diethyl ether and isoflurane on the water distribution in lipid bilayers by combining all-atom molecular dynamics simulations and neutron diffraction experiments. The simulations show strong membrane-drug interactions with partitioning coefficients of 38%, 92% and 100% for ethanol, diethyl ether and isoflurane, respectively, and provide evidence for an increased water partitioning in the membrane core. The amount of intramembrane water molecules was experimentally determined by selectively deuterium labeling lipids, anesthetic drug and water molecules in neutron diffraction experiments. Four additional water molecules per lipid were observed in the presence of ethanol. Diethyl ether and isoflurane were found to significantly increase the amount of intramembrane water by 25% (8 water molecules). This increase in intramembrane water may contribute to the non-specific interactions between anesthetics and lipid membranes.

Not just a fluidifying effect: omega-3 phospholipids induce formation of non-lamellar structures in biomembranes.

Polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA) is found in very high concentrations in a few peculiar tissues, suggesting that it must have a specialized role. DHA was proposed to affect the function of the cell membrane and related proteins through an indirect mechanism of action, based on the DHA-phospholipid effects on the lipid bilayer structure. In this respect, most studies have focused on its influence on lipid-rafts, somehow neglecting the analysis of effects on liquid disordered phases that constitute most of the cell membranes, by reporting in these cases only a general fluidifying effect. In this study, by combining neutron reflectivity, cryo-transmission electron microscopy, small angle neutron scattering, dynamic light scattering and electron paramagnetic resonance spectroscopy, we characterize liquid disordered bilayers formed by the naturally abundant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and different contents of a di-DHA glycero-phosphocholine, 22:6-22:6PC, from both a molecular/microscopic and supramolecular/mesoscopic viewpoint. We show that, below a threshold concentration of about 40% molar percent, incorporation of 22:6-22:6PC in the membrane increases the lipid dynamics slightly but sufficiently to promote the membrane deformation and increase of multilamellarity. Notably, beyond this threshold, 22:6-22:6PC disfavours the formation of lamellar phases, leading to a phase separation consisting mostly of small spherical particles that coexist with a minority portion of a lipid blob with water-filled cavities. Concurrently, from a molecular viewpoint, the polyunsaturated acyl chains tend to fold and expose the termini to the aqueous medium. We propose that this peculiar tendency is a key feature of the DHA-phospholipids making them able to modulate the local morphology of biomembranes.

Aß Beyond the AD Pathology: Exploring the Structural Response of Membranes Exposed to Nascent Aß Peptide.

The physiological and pathological roles of nascent amyloid beta (Aß) monomers are still debated in the literature. Their involvement in the pathological route of Alzheimer's Disease (AD) is currently considered to be the most relevant, triggered by their aggregation into structured oligomers, a toxic species. Recently, it has been suggested that nascent Aß, out of the amyloidogenic pathway, plays a physiological and protective role, especially in the brain. In this emerging perspective, the study presented in this paper investigated whether the organization of model membranes is affected by contact with Aß in the nascent state, as monomers. The outcome is that, notably, the rules of engagement and the resulting structural outcome are dictated by the composition and properties of the membrane, rather than by the Aß variant. Interestingly, Aß monomers are observed to favor the tightening of adjacent complex membranes, thereby affecting a basic structural event for cell-cell adhesion and cell motility.

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers.

Understanding the molecular mechanisms governing nanoparticle-membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse-grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di-stearoyl-phosphatidylcholine (DSPC) and anionic di-stearoyl-phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC-DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP-membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse-grained MD simulations as a lipid-crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.

Atom-scale depth localization of biologically important chemical elements in molecular layers.

In nature, biomolecules are often organized as functional thin layers in interfacial architectures, the most prominent examples being biological membranes. Biomolecular layers play also important roles in context with biotechnological surfaces, for instance, when they are the result of adsorption processes. For the understanding of many biological or biotechnologically relevant phenomena, detailed structural insight into the involved biomolecular layers is required. Here, we use standing-wave X-ray fluorescence (SWXF) to localize chemical elements in solid-supported lipid and protein layers with near-Ångstrom precision. The technique complements traditional specular reflectometry experiments that merely yield the layers' global density profiles. While earlier work mostly focused on relatively heavy elements, typically metal ions, we show that it is also possible to determine the position of the comparatively light elements S and P, which are found in the most abundant classes of biomolecules and are therefore particularly important. With that, we overcome the need of artificial heavy atom labels, the main obstacle to a broader application of high-resolution SWXF in the fields of biology and soft matter. This work may thus constitute the basis for the label-free, element-specific structural investigation of complex biomolecular layers and biological surfaces.

Reflectometry Reveals Accumulation of Surfactant Impurities at Bare Oil/Water Interfaces.

Bare interfaces between water and hydrophobic media like air or oil are of fundamental scientific interest and of great relevance for numerous applications. A number of observations involving water/hydrophobic interfaces have, however, eluded a consensus mechanistic interpretation so far. Recent theoretical studies ascribe these phenomena to an interfacial accumulation of charged surfactant impurities in water. In the present work, we show that identifying surfactant accumulation with X-ray reflectometry (XRR) or neutron reflectometry (NR) is challenging under conventional contrast configurations because interfacial surfactant layers are then hardly visible. On the other hand, both XRR and NR become more sensitive to surfactant accumulation when a suitable scattering length contrast is generated by using fluorinated oil. With this approach, significant interfacial accumulation of surfactant impurities at the bare oil/water interface is observed in experiments involving standard cleaning procedures. These results suggest that surfactant impurities may be a limiting factor for the investigation of fundamental phenomena involving water/hydrophobic interfaces.

