Neutron Reflectometry Research Articles

Sterol Structural Features' Impact on the Spontaneous Membrane Insertion of CLIC1 into Artificial Lipid Membranes.

Background: A membrane protein interaction with lipids shows distinct specificity in terms of the sterol structure. The structure of the sterol's polar headgroup, steroidal rings, and aliphatic side chains have all been shown to influence protein membrane interactions, including the initial binding and subsequent oligomerization to form functional channels. Previous studies have provided some insights into the regulatory role that cholesterol plays in the spontaneous membrane insertion of the chloride intracellular ion channel protein, CLIC1. However, the manner in which cholesterol interacts with CLIC1 is yet largely unknown. Method: In this study, the CLIC1 interaction with different lipid:sterol monolayers was studied using the Langmuir trough and neutron reflectometry in order to investigate the structural features of cholesterol essential for the spontaneous membrane insertion of the CLIC1 protein. Molecular docking simulations were also performed to study the binding affinities between CLIC1 and the different sterol molecules. Results: This study, for the first time, highlights the vital role of the free sterol 3β-OH group as an essential structural requirement for the interaction of CLIC1 with cholesterol. Furthermore, the presence of additional hydroxyl groups, methylation of the sterol skeleton, and the structure of the sterol alkyl side chain have also been shown to modulate the magnitude of CLIC1 interaction with sterols and hence their spontaneous membrane insertion. This study also reports the ability of CLIC1 to interact with other naturally existing sterol molecules. General Significance: Like the sterol molecules, CLIC proteins are evolutionarily conserved with almost all vertebrates expressing six CLIC proteins (CLIC1-6), and CLIC-like proteins are also present in invertebrates and have also been reported in plants. This discovery of CLIC1 protein interaction with other natural sterols and the sterol structural requirements for CLIC membrane insertion provide key information to explore the feasibility of exploiting these properties for therapeutic and prophylactic purposes.

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.

Tuning the Properties of Thin-Film TaRu for Hydrogen-Sensing Applications

Accurate, cost-efficient, and safe hydrogen sensors will play a key role in the future hydrogen economy. Optical hydrogen sensors based on metal hydrides are attractive owing to their small size and costs and the fact that they are intrinsically safe. These sensors rely on suitable sensing materials, of which the optical properties change when they absorb hydrogen if they are in contact with a hydrogen-containing environment. Here, we illustrate how we can use alloying to tune the properties of hydrogen-sensing materials by considering thin films consisting of tantalum doped with ruthenium. Using a combination of optical transmission measurements, ex situ and in situ X-ray diffraction, and neutron and X-ray reflectometry, we show that introducing Ru in Ta results in a solid solution of Ta and Ru up to at least 30% Ru. The alloying has two major effects: the compression of the unit cell with increasing Ru doping modifies the enthalpy of hydrogenation and thereby shifts the pressure window in which the material absorbs hydrogen to higher hydrogen concentrations, and it reduces the amount of hydrogen absorbed by the material. This allows one to tune the pressure/concentration window of the sensor and its sensitivity and makes Ta1-yRuy an ideal hysteresis-free tunable hydrogen-sensing material with a sensing range of >7 orders of magnitude in pressure. In a more general perspective, these results demonstrate that one can rationally tune the properties of metal hydride optical hydrogen-sensing layers by appropriate alloying.

Significantly Enhanced Room-Temperature Ferromagnetism in Multiferroic EuFeO3-δ Thin Films.

