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Unravelling 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. Here, 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. The structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral packing, was 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.
Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.
SARS-CoV2 spike glycoprotein is prime target for vaccines and for diagnostics and therapeutic antibodies against the virus. While anchored in the viral envelope, for effective virulance, the spike needs to maintain structural flexibility to recognize the host cell surface receptors and bind to them, a property that can heavily hinge upon the dynamics of the unresolved domains, most prominently the stalk. Construction of the complete, membrane-bound spike model and the description of its dynamics remain critical steps in understanding the inner working of this key element in viral infection. Using a hybrid approach, combining homology modeling, protein-protein docking and MD simulations, guided by biochemical and glycomics data, we have developed a full-length, membrane-bound, palmitoylated and fully-glycosylated spike structure in a native membrane. Multi-microsecond MD simulations of this model, the longest known trajectory of the full-spike, reveals conformational dynamics employed by the protein to explore the crowded surface of the host cell. In agreement with cryoEM, three flexiblele hinges in stalk allow for global conformational heterogeneity of spike in the fully-glycosyslated system mediated by glycan-glycan and glycan-lipid interactions. Dynamical range of spike is considerably reduced in its non-glycosylated form, confining the area explored by the spike on the host cell surface. Furthermore, palmitoylation of the membrane domain amplify the local curvature that may prime the fusion. We show that the identified hinge regions are highly conserved in SARS coronaviruses, highlighting their functional importance in enhancing viral infection, and thereby provide novel points for discovery of alternative therapeutics against the virus.
SARS-CoV2 Spike protein, which forms the basis for high pathogenicity and transmissibility of the virus, is also prime target for the development of both diagnostics and vaccines for the debilitating disease caused by the virus. We present a full model of spike methodically crafted and used to study its atomic-level dynamics by multiple- µ s simulations. The results shed new light on the impact of posttranslational modifications in the pathogenicity of the virus. We show how glycan-glycan and glycan-lipid interactions broaden the protein’s dynamical range, and thereby, its effective interaction with the surface receptors on the host cell. Palmitoylation of spike membrane domain, on the other hand, results in a unique deformation pattern that might prime the membrane for fusion.
SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.
Fluid interfaces, i.e. the boundary layer of two liquids or a liquid and a gas, play a vital role in physiological processes as diverse as visual perception, oral health and taste, lipid metabolism, and pulmonary breathing. These fluid interfaces exhibit a complex composition, structure, and rheology tailored to their individual physiological functions. Advances in interfacial thin film techniques have facilitated the analysis of such complex interfaces under physiologically relevant conditions. This allowed new insights on the origin of their physiological functionality, how deviations may cause disease, and has revealed new therapy strategies. Furthermore, the interactions of physiological fluid interfaces with exogenous substances is crucial for understanding certain disorders and exploiting drug delivery routes to or across fluid interfaces. Here, we provide an overview on fluid interfaces with physiological relevance, namely tear films, interfacial aspects of saliva, lipid droplet digestion and storage in the cell, and the functioning of lung surfactant. We elucidate their structure-function relationship, discuss diseases associated with interfacial composition, and describe therapies and drug delivery approaches targeted at fluid interfaces.
Statement of Significance
Fluid interfaces are inherent to all living organisms and play a vital role in various physiological processes. Examples are the eye tear film, saliva, lipid digestion & storage in cells, and pulmonary breathing. These fluid interfaces exhibit complex interfacial compositions and structures to meet their specific physiological function. We provide an overview on physiological fluid interfaces with a focus on interfacial phenomena. We elucidate their structure-function relationship, discuss diseases associated with interfacial composition, and describe novel therapies and drug delivery approaches targeted at fluid interfaces. This sets the scene for ocular, oral, or pulmonary surface engineering and drug delivery approaches.
