Effect of amphiphilic environment on the solution structure of mouse TSPO translocator protein.

The translocator protein (TSPO) is a ubiquitous transmembrane protein of great pharmacological interest thanks to its high affinity to many drug ligands. The only high-resolution 3D-structure known for mammalian TSPO was obtained by NMR for the mouse mTSPO in DPC detergent only in presence of the high-affinity PK 11195 ligand. An atomic structure of free-ligand mTSPO is still missing to better understand the interaction of ligands with mTSPO and their effects on the protein conformation. Here, we decipher the solution structures of the recombinant mTSPO without ligand both in (i) SDS, the detergent used to extract and purify the protein from E. coli inclusion bodies, and (ii) DPC, the detergent used to solve the PK 11195-binding mTSPO NMR structure. We report partially refolded and less flexible mTSPO helices in DPC compared to SDS. Besides, DPC stabilizes the tertiary structure of mTSPO, as shown by a higher intrinsic Trp fluorescence and changes in indole environment. We evaluate by SEC-MALLS that ∼135 SDS and ∼100 DPC molecules are bound to mTSPO. SEC-small-angle X-ray (SAXS) and neutron (SANS) scattering confirm a larger mTSPO-detergent complex in SDS than in DPC. Using the contrast-matching technique in SEC-SANS, we demonstrate that mTSPO conformation is more compact and less flexible in DPC than in SDS. Combining ab initio modeling with SANS, we confirm that mTSPO conformation is less elongated in DPC than in SDS. However, the free-ligand mTSPO envelope in DPC is not as compact as the PK 11195-binding protein NMR structure, the ligand stiffening the protein.

Dysmyelination and glycolipid interference caused by phenylalanine in phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder connected to an excess of phenylalanine (Phe) in the blood and tissues, with neurological consequences. The disease's molecular bases seem to be related to the accumulation of Phe at the cell membrane surface. Radiological outcomes in the brain demonstrate decreased water diffusivity in white matter, involving axon dysmyelination of not yet understood origin. We used a biophysical approach and model membranes to extend our knowledge of Phe-membrane interaction by clarifying Phe's propensity to affect membrane structure and dynamics based on lipid composition, with emphasis on modulating cholesterol and glycolipid components to mimic raft domains and myelin sheath membranes. Phe showed affinity for the investigated membrane mimics, mainly affecting the Phe-facing membrane leaflet. The surfaces of our neuronal membrane raft mimics were strong anchoring sites for Phe, showing rigidifying effects. From a therapeutic perspective, we further investigated the role of doxycycline, known to disturb Phe packing, unveiling its action as a competitor in Phe interactions with the membrane, suggesting its potential for treatment in the early stages of PKU. Our results suggest how Phe accumulation in extracellular fluids can impede normal growth of myelin sheaths by interfering with membrane slipping and by remodulating free water and myelin-associated water contents.

Order vs. Disorder: Cholesterol and Omega-3 Phospholipids Determine Biomembrane Organization.

Lipid structural diversity strongly affects biomembrane chemico-physical and structural properties in addition to membrane-associated events. At high concentrations, cholesterol increases membrane order and rigidity, while polyunsaturated lipids are reported to increase disorder and flexibility. How these different tendencies balance in composite bilayers is still controversial. In this study, electron paramagnetic resonance spectroscopy, small angle neutron scattering, and neutron reflectivity were used to investigate the structural properties of cholesterol-containing lipid bilayers in the fluid state with increasing amounts of polyunsaturated omega-3 lipids. Either the hybrid 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine or the symmetric 1,2-docosahexaenoyl-sn-glycero-3-phosphocholine were added to the mixture of the naturally abundant 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and cholesterol. Our results indicate that the hybrid and the symmetric omega-3 phospholipids affect the microscopic organization of lipid bilayers differently. Cholesterol does not segregate from polyunsaturated phospholipids and, through interactions with them, is able to suppress the formation of non-lamellar structures induced by the symmetric polyunsaturated lipid. However, this order/disorder balance leads to a bilayer whose structural organization cannot be ascribed to either a liquid ordered or to a canonical liquid disordered phase, in that it displays a very loose packing of the intermediate segments of lipid chains.

Carbohydrate-carbohydrate interaction drives the preferential insertion of dirhamnolipid into glycosphingolipid enriched membranes.

