Unexpected asymmetric distribution of cholesterol and phospholipids in equilibrium model membranes.

Lipid compositional asymmetry across the leaflets of the plasma membrane is an ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long-tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly-taking many hours or days. However, a lipid like cholesterol-which is the most abundant lipid in the plasma membrane of animal cells-has been harder to pinpoint in terms of its favored side. In this work we show that, when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long-tail phospholipids organize asymmetrically across the membrane's leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid-dipalmitoylphosphatidylcholine or sphingomyelin-segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight phospholipid number imbalance favoring the outer leaflet and thus opposite to where cholesterol and the saturated lipids preferentially partitioned. These results were obtained using magic-angle spinning nuclear magnetic resonance (MAS NMR) in combination with small-angle neutron scattering (SANS) using isotope labeling to differentiate lipid species. We suggest that sidedness in membranes can be driven by thermodynamic processes. In addition, our MAS NMR results show that the lower bound for cholesterol's flip-flop half-time at 45°C is 10 ms, which is at least two orders of magnitude slower than current MD simulations predict. This result stands in stark contrast to previous work that suggested that cholesterol's flip-flop half-time at 37°C has an upper bound of 10 ms.

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

Micellar Nanogels from Alginate-Based Diblock Copolysaccharides.

Alginates are marine polysaccharides known for their ability to selectively bind calcium ions and form hydrogels. They are widely used in biomedical applications but are challenging to produce as nanogels. Here we introduce a self-assembly route to create stable alginate-based nanogels under near-equilibrium conditions. Guluronate (G) blocks, which interact with divalent cations such as Ca2+, Ba2+, and Sr2+, were extracted from alginates and covalently linked through their reducing end to the reducing end of dextran (Dex) chains, forming linear block copolymers that self-assemble into micellar nanogels with a core-corona structure in the presence of these ions. Real-time dynamic light scattering (DLS) and small-angle neutron scattering (SANS) were used to study the self-assembly mechanism of the copolymer during dialysis against divalent ions. For the G12-b-Dex51 copolymer, we achieved spherical micelles with an 8 nm radius and an aggregation number of around 20. Although the type of divalent cation affected micelle stability, it did not influence their size. Micellar nanogels are dynamic structures, capable of ion exchange, and can disassemble with chelating agents like ethylenediamine tetraacetic acid (EDTA).

Are SAXS and SANS suitable to extract information on the role of water for electric-double-layer formation at the carbon-aqueous-electrolyte interface?

This study reports on the applicability of X-ray transmission (XRT), small- and wide-angle X-ray scattering (SAXS/WAXS) and small-angle neutron scattering (SANS) for investigating fundamental processes taking place in the working electrode of an electric double-layer capacitor with 1 M RbBr aqueous electrolyte at different applied potentials. XRT and incoherent neutron scattering are employed to determine global ion- and water-concentration changes and associated charge-balancing mechanisms. We showcase the suitability of SAXS and SANS, respectively, to get complementary information on local ion and solvent rearrangement in nanoconfinement, but also underscore the limitations of simple qualitative models, asking for more quantitative descriptions of water-water and ion-water interactions via detailed atomistic modelling approaches.

Structural characterisation of α-synuclein-membrane interactions and the resulting aggregation using small angle scattering.

The presence of amyloid fibrils is a hallmark of several neurodegenerative diseases. Some amyloidogenic proteins, such as α-synuclein and amyloid ß, interact with lipids, and this interaction can strongly favour the formation of amyloid fibrils. In particular the primary nucleation step, i.e. the de novo formation of amyloid fibrils, has been shown to be accelerated by lipids. However, the exact mechanism of this acceleration is still mostly unclear. Here we use a range of scattering methods, such as dynamic light scattering (DLS) and small angle X-ray and neutron scattering (SAXS and SANS) to obtain structural information on the binding of α-synuclein to model membranes formed from negatively charged lipids and their co-assembly into amyloid fibrils. We find that the model membranes take an active role in the reaction. The binding of α synuclein to the model membranes immediately induces a major structural change in the lipid assembly, which leads to a break-up into small and mostly disc- or rod-like lipid-protein particles. This transition can be reversed by temperature changes or proteolytic protein removal. Incubation of the small lipid-α-synuclein particles for several hours, however, leads to amyloid fibril formation, whereby the lipids are incorporated into the amyloid fibrils.

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering.

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Solution Structures of Two Different FRP-OCP Complexes as Revealed via SEC-SANS.