Regulating the magnetic properties of multiferroics lays the foundation for their prospective application in spintronic devices. Single-phase multiferroics, such as rare-earth ferrites, are promising candidates; however, they typically exhibit weak magnetism at room temperature (RT). Here, we significantly boosted the RT ferromagnetism of a representative ferrite, EuFeO3, by oxygen defect engineering. Polarized neutron reflectometry and magnetometry measurements reveal that saturation magnetization reaches 0.04 μB/Fe, which is approximately 5 times higher than its bulk phase. Combining the annular bright-field images with theoretical assessment, we unravel the underlying mechanism for magnetic enhancement, in which the decrease in Fe-O-Fe bond angles caused by oxygen vacancies (VO) strengthens magnetic interactions and tilts Fe spins. Furthermore, the internal relationship between magnetism and VO was established by illustrating how the magnetic structure and magnitude change with VO configuration and concentration. Our strategy for regulating magnetic properties can be applied to numerous functional oxide materials.

Pulmonary surfactant structure as solved by neutron reflectometry and atomic force microscopy.

In order to sustain breathing mechanics, the alveolar surface needs to be covered by pulmonary surfactant, a lipid/protein system that provides with the required surface tension reduction to avoid alveolar collapse at the end of expiration. Any disruption of this system could impair its function leading to severe respiratory pathologies. Pulmonary surfactant is composed by 90 % lipids and 10 % hydrophilic (SP-A and SP-D) and hydrophobic (SP-B and SP-C) proteins. After secretion, surfactant lipids form a complex structure at the alveolar air-liquid interface consisting of an adsorbed monolayer connected to a membranous reservoir in the subphase maintained thanks to the action of both hydrophobic proteins. The molecular mechanism of surfactant proteins is still not fully understood, making the finding of treatments for some respiratory pathologies a challenge. Model lipid systems mimicking pulmonary surfactant composition (DPPC/POPC/POPG 50:25:15 w:w:w) in the presence of SP-B and/or SP-C, as well as a native surfactant isolated from animal lungs, have been used as models for the study of the structure and function of pulmonary surfactant films. In this work, we have characterized these models with different biophysical technics such as neutron reflectometry and atomic force microscopy. The results point to the importance of hydrophobic proteins in the formation of three-dimensional structures associated to highly compressed surfactant films. Furthermore, a recently isolated human amniotic fluid surfactant has also been studied. This material is thought to maintain the properties of a freshly synthetized and secreted surfactant that could cover the air-liquid interface in a more efficient way, with physiological importance to develop new surfactant therapies. Here we show that amniotic fluid surfactant exhibits a similar behavior than animal-derived surfactant once it coats the interface.

Insight into Bcl-2 proteins' functioning at mitochondrial membrane level.

Programmed cell death (apoptosis) is essential in life. In its intrinsic apoptotic pathway opposing members of the B-cell lymphoma 2 (Bcl-2) protein family control the permeability of the mitochondrial outer membrane (MOM) and the release of apoptotic factors such as cytochrome c. Any misregulation of this process can cause disorders most prominently cancer, where often upregulation of cell protecting (anti-apoptotic) Bcl-2 members such as the Bcl-2 membrane protein itself plays a notorious role by blocking MOM perforation by - often drug induced - apoptotic proteins such as Bax which would cause cancer cell death normally. Here, we apply neutron reflectometry (NR) on supported lipid bilayers which mimic MOM environment and solid state/liquid state NMR spectroscopy to unravel the molecular basis driving opposing proteins to interact with each other at the MOM; a mechanism which is not really understood yet due to lack of high-resolution structural insight. Based on our central hypothesis that Bcl-2 drives its cell-protecting function at a membrane-embedded location as revealed by NR (1), we focus i) to determine the structure of human Bcl-2 protein in its membrane setting by combining solution and solid-state NMR; ii) use NR to study the kinetics and lipid/protein pore assemblied upon binding of Bax to mitochondrial membranes and its membrane destroying activities there; and iii) unravel the nature of direct interaction between Bcl-2 and Bax to neutralize each other. Knowledge generated here, will be indispensable in understanding the regulative function of the Bcl-2 family at mitochondrial membranes. 1. A. Ul Mushtaq, J. Åden, L.A. Clifton, H. Wacklin-Knecht, M. Campana, A.P.G. Dingeldein, C. Persson, T. Sparrman, G. Gröbner. Neutron reflectometry and NMR spectroscopy of full length Bcl-2 protein reveal its membrane localization and conformation. Communications Biology 4 (2021) 507.