Rhamnolipids (RLs) are among the most important biosurfactants produced by microorganisms, and have been widely investigated because of their multiple biological activities. Their action appears to depend on their structural interference with lipid membranes, therefore several studies have been performed to investigate this aspect. We studied by X-ray scattering, neutron reflectometry and molecular dynamic simulations the insertion of dirhamnolipid (diRL), the most abundant RL, in model cellular membranes made of phospholipids and glycosphingolipids. In our model systems the affinity of diRL to the membrane is highly promoted by the presence of the glycosphingolipids and molecular dynamics simulations unveil that this evidence is related to sugar-sugar attractive interactions at the membrane surface. Our results improve the understanding of the plethora of activities associated with RLs, also opening new perspectives in their selective use for pharmaceutical and cosmetics formulations. Additionally, they shed light on the still debated role of carbohydrate-carbohydrate interactions as driving force for molecular contacts at membrane surface.

Open-Bundle Structure as the Unfolding Intermediate of Cytochrome c' Revealed by Small Angle Neutron Scattering.

The dynamic structure changes, including the unfolding, dimerization, and transition from the compact to the open-bundle unfolding intermediate structure of Cyt c', were detected by a small-angle neutron scattering experiment (SANS). The structure of Cyt c' was changed into an unstructured random coil at pD = 1.7 (Rg = 25 Å for the Cyt c' monomer). The four-α-helix bundle structure of Cyt c' at neutral pH was transitioned to an open-bundle structure (at pD ~13), which is given by a numerical partial scattering function analysis as a joint-clubs model consisting of four clubs (α-helices) connected by short loops. The compactly folded structure of Cyt c' (radius of gyration, Rg = 18 Å for the Cyt c' dimer) at neutral or mildly alkaline pD transited to a remarkably larger open-bundle structure at pD ~13 (Rg = 25 Å for the Cyt c' monomer). The open-bundle structure was also supported by ab initio modeling.

Modulation of plant plasma membrane structure by exogenous fatty acid hydroperoxide is a potential perception mechanism for their eliciting activity.

Oxylipins are lipid-derived molecules that are ubiquitous in eukaryotes and whose functions in plant physiology have been widely reported. They appear to play a major role in plant immunity by orchestrating reactive oxygen species (ROS) and hormone-dependent signalling pathways. The present work focuses on the specific case of fatty acid hydroperoxides (HPOs). Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this study, the potential of 13(S)-hydroperoxy-(9Z, 11E)-octadecadienoic acid (13-HPOD) and 13(S)-hydroperoxy-(9Z, 11E, 15Z)-octadecatrienoic acid (13-HPOT), as plant defence elicitors and the underlying mechanism of action is investigated. Arabidopsis thaliana leaf resistance to Botrytis cinerea was observed after root application with HPOs. They also activate early immunity-related defence responses, like ROS. As previous studies have demonstrated their ability to interact with plant plasma membranes (PPM), we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. The relationship between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.

anaklasis: a compact software package for model-based analysis of specular neutron and X-ray reflectometry data sets.

anaklasis constitutes a set of open-source Python scripts that facilitate a range of specular neutron and X-ray reflectivity calculations, involving the generation of theoretical curves and the comparison/fitting of interfacial model reflectivity against experimental data sets. The primary focus of the software is twofold: on one hand to offer a more natural framework for model definition, requiring minimum coding literacy, and on the other hand to include advanced analysis methods that have been proposed in recent work. Particular attention is given to the ability to co-refine reflectivity data and to the estimation of model-parameter uncertainty and covariance using bootstrap analysis and Markov chain Monte Carlo sampling. The compactness and simplicity of model definition together with the streamlined analysis do not present a steep learning curve for the user, an aspect that may accelerate the generation of reproducible, easily readable and statistically accurate reports in future neutron and X-ray reflectivity related literature.

Migration Kinetics of Surface Ions in Oxygen-Deficient Perovskite During Topotactic Transitions.

