Incorporation and localisation of alkanes in a protomembrane model by neutron diffraction

Protomembranes at the origin of life were likely composed of short-chain lipids, readily available on the early Earth. Membranes formed by such lipids are less stable and more permeable under extreme conditions, so a novel membrane architecture was suggested to validate the accuracy of this assumption. The model membrane includes the presence of a layer of alkanes in the mid-plane of the protomembrane in between the two monolayer leaflets and lying perpendicular to the lipid acyl chains. Here, we investigated such a possibility experimentally for membranes formed by the short-chain phospholipid 1,2-didecanoyl-sn-glycero-3-phophocholine, including or not the alkanes eicosane, squalane or triacontane by means of neutron membrane diffraction and contrast variation. We found strong indications for incorporation of two of the three alkanes in the membrane mid-plane through the determination of neutron scattering length density profiles with hydrogenated vs deuterated alkanes and membrane swelling at various relative humidities indicating a slightly increased bilayer thickness when the alkanes are incorporated into the bilayers. The selectivity of the incorporation points out the role of the length of the n-alkanes with respect to the capacity of the membrane to incorporate them.     

The dynamical Matryoshka model: 1. Incoherent neutron scattering functions for lipid dynamics in bilayers

Fluid lipid bilayers are the building blocks of biological membranes. Although there is a large amount of experimental data using incoherent quasi-elastic neutron scattering (QENS) techniques to study membranes, very little theoretical works have been developed to study the local dynamics of membranes. The main objective of this work is to build a theoretical framework to study and describe the local dynamics of lipids and derive analytical expressions of intermediate scattering functions (ISF) for QENS. As results, we developed the dynamical Matryoshka model which describes the local dynamics of lipid molecules in membrane layers as a nested hierarchical convolution of three motional processes: (i) individual motions described by the vibrational motions of H-atoms; (ii) internal motions including movements of the lipid backbone, head groups and tails, and (iii) molecule movements of the lipid molecule as a whole. The analytical expressions of the ISF associated with these movements are all derived. For use in analyzing the QENS experimental data, we also derived an analytical expression for the aggregate ISF of the Matryoshka model which involves an elastic term plus three inelastic terms of well-separated time scales and whose amplitudes and rates are functions of the lipid motions. And as an illustrative application, we used the aggregated ISF to analyze the experimental QENS data on a lipid sample of multilamellar bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). It is clear from this analysis that the dynamical Matryoshka model describes very well the experimental data and allow extracting the dynamical parameters of the studied system.     

Hydration dependence of chain dynamics and local diffusion in L-alpha-dipalmitoylphosphtidylcholine multilayers studied by incoherent quasi-elastic neutron scattering.

Incoherent quasi-elastic neutron scattering is applied to study the local diffusion and chain dynamics of L-alpha-diplamiotylphosphatidylcholine molecules in oriented model membranes. Different motions are distinguished by changing the hydration of the multilayers as well as by measuring below and above the gel-to-liquid crystalline phase transition. The time range of the utilized time-of-flight spectrometer permits to observe two types of motion to be observed more closely: chain defect motions and the local diffusion of the whole molecule in its solvation cage. Oriented lipid membranes are a useful system for the observation of chain defects, as they can be macroscopically oriented, in contrast to most polymers. As a representative model for a chain defect a kink is chosen and the corresponding scattering functions are derived. The kink motion can explain the entire dynamics seen in the gel phase, and the lifetime of such a defect was found to be 10-15 ps, in good agreement with theoretical predictions. On the other hand the dynamics in the liquid crystalline phase cannot be explained even by a superposition of several kinks and thus requires the consideration of an additional motion: the local diffusion of the molecule in its solvation cage. The size of the solvation cage is increasing with multilayer hydration and reduced temperature. Particularly interesting in view of recent discussions about the origin of the short-range repulsive forces between membranes is the experimental finding of an out-of-plane motion with an amplitude of 1-1.5 A, which cannot be explained by the undulation of the whole membrane.     

Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated Escherichia coli Lipids

Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of Escherichia coli by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of Escherichia coli allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.     

Localisation of partially deuterated cholesterol in quaternary SC lipid model membranes: a neutron diffraction study

This letter presents our first results in using the benefit of selective deuteration in neutron diffraction studies on stratum corneum (SC) lipid model systems. The SC represents the outermost layer of the mammalian skin and exhibits the main skin barrier. It is essential for studying drug penetration through the SC to know the internal structure and hydration behaviour on the molecular level. The SC intercellular matrix is mainly formed by ceramides (CER), cholesterol (CHOL) and long- chain free fatty acids (FFA). Among them, CHOL is the most abundant individual lipid, but a detailed knowledge about its localisation in the SC lipid matrix is still lacking. The structure of the quaternary SC lipid model membranes composed of either CER[AP]/CHOL-D6/palmitic acid (PA)/cholesterol sulphate (ChS) or CER[AP]/CHOL-D7/PA/ChS is characterized by neutron diffraction. Neutron diffraction patterns from the oriented samples are collected at the V1 diffractometer of the Hahn-Meitner-Institute, Berlin, measured at 32°C, 60% humidity and at different D₂O contents. The neutron scattering length density profile in the direction normal to the surface is restored by Fourier synthesis from the experimental diffraction patterns. The analysis of scattering length density profile is a suitable tool for investigating the internal structure of the SC lipid model membranes. The major finding is the experimental proof of the CHOL localisation in SC model membrane by deuterium labelling at prominent positions in the CHOL molecules.     

Alzheimer's disease amyloid-β peptide analogue alters the ps-dynamics of phospholipid membranes

We have investigated the influence of the neurotoxic Alzheimer's disease peptide amyloid-β (25-35) on the dynamics of phospholipid membranes by means of quasi-elastic neutron scattering in the picosecond time-scale. Samples of pure phospholipids (DMPC/DMPS) and samples with amyloid-β (25-35) peptide included have been compared. With two different orientations of the samples the directional dependence of the dynamics was probed. The sample temperature was varied between 290 K and 320 K to cover both the gel phase and the liquid-crystalline phase of the lipid membranes. The model for describing the dynamics combines a long-range translational diffusion of the lipid molecules and a spatially restricted diffusive motion. Amyloid-β (25-35) peptide affects significantly the ps-dynamics of oriented lipid membranes in different ways. It accelerates the lateral diffusion especially in the liquid-crystalline phase. This is very important for all kinds of protein-protein interactions which are enabled and strongly influenced by the lateral diffusion such as signal and energy transducing cascades. Amyloid-β (25-35) peptide also increases the local lipid mobility as probed by variations of the vibrational motions with a larger effect in the out-of-plane direction. Thus, the insertion of amyloid-β (25-35) peptide changes not only the structure of phospholipid membranes as previously demonstrated by us employing neutron diffraction (disordering effect on the mosaicity of the lipid bilayer system) but also the dynamics inside the membranes. The amyloid-β (25-35) peptide induced membrane alteration even at only 3 mol% might be involved in the pathology of Alzheimer's disease as well as be a clue in early diagnosis and therapy.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.     

Basic Nanostructure of Stratum Corneum Lipid Matrices Based on Ceramides [EOS] and [AP]: A Neutron Diffraction Study

The goal of this study was to investigate the nanostructure of SC lipid model membranes comprising the most relevant SC lipids such as the unique-structured ω-acylceramide [EOS] in a near natural ratio with neutron diffraction. In models proposed recently the presence of ceramide [EOS] and FFA are necessary for the formation of one of the two existent crystalline lamellar phases of the SC lipids, the long-periodicity phase as well as for the normal barrier function of the SC. The focus of this study was placed on the influence of the FFA BA on the membrane structure and its localization within the membrane based on the ceramides [EOS] and [AP]. The internal nanostructure of such membranes was obtained by Fourier synthesis from the experimental diffraction patterns. The resulting neutron scattering length density profiles showed that the exceptionally long ceramide [EOS] is arranged in a short-periodicity phase created by ceramide [AP] by spanning through the whole bilayer and extending even further into the adjacent bilayer. Specifically deuterated BA allowed us to determine the exact position of this FFA inside this SC lipid model membrane. Furthermore, hydration experiments showed that the presented SC mimic system shows an extremely small intermembrane hydration of ∼1 Å, consequently the headgroups of the neighboring leaflets are positioned close to each other.     