Advice on describing Bayesian analysis of neutron and X-ray reflectometry.

As a result of the availability of modern software and hardware, Bayesian analysis is becoming more popular in neutron and X-ray reflectometry analysis. The understandability and replicability of these analyses may be harmed by inconsistencies in how the probability distributions central to Bayesian methods are represented in the literature. Herein advice is provided on how to report the results of Bayesian analysis as applied to neutron and X-ray reflectometry. This includes the clear reporting of initial starting conditions, the prior probabilities, the results of any analysis and the posterior probabilities that are the Bayesian equivalent of the error bar, to enable replicability and improve understanding. It is believed that this advice, grounded in the authors' experience working in the field, will enable greater analytical reproducibility in the work of the reflectometry community, and improve the quality and usability of results.

Passivation of Ni-Cr and Ni-Cr-Mo Alloys in Low and High pH Sulfate Solutions

The kinetics of passive oxide film formation, its thickening, and composition on Ni-22Cr and Ni-22Cr-6Mo wt% alloys were investigated at selected anodic potentials. Experiments were performed in acidic and alkaline sulfate environments using a number of characterization techniques including a combination of potentiodynamic polarization, on-line atomic emission spectro-electrochemistry (AESEC), in situ potentiostatic passive film growth, along with in situ neutron reflectometry (NR) and ex situ X-ray photoelectron spectroscopy (XPS). The roles of solution pH and Mo on the passivation behavior were discussed in terms of thermodynamic and kinetic factors governing passivation. The pH was found to have an impact on the relative chemical compositions of passive film of the Ni-22Cr alloy but not noticeably for the Ni-22Cr-6Mo alloy. Ni-rich films formed early during the passivation process while Cr(III) enrichment was observed at longer times, albeit less extensively than observed previously in Cl− solutions. The fraction of Cr(III) cations also increased with alloying of Mo at low and high pH demonstrating a strong effect of Mo on Cr(III) content during aqueous passivation in Ni based superalloys, however, a larger effect is seen in Cl−. This is a noteworthy finding as Mo is usually assumed to mainly influence pit and crevice stabilization.

Protein-Nanoparticle Interactions Govern the Interfacial Behavior of Polymeric Nanogels: Study of Protein Corona Formation at the Air/Water Interface.

Biomedical applications of nanoparticles require a fundamental understanding of their interactions and behavior with biological interfaces. Protein corona formation can alter the morphology and properties of nanomaterials, and knowledge of the interfacial behavior of the complexes, using in situ analytical techniques, will impact the development of nanocarriers to maximize uptake and permeability at cellular interfaces. In this study we evaluate the interactions of acrylamide-based nanogels, with neutral, positive, and negative charges, with serum-abundant proteins albumin, fibrinogen, and immunoglobulin G. The formation of a protein corona complex between positively charged nanoparticles and albumin is characterized by dynamic light scattering, circular dichroism, and surface tensiometry; we use neutron reflectometry to resolve the complex structure at the air/water interface and demonstrate the effect of increased protein concentration on the interface. Surface tensiometry data suggest that the structure of the proteins can impact the interfacial properties of the complex formed. These results contribute to the understanding of the factors that influence the bio-nano interface, which will help to design nanomaterials with improved properties for applications in drug delivery.

Neutral polymers partial exclusion in ionic solutions near charged membranes.