Oxygen diffusivity and surface exchange kinetics underpin the ionic, electronic, and catalytic functionalities of complex multivalent oxides. Towards understanding and controlling the kinetics of oxygen transport in emerging technologies, it is highly desirable to reveal the underlying lattice dynamics and ionic activities related to oxygen variation. In this study, the evolution of oxygen content is identified in real-time during the progress of a topotactic phase transition in La0.7 Sr0.3 MnO3-δ epitaxial thin films, both at the surface and throughout the bulk. Using polarized neutron reflectometry, a quantitative depth profile of the oxygen content gradient is achieved, which, alongside atomic-resolution scanning transmission electron microscopy, uniquely reveals the formation of a novel structural phase near the surface. Surface-sensitive X-ray spectroscopies further confirm a significant change of the electronic structure accompanying the transition. The anisotropic features of this novel phase enable a distinct oxygen diffusion pathway in contrast to conventional observation of oxygen motion at moderate temperatures. The results provide insights furthering the design of solid oxygen ion conductors within the framework of topotactic phase transitions.

Mutually Beneficial Combination of Molecular Dynamics Computer Simulations and Scattering Experiments.

We showcase the combination of experimental neutron scattering data and molecular dynamics (MD) simulations for exemplary phospholipid membrane systems. Neutron and X-ray reflectometry and small-angle scattering measurements are determined by the scattering length density profile in real space, but it is not usually possible to retrieve this profile unambiguously from the data alone. MD simulations predict these density profiles, but they require experimental control. Both issues can be addressed simultaneously by cross-validating scattering data and MD results. The strengths and weaknesses of each technique are discussed in detail with the aim of optimizing the opportunities provided by this combination.

Adhesion Process of Biomimetic Myelin Membranes Triggered by Myelin Basic Protein.

The myelin sheath-a multi-double-bilayer membrane wrapped around axons-is an essential part of the nervous system which enables rapid signal conduction. Damage of this complex membrane system results in demyelinating diseases such as multiple sclerosis (MS). The process in which myelin is generated in vivo is called myelination. In our study, we investigated the adhesion process of large unilamellar vesicles with a supported membrane bilayer that was coated with myelin basic protein (MBP) using time-resolved neutron reflectometry. Our aim was to mimic and to study the myelination process of membrane systems having either a lipid-composition resembling that of native myelin or that of the standard animal model for experimental autoimmune encephalomyelitis (EAE) which represents MS-like conditions. We were able to measure the kinetics of the partial formation of a double bilayer in those systems and to characterize the scattering length density profiles of the initial and final states of the membrane. The kinetics could be modeled using a random sequential adsorption simulation. By using a free energy minimization method, we were able to calculate the shape of the adhered vesicles and to determine the adhesion energy per MBP. For the native membrane the resulting adhesion energy per MBP is larger than that of the EAE modified membrane type. Our observations might help in understanding myelination and especially remyelination-a process in which damaged myelin is repaired-which is a promising candidate for treatment of the still mostly incurable demyelinating diseases such as MS.

Insertion and activation of functional Bacteriorhodopsin in a floating bilayer.

The proton pump transmembrane protein bacteriorhodopsin was successfully incorporated into planar floating lipid bilayers in gel and fluid phases, by applying a detergent-mediated incorporation method. The method was optimized on single supported bilayers by using quartz crystal microbalance, atomic force and fluorescence microscopy techniques. Neutron and X-ray reflectometry were used on both single and floating bilayers with the aim of determining the structure and composition of this membrane-protein system before and after protein reconstitution at sub-nanometer resolution. Lipid bilayer integrity and protein activity were preserved upon the reconstitution process. Reversible structural modifications of the membrane, induced by the bacteriorhodopsin functional activity triggered by visible light, were observed and characterized at the nanoscale.

Soliton-Mediated Magnetic Reversal in an All-Oxide-Based Synthetic Antiferromagnetic Superlattice.

All-oxide-based synthetic antiferromagnets (SAFs) are attracting intense research interest due to their superior tunability and great potentials for antiferromagnetic spintronic devices. In this work, using the La2/3Ca1/3MnO3/CaRu1/2Ti1/2O3 (LCMO/CRTO) superlattice as a model SAF, we investigated the layer-resolved magnetic reversal mechanism by polarized neutron reflectivity. We found that the reversal of LCMO layer moments is mediated by nucleation, expansion, and shrinkage of a magnetic soliton. This unique magnetic reversal process creates a reversed magnetic configuration of the SAF after a simple field cycling. Therefore, it can enable vertical data transfer from the bottom to the top of the superlattice. The physical origin of this intriguing magnetic reversal process could be attributed to the cooperation of the surface spin-flop effect and enhanced uniaxial magnetic anisotropy of the bottom LCMO layer. This work may pave a way to utilize all-oxide-based SAFs for three-dimensional spintronic devices with vertical data transfer and high-density data storage.