The dynamical Matryoshka model: 3. Diffusive nature of the atomic motions contained in a new dynamical model for deciphering local lipid dynamics

In accompanying papers [Bicout et al., BioRxiv https://doi.org/10.1101/2021.09.21.461198 (2021); Cissé et al., BioRxiv https://doi.org/10.1101/2022.03.30.486370 (2022)], a new model called Matryoshka model has been proposed to describe the geometry of atomic motions in phospholipid molecules in bilayers and multilamellar vesicles based on their quasielastic neutron scattering (QENS) spectra. Here, in order to characterize the relaxational aspects of this model, the energy widths of the QENS spectra of the samples were analyzed first in a model-free way. The spectra were decomposed into three Lorentzian functions, which are classified as slow, intermediate, and fast motions depending on their widths. The analysis provides the diffusion coefficients, residence times, and geometrical parameters for the three classes of motions. The results corroborate the parameter values such as the amplitudes and the mobile fractions of atomic motions obtained by the application of the Matryoshka model to the same samples. Since the current analysis was carried out independently of the development of the Matryoshka model, the present results enhance the validity of the model. The model will serve as a powerful tool to decipher the dynamics of lipid molecules not only in model systems, but also in more complex systems such as mixtures of different kinds of lipids or natural cell membranes.     

Lipophilic penetration enhancers and their impact to the bilayer structure of stratum corneum lipid model membranes: neutron diffraction studies based on the example oleic acid

The present study analyzes the effect of the lipophilic penetration enhancer oleic acid on the bilayer structure of stratum corneum (SC) lipid model membranes based on Ceramide AP by using the neutron diffraction technique. Our results indicate the formation of a single lamellar phase in the presence of oleic acid under the chosen experimental conditions; a separated fluid-like oleic acid-rich phase was not detected in the present study. By comparing the internal membrane structure received from Fourier synthesis with the model system lacking oleic acid, considerable structural changes in terms of impairment of the lamellar order were found after incorporation of the penetration enhancer into the bilayers. In addition, by using specifically deuterated oleic acid we were able to prove the integration of the enhancer molecules into the model bilayers and moreover, to determine the exact position of oleic acid inside the SC lipid model membrane. From the present results we conclude a strong perturbation of lamellar SC lipid arrangement due to the intercalated penetration enhancer which can account for the promoting effects on drug penetration across the SC known for oleic acid.     

Squalane is in the midplane of the lipid bilayer: implications for its function as a proton permeability barrier

A recently proposed model for proton leakage across biological membranes [Prog. Lipid Res. 40 (2001) 299] suggested that hydrocarbons specifically in the center of the lipid bilayer inhibit proton leaks. Since cellular membranes maintain a proton electrochemical gradient as a principal energy transducer, proton leakage unproductively consumes cellular energy. Hydrocarbons in the bilayer are widespread in membranes that sustain such gradients. The alkaliphiles are unique in that they contain up to 40 mol% isoprenes in their membranes including 10-11 mol% squalene [J. Bacteriol. 168 (1986) 334]. Squalene is a polyisoprene hydrocarbon without polar groups. Localizing hydrocarbons in lipid bilayers has not been trivial. A myriad of physical methods including fluorescence spectroscopy, electron-spin resonance, nuclear magnetic resonance as well as X-ray and neutron diffraction have been used to explore this question with various degrees of success and often contradictory results. Seeking unambiguous evidence for the localization of squalene in membranes or lipid bilayers, we employed neutron diffraction. We incorporated 10 mol% perdeuterated or protonated squalane, an isosteric analogue of squalene, into stacked bilayers of dioleoyl phosphatidyl choline (DOPC) doped with dioleoyl phosphatidyl glycerol (DOPG) to simulate the negative charges found on natural membranes. The neutron diffraction data clearly show that the squalane lies predominantly in the bilayer center, parallel to the plane of the membrane.     