Neutral polymers have become a useful tool in biophysics. They are currently used in a variety of single-channel conductance measurements, to change solution conductivity and viscosity, to induce osmotic stress in polymer-excluded regions, or simply as crowding agents. Many experiments involve the proximity of neutral polymers to charged membranes or their confinement in aqueous pores formed by protein channels with charged residues. A well-known phenomenon relevant to this problem is the polarization of a neutral particle with dielectric properties different from the surrounding medium under the effect of an electric field. This polarization causes a dielectrophoretic force when inhomogeneous electric fields act on the particle and it has been extensively used in many biomedical applications involving particle trapping and cell separation, among others. In addition, whenever a neutral particle perturbs the equilibrium electric double layer near a charged surface, the osmotic pressure gradient acts as a net repulsive force that pushes the particle away from that surface. Both the dielectrophoretic force and the hydrostatic force contribute to exclude a neutral particle from a charged surface, provided the polarizability is lower in the particle than in the solution. Here we report analytical predictions and simulations of the exclusion of polyethylene glycol (PEG) molecules of different molecular weight from charged surfaces. Molecular dynamics of PEG near charged DPPS membranes and neutral DPPC membranes show a region of PEG exclusion near the membrane-solution interface for charged membranes. The simulations also point to a preferential alignment of PEG molecules near the charged membrane. The simulations are confirmed by preliminary neutron reflectometry data, which are consistent with an exclusion region of concentrated PEG solutions near the highly charged silicon dioxide/water interface.

Biologically inspired reinforcement using polydopamine of polymer bound composites

Interfacial strength plays a critical role in the mechanical properties of a composite system. In this work, a proven mock high explosive (HE), 5-iodo-2’-deoxyuridine (IDOX) was coated by the adhesive promoting polymer polydopamine (PDA), then formulated with an Estane binder system to form an improved HE mock composite. An extensive comparison study of mechanical properties was performed to determine the effects, if any, of PDA on the composite system. To aid in interpretation of the mechanical test data, neutron reflectometry experiments were performed to determine the thickness and structure of the as deposited PDA films. The mechanical performance of PDA-treated IDOX was then compared to untreated specimens by compression testing in the Brazilian disk geometry. The results strongly suggested that the structure of PDA consists of two layers: one low in density caused by polymer agglomerates attaching to the substrate during polymerization, and the second high in density caused by controlled linear deposition of polydopamine. The mechanical testing showed that PDA greatly increased the stiffness and yield strength of IDOX based mocks without causing any disruption to the underlying crystal system.

Topical Review of Quantum Materials and Heterostructures Studied by Polarized Neutron Reflectometry

A review of the applications of polarized neutron reflectometry (PNR) for the investigation of quantum materials is provided. Recent studies of superconductors, strongly correlated oxides, hydrogen‐induced modifications, topological insulators and chiral magnets are highlighted. The PNR technique uses a quantum beam of spin‐polarized neutrons to measure the nanomagnetic structure of thin films and heterostructures, with a sensitivity to magnetization at the scale of 10–2000 emu cm−3 and a vertical spatial resolution of 1–500 nm. From simple beginnings studying the magnetic flux penetration at superconducting surfaces, today the PNR technique is widely used for investigating many different types of thin film structures, surfaces, interfaces, and 2D materials. PNR measurements can reveal a number of details about magnetic, electronic, and superconducting properties, in tandem with chemical information including the stoichiometry of light elements such as oxygen and hydrogen.

Investigation on the relationship between lipid composition and structure in model membranes composed of extracted natural phospholipids

HypothesisUnravelling the structural diversity of cellular membranes is a paramount challenge in life sciences. In particular, lipid composition affects the membrane collective behaviour, and its interactions with other biological molecules. ExperimentsHere, the relationship between membrane composition and resultant structural features was investigated by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry on in vitro membrane models of the mammalian plasma and endoplasmic-reticulum-Golgi intermediate compartment membranes in the form of Langmuir monolayers. Natural extracted yeast lipids were used because, unlike synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar. FindingsThe structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral packing, were found to depend strongly on their specific composition, with cholesterol having a major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquid-disorder phases.