Magnetic Particle Self-Assembly at Functionalized Interfaces.

We study the assembly of magnetite nanoparticles in water-based ferrofluids in wetting layers close to silicon substrates with different functionalization without and with an out-of-plane magnetic field. For particles of nominal sizes 5, 15, and 25 nm, we extract density profiles from neutron reflectivity measurements. We show that self-assembly is only promoted by a magnetic field if a seed layer is formed at the silicon substrate. Such a layer can be formed by chemisorption of activated N-hydroxysuccinimide ester-coated nanoparticles at a (3-aminopropyl)triethoxysilane functionalized surface. Less dense packing is reported for physisorption of the same particles at a piranha-treated (strongly hydrophilic) silicon wafer, and no wetting layer is found for a self-assembled monolayer of octadecyltrichlorosilane (strongly hydrophobic) at the interface. We show that once the seed layer is formed and under an out-of-plane magnetic field further wetting layers assemble. These layers become denser with time, larger magnetic fields, higher particle concentrations, and larger moment of the nanoparticles.

Membrane stiffness and myelin basic protein binding strength as molecular origin of multiple sclerosis.

Myelin basic protein (MBP) and its interaction with lipids of the myelin sheath plays an important part in the pathology of multiple sclerosis (MS). Previous studies observed that changes in the myelin lipid composition lead to instabilities and enhanced local curvature of MBP-lipid multilayer structures. We investigated the molecular origin of the instability and found that the diseased lipid membrane has a 25% lower bending rigidity, thus destabilizing smooth [Formula: see text]µm curvature radius structures such as in giant unilamellar vesicles. MBP-mediated assembling of lipid bilayers proceeds in two steps, with a slow second step occurring over many days where native lipid membranes assemble into well-defined multilayer structures, whereas diseased lipid membranes form folded assemblies with high local curvature. For both native and diseased lipid mixtures we find that MBP forms dense liquid phases on top of the lipid membranes mediating attractive membrane interactions. Furthermore, we observe MBP to insert into its bilayer leaflet side in case of the diseased lipid mixture, whereas there is no insertion for the native mixture. Insertion increases the local membrane curvature, and could be caused by a decrease of the sphingomyelin content of the diseased lipid mixture. These findings can help to open a pathway to remyelination strategies.

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.

Mucin Thin Layers: A Model for Mucus-Covered Tissues.

The fate of macromolecules of biological or pharmacological interest that enter the mucus barrier is a current field of investigation. Studies of the interaction between the main constituent of mucus, mucins, and molecules involved in topical transmucoidal drug or gene delivery is a prerequisite for nanomedicine design. We studied the interaction of mucin with the bio-inspired arginine-derived amphoteric polymer d,l-ARGO7 by applying complementary techniques. Small angle X-ray scattering in bulk unveiled the formation of hundreds of nanometer-sized clusters, phase separated from the mucin mesh. Quartz microbalance with dissipation and neutron reflectometry measurements on thin mucin layers deposited on silica supports highlighted the occurrence of polymer interaction with mucin on the molecular scale. Rinsing procedures on both experimental set ups showed that interaction induces alteration of the deposited hydrogel. We succeeded in building up a new significant model for epithelial tissues covered by mucus, obtaining the deposition of a mucin layer 20 Å thick on the top of a glycolipid enriched phospholipid single membrane, suitable to be investigated by neutron reflectometry. The model is applicable to unveil the cross structural details of mucus-covered epithelia in interaction with macromolecules within the Å discreteness.

Long-range excitations in phospholipid membranes.

We investigated the existence of long-range excitations and correlated structures in phospholipid membranes by means of grazing-incidence neutron spin echo spectroscopy, grazing-incidence small-angle neutron scattering, and corresponding theoretical calculations inspired by smectic-membrane theory. All these methods confirmed the existence of thermal excitations in the plane of the surface of the phospholipid membranes or the corresponding structures, respectively. Also, these measurements revealed a temperature dependence of these excitations. These excitations are associated with 100 nm in-plane correlations around physiological temperatures and of 75 nm at 16 °C. A single excitation has an energy around the µeV-regime. A temperature series revealed a high abundance at physiological temperatures and pronounced long-range in-plane structures, which are strongly suppressed at temperatures below 20 °C. From the length-scales and energy transfers involved we surmise that these excitations may play a role in several functions of the cell membranes such as stability and energy dissipation along the membrane. From a fundamental point of view, the observed behavior of those excitations is congruent with that of a quasi-particle (surface mode phonon, smomon) that exists in the plane of phospholipid membranes.