Photosynthetic organisms have established photoprotective mechanisms in order to dissipate excess light energy into heat, which is commonly known as non-photochemical quenching. Cyanobacteria utilize the orange carotenoid protein (OCP) as a high-light sensor and quencher to regulate the energy flow in the photosynthetic apparatus. Triggered by strong light, OCP undergoes conformational changes to form the active red state (OCPR). In many cyanobacteria, the back conversion of OCP to the dark-adapted state is assisted by the fluorescence recovery protein (FRP). However, the exact molecular events involving OCP and its interaction with FRP remain largely unraveled so far due to their metastability. Here, we use small-angle neutron scattering combined with size exclusion chromatography (SEC-SANS) to unravel the solution structures of FRP-OCP complexes using a compact mutant of OCP lacking the N-terminal extension (∆NTEOCPO) and wild-type FRP. The results are consistent with the simultaneous presence of stable 2:2 and 2:1 FRP-∆NTEOCPO complexes in solution, where the former complex type is observed for the first time. For both complex types, we provide ab initio low-resolution shape reconstructions and compare them to homology models based on available crystal structures. It is likely that both complexes represent intermediate states of the back conversion of OCP to its dark-adapted state in the presence of FRP, which are of transient nature in the photocycle of wild-type OCP. This study demonstrates the large potential of SEC-SANS in revealing the solution structures of protein complexes in polydisperse solutions that would otherwise be averaged, leading to unspecific results.

Distributing aminophospholipids asymmetrically across leaflets causes anomalous membrane stiffening.

We studied the mechanical leaflet coupling of prototypic mammalian plasma membranes using neutron spin-echo spectroscopy. In particular, we examined a series of asymmetric phospholipid vesicles with phosphatidylcholine and sphingomyelin enriched in the outer leaflet and inner leaflets composed of phosphatidylethanolamine/phosphatidylserine mixtures. The bending rigidities of most asymmetric membranes were anomalously high, exceeding even those of symmetric membranes formed from their cognate leaflets. Only asymmetric vesicles with outer leaflets enriched in sphingolipid displayed bending rigidities in conformity with these symmetric controls. We performed complementary small-angle neutron and x-ray experiments on the same vesicles to examine possible links to structural coupling mechanisms, which would show up in corresponding changes in membrane thickness. In addition, we estimated differential stress between leaflets originating either from a mismatch of their lateral areas or spontaneous curvatures. However, no correlation with asymmetry-induced membrane stiffening was observed. To reconcile our findings, we speculate that an asymmetric distribution of charged or H-bond forming lipids may induce an intraleaflet coupling, which increases the weight of hard undulatory modes of membrane fluctuations and hence the overall membrane stiffness.

Composition and temperature effects on the solution structure of SDS/octanol/brine by SANS, NMR and microscopy.

We investigate the solution structures of model sodium dodecyl sulfate/octanol/brine ternary mixtures across the lamellar (Lα), vesicle (L4) and micellar (L1) phases employing small angle neutron scattering (SANS), optical microscopy and nuclear magnetic resonance (NMR). Specifically, we examine the effect of co-surfactant octanol (0.2-9.48 w/v%) and temperature (25-65 °C) along dilution lines at fixed octanol : SDS ratios (0.08-1.21). A transition from Lα to sponge phase (L3) above 35 °C is found along the octanol : SDS = 1.21 isopleth, with phase coexistence above ϕ ≈ 0.14 weight fraction of surfactant and co-surfactant. The lamellar bilayers swell upon dilution, with an approximately linear increase of d-spacing, accompanied by a decrease of the Caillé parameter, indicative of greater membrane rigidity. At a lower octanol : SDS ratio of 0.62, coexistence of oblate micelles and vesicles is observed with preferential formation of vesicles at low concentrations. Dilution of the L1 phase, along octanol : SDS = 0.08, results in elongated micelles, as the NaCl : SDS ratio increases, while higher temperatures favour the formation of less elongated micelles. Our results provide a detailed map of the equilibrium structures found in the Lα vicinity of this extensively investigated flow-responsive surfactant system.

Spatial mapping and scaling of the shear-induced transformation from bicontinuous microemulsions towards lamellar structures by coupling microfluidics and SANS.

Coupling microfluidics and small-angle neutron scattering (SANS), we investigate the influence of shear flow on a model bicontinuous microemulsion of D2O/n-octane/C10E4, examining the role of membrane volume fraction in the transformation towards a lamellar structure. We employ a contraction-expansion geometry with flow velocities in excess of 10 m s-1 and spatially map the microfluidic field using a small SANS beam, illuminating down to 10 nL sample volumes. The shear-induced, progressive, bicontinuous-to-lamellar transition is found to be promoted by additional extensional flow (>103 s-1), while fast relaxation kinetics (<2 ms) return the scattering pattern to isotropic shortly after the constriction. Further, increasing the domain size of the bicontinuous structure (determined by the membrane volume fraction) appears to amplify its response to shear. Hence, the structural changes within the dilute bicontinuous microemulsions simply scale with the volume fraction of the membrane. By contrast, the stronger response of the microemulsion with the smallest domain size, located near the bicontinuous/lamellar coexistence, indicates an influence of an already more ordered structure with fewer passages. Our findings provide insight into the high shear behaviour of microemulsions of both academic and industrial relevance.