Interactions of two O-phosphorylresveratrol derivatives with model membranes

The hydrosoluble resveratrol derivative 3-O-phosphorylresveratrol was shown to be more cytotoxic against DU 145 prostate cancer cells than its analog 4'-O-phosphorylresveratrol. In an attempt to unveil the molecular determinants that lye at the root of their different biological effects, here we investigate the interactions of the two resveratrol derivatives with DMPC model membranes by using DSC, membrane permeation/poration assays and molecular dynamics. The results show that the 3-O-derivative interacts with DMPC membranes and diffuses across them. The 4'-O-derivative lies preferentially onto the surface of membrane. The MD simulations provide a molecular interpretation of the experiments and highlight that, in order to maximize the apolar interactions, the 3-O-derivative is embedded in the lipid hydrophobic region. This topographical position of the 3-O resveratrol analog perturbs the liquid-crystalline order of the lipid bilayer promoting membrane curvature and partial lipid loss from the vesicle. This finding reconciles with the lowering of the enthalpy of the lipid phase transition and the ability of the molecule to diffuse across membranes. The present data contribute to explain the different biological activity of the two molecules and evidence that membrane permeability is a key requirement for effective design of resveratrol derivatives to be used for therapeutic purposes.     

Lateral forces acting between particles in liquid films or lipid membranes

To investigate the mechanism of formation of 2D arrays of protein macromolecules in liquid films we carried out model experiments with μm-sized latex particles. The direct observations revealed that the process of ordering is triggered by attractive lateral capillary forces due to the overlap of the menisci formed around the particles. Two types of lateral capillary forces, flotation and immersion, can be distinguished, and a theory of these interactions is developed. Similar forces are operative between inclusions (proteins) incorporated in lipid membranes. We develop an appropriate model of a lipid bilayer, which is described as an elastic layer (the hydrocarbon chain region) sandwiched between two Gibbs dividing surfaces (the two headgroup regions). The range of the interaction between two cylindrical inclusions turns out to be of the order of several inclusion radii. The results, which are in qualitative agreement with the experimental observations, can be applied to the interpretation of membrane processes and mechanisms such as protein aggregation in lipid membranes.     

Interaction of the full-length Bax protein with biomimetic mitochondrial liposomes: a small-angle neutron scattering and fluorescence study

In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of several mitochondrial components, and sealing the cell's fate. To study the binding of Bax to membranes, we used an in vitro system consisting of 50nm diameter liposomes prepared with a lipid composition mimicking that of mitochondrial membranes in which recombinant purified full-length Bax was inserted via activation with purified tBid. We detected the association of the protein with the membrane using fluorescence fluctuation methods, and found that it could well be described by an equilibrium between soluble and membrane-bound Bax and that at a high protein-to-liposome ratio the binding seemed to saturate at about 15 Bax proteins per 50nm diameter liposome. We then obtained structural data for samples in this saturated binding regime using small-angle neutron scattering under different contrast matching conditions. Utilizing a simple model to fit the neutron data, we observed that a significant amount of the protein mass protrudes above the membrane, in contrast to the conjecture that all of the membrane-associated Bax states are umbrella-like. Upon protein binding, we also observed a thinning of the lipid bilayer accompanied by an increase in liposome radius, an effect reminiscent of the action of antimicrobial peptides on membranes.     

Characterization of the interaction of diacylglycerol acyltransferase-2 with the endoplasmic reticulum and lipid droplets

Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.     