Conformation of Single and Interacting Lipopolysaccharide Surfaces Bearing O-Side Chains.

The outer surfaces of Gram-negative bacteria are composed of lipopolysaccharide (LPS) molecules exposing oligo- and polysaccharides to the aqueous environment. This unique, structurally complex biological interface is of great scientific interest as it mediates the interaction of bacteria with antimicrobial agents as well as with neighboring bacteria in colonies and biofilms. Structural studies on LPS surfaces, however, have so far dealt almost exclusively with rough mutant LPS of reduced molecular complexity and limited biological relevance. Here, by using neutron reflectometry, we structurally characterize planar monolayers of wild-type LPS from Escherichia coli O55:B5 featuring strain-specific O-side chains in the presence and absence of divalent cations and under controlled interaction conditions. The model used for the reflectivity analysis is self-consistent and based on the volume fraction profiles of all chemical components. The saccharide profiles are found to be bimodal, with dense inner oligosaccharides and more dilute, extended O-side chains. For interacting LPS monolayers, we establish the pressure-distance curve and determine the distance-dependent saccharide conformation.

End Point Versus Backbone Specificity Governs Characteristics of Antibody Binding to Poly(ethylene glycol) Brushes.

End-grafted poly(ethylene glycol) (PEG) brushes are widely used in order to suppress undesired protein adsorption to surfaces exposed to blood or other biological fluids. The specific adsorption of antibodies (Abs) to PEG brushes associated with PEG's antigenicity is drawing increasing attention because it can affect clinical applications. Here, the adsorption to PEG brushes of two Ab types, specifically binding the polymer backbone and the polymer endpoints, is structurally characterized by neutron reflectometry. The measurements yield volume fraction profiles of PEG and of the adsorbed Abs with sub-nanometer resolution perpendicular to the surface. For all brush parameters in terms of grafting density and polymerization degree, the Ab profiles clearly differ between backbone binders and endpoint binders. The adsorbed Ab amount per unit area is substantial for both Ab types and for all brush parameters investigated, even for dense brushes, which impose a considerable osmotic barrier to Ab insertion. The results therefore indicate that variation of brush parameters alone is insufficient to prevent undesired Ab adsorption. Instead, our work motivates further efforts in the search for nonantigenic brush chemistry.

Membrane Permeation versus Amyloidogenicity: A Multitechnique Study of Islet Amyloid Polypeptide Interaction with Model Membranes.

Islet amyloid polypeptide (IAPP) is responsible for cell depletion in the pancreatic islets of Langherans, and for multiple pathological consequences encountered by patients suffering from type 2 Diabetes Mellitus. We have examined the amyloidogenicity and cytotoxic mechanisms of this peptide by investigating model-membrane permeation and structural effects of fragments of the human IAPP and several rat IAPP mutants. In vitro experiments and molecular dynamics simulations reveal distinct physical segregation, membrane permeation, and amyloid aggregation processes that are mediated by two separate regions of the peptide. These observations suggest a "detergent-like" mechanism, where lipids are extracted from the bilayer by the N-terminus of IAPP, and integrated into amyloid aggregates. The amyloidogenic aggregation would kinetically compete with the process of membrane permeation and, therefore, inhibit it. This hypothesis represents a new perspective on the mechanism underlying the membrane disruption by amyloid peptides, and could influence the development of new therapeutic strategies.

Membrane restructuring following in situ sialidase digestion of gangliosides: Complex model bilayers by synchrotron radiation reflectivity.

Synchrotron radiation reflectometry was used to access the transverse structure of model membranes under the action of the human sialidase NEU2, down to the Ångström length scale. Model membranes were designed to mimic the lipid composition of so-called Glycosphingolipids Enriched Microdomains (GEMs), which are membrane platforms specifically enriched in cholesterol and sphingolipids, and where also typical signalling molecules are hosted. Gangliosides, glycosphingolipids containing one or more sialic acid residues, are asymmetrically embedded in GEMs, in the outer membrane leaflet where gangliosides are claimed to interact directly with growth-factor receptors, modulating their activation and then the downstream intracellular signalling pathways. Thus, membrane dynamics and signalling could be strongly influenced by the activity of enzymes regulating the membrane ganglioside composition, including sialidases. Our results, concerning the structure of single membranes undergoing in-situ enzymatic digestion, show that the outcome of the sialidase action is not limited to the emergence of lower-sialylated ganglioside species. In fact, membrane reshaping occurs, involving a novel arrangement of the headgroups on its surface. Thus, sialidase activity reveals to be a potential tool to control dynamically the structural properties of the membrane external leaflet of living cells, influencing both the morphology of the close environment and the extent of interaction among active molecules belonging to signalling platforms.

Neutron Reflectometry Elucidates Protein Adsorption from Human Blood Serum onto PEG Brushes.

Poly(ethylene glycol) (PEG) brushes are reputed for their ability to prevent undesired protein adsorption to material surfaces exposed to biological fluids. Here, protein adsorption out of human blood serum onto PEG brushes anchored to solid-supported lipid monolayers was characterized by neutron reflectometry, yielding volume fraction profiles of lipid headgroups, PEG, and adsorbed proteins at subnanometer resolution. For both PEGylated and non-PEGylated lipid surfaces, serum proteins adsorb as a thin layer of approximately 10 Å, overlapping with the headgroup region. This layer corresponds to primary adsorption at the grafting surface and resists rinsing. A second diffuse protein layer overlaps with the periphery of the PEG brush and is attributed to ternary adsorption due to protein-PEG attraction. This second layer disappears upon rinsing, thus providing a first observation of the structural effect of rinsing on protein adsorption to PEG brushes.