Human myelin proteolipid protein structure and lipid bilayer stacking.

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.

Effective Parameters Controlling Sterol Transfer: A Time-Resolved Small-Angle Neutron Scattering Study.

Though cholesterol is the most prevalent and essential sterol in mammalian cellular membranes, its precursors, post-synthesis cholesterol products, as well as its oxidized derivatives play many other important physiological roles. Using a non-invasive in situ technique, time-resolved small angle neutron scattering, we report on the rate of membrane desorption and corresponding activation energy for this process for a series of sterol precursors and post-synthesis cholesterol products that vary from cholesterol by the number and position of double bonds in B ring of cholesterol's steroid core. In addition, we report on sterols that have oxidation modifications in ring A and ring B of the steroid core. We find that sterols that differ in position or the number of double bonds in ring B have similar time and energy characteristics, while oxysterols have faster transfer rates and lower activation energies than cholesterol in a manner generally consistent with known sterol characteristics, like Log P, the n-octanol/water partitioning coefficient. We find, however, that membrane/water partitioning which is dependent on lipid-sterol interactions is a better predictor, shown by the correlation of the sterols' tilt modulus with both the desorption rates and activation energy.

Interdigitation-Induced Order and Disorder in Asymmetric Membranes.

We studied the transleaflet coupling of compositionally asymmetric liposomes in the fluid phase. The vesicles were produced by cyclodextrin-mediated lipid exchange and contained dipalmitoyl phosphatidylcholine (DPPC) in the inner leaflet and different mixed-chain phosphatidylcholines (PCs) as well as milk sphingomyelin (MSM) in the outer leaflet. In order to jointly analyze the obtained small-angle neutron and X-ray scattering data, we adapted existing models of trans-bilayer structures to measure the overlap of the hydrocarbon chain termini by exploiting the contrast of the terminal methyl ends in X-ray scattering. In all studied systems, the bilayer-asymmetry has large effects on the lipid packing density. Fully saturated mixed-chain PCs interdigitate into the DPPC-containing leaflet and evoke disorder in one or both leaflets. The long saturated acyl chains of MSM penetrate even deeper into the opposing leaflet, which in turn has an ordering effect on the whole bilayer. These results are qualitatively understood in terms of a balance of entropic repulsion of fluctuating hydrocarbon chain termini and van der Waals forces, which is modulated by the interdigitation depth. Monounsaturated PCs in the outer leaflet also induce disorder in DPPC despite vestigial or even absent interdigitation. Instead, the transleaflet coupling appears to emerge here from a matching of the inner leaflet lipids to the larger lateral lipid area of the outer leaflet lipids.

Effect of salt on the lamellar Lα-to-MLV transformation in SDS/octanol/water under microfluidic flow.

We investigate the effect of added (NaCl) salt and varying flow rate on the phase behaviour and flow response of a model surfactant Lα phase, sodium dodecyl sulfate (SDS)/octanol/water, using small angle neutron scattering (SANS) and polarised optical microscopy in microfluidics, supported by NMR, viscosity, conductivity and zeta potential measurements. A long (∼3 m) tubular microchannel device is employed to quantify the spatiotemporal structural evolution of the system towards multilamellar vesicles (MLV). The effect of salt is rationalised in terms of changes in membrane bending rigidity and phase stability. It is shown that ∼1.8 w/w% NaCl addition results in MLV formation within the shortest time (or equivalent lengthscale) and yields near-centrosymmetric scattering profiles characteristic of MLVs (at a reference 1 mL h-1 flow rate and ≃90 s-1 shear rate). Further salt addition yields biphasic systems that remain strongly aligned under flow, while lower salt content also increases scattering anisotropy, accompanied by higher membrane rigidity and solution viscosity. Increasing flow rate causes greater initial Lα alignment, and thus flow anisotropy, but also faster evolution towards isotropy and MLV formation.

Quantifying the Solution Structure of Metal Nanoclusters Using Small-Angle Neutron Scattering.