Structural Characterization of Nanoparticle-Supported Lipid Bilayer Arrays by Grazing Incidence X-ray and Neutron Scattering

Arrays of nanoparticle-supported lipid bilayers (nanoSLB) are lipid-coated nanopatterned interfaces that provide a platform to study curved model biological membranes using surface-sensitive techniques. We combined scattering techniques with direct imaging, to gain access to sub-nanometer scale structural information on stable nanoparticle monolayers assembled on silicon crystals in a noncovalent manner using a Langmuir-Schaefer deposition. The structure of supported lipid bilayers formed on the nanoparticle arrays via vesicle fusion was investigated using a combination of grazing incidence X-ray and neutron scattering techniques complemented by fluorescence microscopy imaging. Ordered nanoparticle assemblies were shown to be suitable and stable substrates for the formation of curved and fluid lipid bilayers that retained lateral mobility, as shown by fluorescence recovery after photobleaching and quartz crystal microbalance measurements. Neutron reflectometry revealed the formation of high-coverage lipid bilayers around the spherical particles together with a flat lipid bilayer on the substrate below the nanoparticles. The presence of coexisting flat and curved supported lipid bilayers on the same substrate, combined with the sub-nanometer accuracy and isotopic sensitivity of grazing incidence neutron scattering, provides a promising novel approach to investigate curvature-dependent membrane phenomena on supported lipid bilayers.

Insight on hydrogen injection and GdOx/Co interface chemistry from in operando neutron reflectometry and secondary ion mass spectrometry

Ionic and redox control of magnetism can produce large changes to a variety of magnetic properties using a relatively small voltage. A model structure that continues to be of interest is Pt/Co/GdOxHy/Au, where the Co magnetic layer oxidation state and perpendicular magnetic anisotropy can be toggled using voltage control of proton transport through the GdOxHy electrolyte layer. The hydration of the oxide layer to form a hydroxide phase is the key to improve the speed of these magneto-ionic devices, but there is insufficient understanding of protonic defect incorporation and transport during hydration and electrical gating. In this work, we use polarized neutron reflectometry (PNR) to observe the effects of hydration and electrical gating by scanning in an as-grown state, a hydrated state, and in operando during electrical gating. We directly measure the depth profile of hydrogen and confirm the transformation from oxide (Gd2O3) to hydroxide [Gd(OH)3]. We observe the accumulation of H in the Co magnetic layer and the effects of gating on the structure and hydrogen content of the other layers in the device stack. Using PNR and secondary ion mass spectrometry, we find evidence for much more complex chemistry at the Co/GdOxHy interface than was previously assumed, including evidence for persistent CoO phases and CoOxHy phases. We offer insight on using PNR to observe relatively fast proton dynamics in the system and fitting a rather complex set of parameters to achieve a physical result for the fit spectrum and scattering length density profiles.

Effect of Magnesium Salts with Chaotropic Anions on the Swelling Behavior of PNIPMAM Thin Films

Poly(N-isopropylmethacrylamide) (PNIPMAM) is a stimuli-responsive polymer, which in thin film geometry exhibits a volume-phase transition upon temperature increase in water vapor. The swelling behavior of PNIPMAM thin films containing magnesium salts in water vapor is investigated in view of their potential application as nanodevices. Both the extent and the kinetics of the swelling ratio as well as the water content are probed with in situ time-of-flight neutron reflectometry. Additionally, in situ Fourier-transform infrared (FTIR) spectroscopy provides information about the local solvation of the specific functional groups, while two-dimensional FTIR correlation analysis further elucidates the temporal sequence of solvation events. The addition of Mg(ClO4)2 or Mg(NO3)2 enhances the sensitivity of the polymer and therefore the responsiveness of switches and sensors based on PNIPMAM thin films. It is found that Mg(NO3)2 leads to a higher relative water uptake and therefore achieves the highest thickness gain in the swollen state.

In Situ Neutron Reflectometry Study of a Tungsten Oxide/Li-Ion Battery Electrolyte Interface.