The location and function of vitamin E in membranes (review)

Vitamin E is a fat-soluble vitamin that consists of a group of tocols and tocotrienols with hydrophobic character, but possessing a hydroxyl substituent that confers an amphipathic character on them. The isomers of biological importance are the tocopherols, of which alpha-tocopherol is the most potent vitamin. Vitamin E partitions into lipoproteins and cell membranes, where it represents a minor constituent of most membranes. It has a major function in its action as a lipid antioxidant to protect the polyunsaturated membrane lipids against free radical attack. Other functions are believed to be to act as membrane stabilizers by forming complexes with the products of membrane lipid hydrolysis, such as lysophospholipids and free fatty acids. The main experimental approach to explain the functions of vitamin E in membranes has been to study its effects on the structure and stability of model phospholipid membranes. This review describes the function of vitamin E in membranes and reviews the current state of knowledge of the effect of vitamin E on the structure and phase behaviour of phospholipid model membranes.     

Functional role of water in membranes updated: A tribute to Träuble

The classical view of a cell membrane is as a hydrophobic slab in which only nonpolar solutes can dissolve and permeate. However, water-soluble non-electrolytes such as glycerol, erythritol, urea and others can permeate lipid membranes in the liquid crystalline state. Moreover, recently polar amino acid's penetration has been explained by means of molecular dynamics in which appearance of water pockets is postulated. According to Träuble (1971), water diffuses across the lipid membranes by occupying holes formed in the lipid matrix due to fluctuations of the acyl chain trans–gauche isomers. These holes, named “kinks” have the molecular dimension of CH₂ vacancies. The condensation of kinks may form aqueous spaces into which molecular species of the size of low molecular weight can dissolve. This molecular view can explain permeability properties considering that water may be distributed along the hydrocarbon chains in the lipid matrix. The purpose of this review is to consolidate the mechanism anticipated by Träuble by discussing recent data in literature that directly correlates the molecular state of methylene groups of the lipids with the state of water in each of them. In addition, the structural properties of water near the lipid residues can be related with the water activity triggering kink formation by changes in the head group conformation that induces the propagation along the acyl chains and hence to the diffusion of water.     

Interaction of membrane proteins and lipids with solubilizing detergents

Detergents are indispensable in the isolation of integral membrane proteins from biological membranes to study their intrinsic structural and functional properties. Solubilization involves a number of intermediary states that can be studied by a variety of physicochemical and kinetic methods; it usually starts by destabilization of the lipid component of the membranes, a process that is accompanied by a transition of detergent binding by the membrane from a noncooperative to a cooperative interaction already below the critical micellar concentration (CMC). This leads to the formation of membrane fragments of proteins and lipids with detergent-shielded edges. In the final stage of solubilization membrane proteins are present as protomers, with the membrane inserted sectors covered by detergent. We consider in detail the nature of this interaction and conclude that in general binding as a monolayer ring, rather than as a micelle, is the most probable mechanism. This mode of interaction is supported by neutron diffraction investigations on the disposition of detergent in 3-D crystals of membrane proteins. Finally, we briefly discuss the use of techniques such as analytical ultracentrifugation, size exclusion chromatography, and mass spectrometry relevant for the structural investigation of detergent solubilized membrane proteins.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Effect of charged lidocaine on static and dynamic properties of model bio-membranes

The effect of the charged lidocaine on the structure and dynamics of DMPC/DMPG (mass fraction of 95/5) unilamellar vesicles has been investigated. Changes in membrane organization caused by the presence of lidocaine were detected through small angle neutron scattering experiments. Our results suggest that the presence of lidocaine in the vicinity of the headgroups of lipid membranes leads to an increase of the area per lipid molecule and to a decrease of membrane thickness. Such changes in membrane structure may induce disordering of the tail group. This scenario explains the reduction of the main transition temperature of lipid membranes, as the fraction of lidocaine per lipid molecules increases, which was evident from differential scanning calorimetry results. Furthermore neutron spin echo spectroscopy was used for the dynamics measurements and the results reveal that presence of charged lidocaine increases the bending elasticity of the lipid membranes in the fluid phase and slows the temperature-dependent change of bending elasticity across the main transition temperature.