Metal nanoclusters are a unique class of synthetic material, as their crystal structures can be resolved using X-ray diffraction, and their chemical formula can be precisely determinated from mass spectroscopy. However, a complete structure characterization by these two techniques is often a challenging task. Here, we utilize small-angle neutron scattering (SANS) to directly quantify the key structure parameters of a series of silver and gold nanoclusters in solution. The results not only correlate well to their crystallographic structures, but also allow the quantification of the counterions layer surrounding charged nanoclusters in solution. Furthermore, when combining with X-ray scattering, it is possible to estimate the molecular weight of both the metal core and the ligand shell of nanoclusters. This work offers an alternative characterization tool for nanoclusters without the requirement of crystallization or gas phase ionization.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Antimicrobial peptide activity in asymmetric bacterial membrane mimics.

We report on the response of asymmetric lipid membranes composed of palmitoyl oleoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylglycerol, to interactions with the frog peptides L18W-PGLa and magainin 2 (MG2a), as well as the lactoferricin derivative LF11-215. In particular we determined the peptide-induced lipid flip-flop, as well as membrane partitioning of L18W-PGLa and LF11-215, and vesicle dye-leakage induced by L18W-PGLa. The ability of L18W-PGLa and MG2a to translocate through the membrane appears to correlate with the observed lipid flip-flop, which occurred at the fastest rate for L18W-PGLa. The higher structural flexibility of LF11-215 in turn allows this peptide to insert into the bilayers without detectable changes of membrane asymmetry. The increased vulnerability of asymmetric membranes to L18W-PGLa in terms of permeability, appears to be a consequence of tension differences between the compositionally distinct leaflets, but not due to increased peptide partitioning.

Interpenetrated biosurfactant-silk fibroin networks - a SANS study.

Silk fibroin (SF) based hydrogels have been exploited for years for their inherent biocompatibility and favorable mechanical properties which makes them interesting for biotechnology applications. In this study we investigate silk based composite hydrogels where pH-sensitive, anionic biosurfactant assemblies (sophorolipids SL-C18 : 1 and SL-C18 : 0), are employed to improve the present properties of SF. Results suggest that the presence of SL surfactant assemblies leads to faster gelling of SF by accelerating the refolding from random coil to ß-sheet as shown by infrared and UV-visible spectroscopy. Small angle neutron scattering (SANS) including contrast matching studies show that SF and SL assemblies coexist in a fibrillary network that is, in the case of SL-C18 : 0, interpenetrating. The resulting overall network structure in composite gels is slightly more affected by SL-C18 : 1 than by SL-C18 : 0, whereas the structure of both SF and surfactant assemblies remains unchanged. No disassembly of SL surfactant structures is observed, which gives a new perspective on SF-surfactant interactions. The hydrophobic effect within SF is favored in the presence of SL, leading to faster refolding of SF into ß-sheet conformation. The presented composite gels, being an interpenetrating network of which one compound (SL-C18 : 0) can be tweaked by pH, open an interesting option towards improved workability and stimuli responsive mechanical properties of SF based hydrogels with possible applications in controlled cell culture and tissue engineering or drug delivery. The presented SANS analysis approach has the potential to be expanded to other protein-surfactant systems and composite hydrogels.

Lamellar-to-MLV transformation in SDS/octanol/brine examined by microfluidic-SANS and polarised microscopy.

The lamellar-to-multilamellar vesicle (MLV) transformation in a model surfactant system, sodium dodecyl sulfate (SDS), octanol and brine, is investigated under continuous and oscillatory microfluidic contraction-expansion flows, employing polarised optical microscopy and small angle neutron scattering (SANS), with sample volume probed down to ≃20 nL. We determine the lamellar-to-MLV transition requirements at varying flow velocity, oscillation amplitude, frequency, and number of oscillatory cycles. The spatio-temporal evolution of the hierarchical fluid structure is elucidated: lamellar sheets initially align with flow direction upon entering a constriction and then perpendicularly upon exiting; the formation of MLVs at the nanoscale is first observed by SANS within a few (<5) oscillatory cycles, followed by the gradual appearance of a regular (albeit not crystalline) MLV arrangement, at the micronscale, by optical microscopy after tens of cycles, under the conditions investigated. Once MLVs form under flow, these remain metastable for several days.

An exact inversion method for extracting orientation ordering by small-angle scattering.

We outline a nonparametric inversion strategy for determining the orientation distribution function (ODF) of sheared interacting rods using small-angle scattering techniques. With the presence of direct inter-rod interaction and fluid mechanical forces, the scattering spectra are no longer characterized by the azimuthal symmetry in the coordinates defined by the principal directions of simple shear conditions, which severely compounds the reconstruction of ODFs based on currently available methods developed for dilute systems. Using a real spherical harmonic expansion scheme, the real-space ODFs are uniquely determined from the anisotropic scattering spectra and their numerical accuracy is verified computationally. Our method can be generalized to extract ODFs of uniaxially anisotropic objects under different flow conditions in a properly transformed reference frame with suitable basis vectors.