The solid electrolyte interface/interphase (SEI) is of great importance to the viable operation of lithium-ion batteries. In the present work, the interface between a tungsten oxide electrode and an electrolyte solution consisting of LiPF6 in a deuterated ethylene carbonate/diethyl carbonate solvent was characterized with in situ neutron reflectometry (NR) at a series of applied electrochemical potentials. NR data were fit to yield neutron scattering length density (SLD) depth profiles in the surface normal direction, from which composition depth profiles were inferred. The goals of this work were to characterize SEI formation on a model transition-metal oxide, an example of a conversion electrode, to characterize the lithiation of WO3, and to help interpret the results of an earlier study of tungsten electrodes without an intentionally grown surface oxide. The WO3 electrode was produced by thermal oxidation of a W thin film. Co-analysis of NR and X-ray reflectivity data indicated that the stoichiometry of the thermal oxide was WO3. As the electrode was polarized to progressively more reducing potentials, starting from open circuit and down to +0.25 V versus Li/Li+, the layer that was originally WO3 expanded and increased in lithium content. The reduced electrode consisted of two to three layers: an inner layer (the evolving conversion electrode) which may have been mixed W and Li2O and unreacted WO3 or LixWO3, a layer rich in protons and/or lithium, possibly corresponding to LiOH or LiH (the inner SEI), and an outermost layer adjacent to the solution with an SLD close to that of the solution, possibly consisting of lower SLD species with solution-filled porosity or deuteron-rich species derived from the solvents (the outer SEI), though the presence of this layer was tenuous. For the steps in the direction of more oxidizing potentials, the evolution of the layer structure was qualitatively the reverse of that seen when stepping toward more negative potentials, though with hysteresis. The SLD gradient suggested that the reaction was not limited by diffusion within the film. No clear phase boundary was evident in the evolving conversion electrode.

Pressure-Induced Phase Transitions of Nonionic Polymer Brushes

While the temperature responsive behavior of nonionic polymers has been extensively studied and is nowadays one of the key mechanisms of smart materials, the pressure response of thin films remains basically unexplored. We investigate the conformational transition of nonionic brushes and semidilute solutions induced by hydrostatic pressure and temperature variations. Interestingly, the pressure–temperature phase diagram for the coil-to-globule transition of brushes, probed by neutron reflectometry, nearly coincides with that in semidilute solutions. We also show that the phase behavior can be understood and predicted with simple thermodynamic concepts employed so far for the denaturation of proteins. Fully atomistic molecular dynamics simulations provide molecular insight into the pressure-responsive behavior. Combining all three approaches allows us to demonstrate that pressure-induced hydration of nonionic polymers at low pressure is universal as it is dictated by water and is polymer-independent. In contrast, the pressure-induced dehydration at high pressure is strongly polymer-specific. The outcomes apply to a wide class of nonionic polymers and can aid the design of responsive coatings with the desired pressure-responsive behavior.

Combined, time-resolved, in situ neutron reflectometry and X-ray diffraction analysis of dynamic SEI formation during electrochemical N2 reduction

In situ neutron reflectometry and GI-XRD reveal the dynamics of SEI formation and layer composition during Li-NRR with current cycling.

The development of neutron reflectometry as a probe of the nanoscale structure of polymer thin film systems - founded on the pioneering work of Professor Thomas P. Russell.

Among Professor Russell's numerous, original, and significant contributions to polymer science are those in which he helped pioneer the application of neutron reflectometry to the study of thin film systems. For this groundbreaking work, along with his support of neutron scattering methods in general, he was awarded the 2020 Clifford G. Shull Prize by the Neutron Scattering Society of America, named after and in honor of the Nobel Prize laureate. This article highlights some of the first applications of neutron reflectometry to probe the nanoscale structure of polymer thin film systems that Professor Russell and his colleagues pioneered. A concise account of the subsequent evolution of even more powerful phase-sensitive reflectometry techniques, following the success of their early work, is then presented. In addition to a general description of this current methodology, several particularly relevant and illustrative examples are given.