Tuning spinterface properties in iron/fullerene thin films.

In ferromagnetic (FM) metal/organic semiconductor (OSC) heterostructures charge transfer can occur which leads to induction of magnetism in the non-magnetic OSC. This phenomenon has been described by the change in the density of states in the OSC which leads to a finite magnetic moment at the OSC interface and it is called the 'spinterface'. One of the main motivations in this field of organic spintronics is how to control the magnetic moment in the spinterface. In this regard, there are several open questions such as (i) which combination of FM and OSC can lead to more moment at the spinterface? (ii) Is the thickness of OSC also important? (iii) How does the spinterface moment vary with the FM thickness? (iv) Does the crystalline quality of the FM matter? (v) What is the effect of spinterface on magnetization reversal, domain structure and anisotropy? In this context, we have tried to answer the last four issues in this paper by studying Fe/C60 bilayers of variable Fe thickness deposited on Si substrates. We find that both the induced moment and thickness of the spinterface vary proportionally with the Fe thickness. Such behavior is explained in terms of the growth quality of the Fe layer on the native oxide of the Si (100) substrate. The magnetization reversal, domain structure and anisotropy of these bilayer samples were studied and compared with their respective reference samples without the C60 layer. It is observed that the formation of spinterface leads to a reduction in uniaxial anisotropy in Fe/C60 on Si (100) in comparison to their reference samples.

Model-independent recovery of interfacial structure from multi-contrast neutron reflectivity data.

Neutron specular reflectivity at soft interfaces provides sub-nanometre information concerning the molecular distribution of thin films, while the application of contrast variation can highlight the scattering from different parts of the system and lead to an overall reduction in fitting ambiguity. Traditional modelling approaches involve the construction of a trial scattering length density profile based on initial speculation and the subsequent refinement of its parameters through minimization of the discrepancy between the calculated and measured reflectivity. In practice this might produce an artificial bias towards specific sets of solutions. On the other hand, direct inversion of reflectivity data, despite its ability to provide a unique solution, is subject to limitations and experimental complications. Presented here is an integrated indirect Fourier transform/simulated annealing method that, when applied to multiple solvent contrast reflectivity data and within the limits of finite spatial resolution, leads to reliable reconstructions of the interfacial structure without the need for any a priori assumptions. The generality of the method permits its straightforward application in common experimental contrast-variation investigations at the solid/liquid and air/liquid interface.

Reversible Control of Physical Properties via an Oxygen-Vacancy-Driven Topotactic Transition in Epitaxial La0.7 Sr0.3 MnO3- δ Thin Films.

The vacancy distribution of oxygen and its dynamics directly affect the functional response of complex oxides and their potential applications. Dynamic control of the oxygen composition may provide the possibility to deterministically tune the physical properties and establish a comprehensive understanding of the structure-property relationship in such systems. Here, an oxygen-vacancy-induced topotactic transition from perovskite to brownmillerite and vice versa in epitaxial La0.7 Sr0.3 MnO3- δ thin films is identified by real-time X-ray diffraction. A novel intermediate phase with a noncentered crystal structure is observed for the first time during the topotactic phase conversion which indicates a distinctive transition route. Polarized neutron reflectometry confirms an oxygen-deficient interfacial layer with drastically reduced nuclear scattering length density, further enabling a quantitative determination of the oxygen stoichiometry (La0.7 Sr0.3 MnO2.65 ) for the intermediate state. Associated physical properties of distinct topotactic phases (i.e., ferromagnetic metal and antiferromagnetic insulator) can be reversibly switched by an oxygen desorption/absorption cycling process. Importantly, a significant lowering of necessary conditions (temperatures below 100 °C and conversion time less than 30 min) for the oxygen reloading process is found. These results demonstrate the potential applications of defect engineering in the design of perovskite-based functional materials.