Langmuir monolayers containing different amounts of cholesterol and cholesteryl stearate were studied at two different temperatures (24 °C and 35 °C). The main goal was to contribute towards the understanding of how the variations in the chemical composition may affect the physico‐chemical properties of these specific lipid monolayers. The model mixture was chosen considering that cholesteryl esters are present in cell membranes and some other biological systems, including human tear lipids. Therefore, an investigation into the effect of the lipid monolayer composition on their interfacial properties may elucidate some of the fundamental reasons for the deficiencies in cell membranes and tear film functioning in vivo. The experimental results have shown that the molar ratio of the mixture plays a crucial role in the modulation of the Langmuir film properties. The condensing effects of the cholesterol and the interactions between the lipids in the monolayer were the main factors altering the monolayer response to dilatational deformation.
The modification of the mixture compositions leads to significant changes in the Langmuir films and the mechanical performance, altering the ability of the monolayer to reduce the surface tension
and the viscoelastic properties of the monolayers. This suggests that subtle modifications of the biomembrane composition may significantly alter its physiological function.
Eukaryotic and prokaryotic cell membranes are difficult to characterize directly with biophysical methods. Membrane model systems, that include fewer molecular species, are therefore often used to reproduce their fundamental chemical and physical properties. In this context, natural lipid mixtures directly extracted from cells are a valuable resource to produce advanced models of biological membranes for biophysical investigations and for the development of drug testing platforms. In this study we focused on single phospholipid classes, i.e. Pichia pastoris phosphatidylcholine (PC) and Escherichia coli phosphatidylglycerol (PG) lipids. These lipids were characterized by a different distribution of their respective acyl chain lengths and number of unsaturations. We produced both hydrogenous and deuterated lipid mixtures. Neutron diffraction experiments at different relative humidities were performed to characterize multilayers from these lipids and investigate the impact of the acyl chain composition on the structural organization. The novelty of this work resides in the use of natural extracts with a single class head-group and a mixture of chain compositions coming from yeast or bacterial cells. The characterization of the PC and PG multilayers showed that, as a consequence of the heterogeneity of their acyl chain composition, different lamellar phases are formed.
Human bestrophin-1 protein (hBest1) is a transmembrane channel associated with the calcium-dependent transport of chloride ions in the retinal pigment epithelium as well as with the transport of glutamate and GABA in nerve cells. Interactions between hBest1, sphingomyelins, phosphatidylcholines and cholesterol are crucial for hBest1 association with cell membrane domains and its biological functions. As cholesterol plays a key role in the formation of lipid rafts, motional ordering of lipids and modeling/remodeling of the lateral membrane structure, we examined the effect of different cholesterol concentrations on the surface tension of hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and hBest1/SM Langmuir monolayers in the presence/absence of Ca2+ ions using surface pressure measurements and Brewster angle microscopy studies. Here, we report that cholesterol: (1) has negligible condensing effect on pure hBest1 monolayers detected mainly in the presence of Ca2+ ions, and; (2) induces a condensing effect on composite hBest1/POPC and hBest1/SM monolayers. These results offer evidence for the significance of intermolecular protein–lipid interactions for the conformational dynamics of hBest1 and its biological functions as multimeric ion channel.
Cholesterol regulates critical cell functions, including lysis, viral budding, and antibiotic resistance, by modifying the bending rigidity of cell membranes; i.e., the ability of membranes to bend or withstand mechanical stresses. A molecular-level understanding of these functions requires knowledge of how cholesterol modifies membrane mechanics over relevant length and time scales. Currently, it is widely accepted that cholesterol has no effect on the mechanical properties of unsaturated lipid membranes, implying that viruses, for example, can bud from regions enriched in (poly)unsaturated lipids. Our observations that cholesterol causes local stiffening in DOPC membranes indicate that a reassessment of existing concepts is necessary. These findings have far-reaching implications in understanding cholesterol’s role in biology and its applications in bioengineering and drug design.
The outbreak of COVID-19 across the world has posed unprecedented and global challenges on multiple fronts. Most of the vaccine and drug development has focused on the spike proteins and viral RNA-polymerases and main protease for viral replication. Using the bioinformatics and structural modelling approach, we modelled the structure of the envelope (E)-protein of novel SARS-CoV-2. The E-protein of this virus shares sequence similarity with that of SARS- CoV-1, and is highly conserved in the N-terminus regions. Incidentally, compared to spike proteins, E proteins demonstrate lower disparity and mutability among the isolated sequences. Using homology modelling, we found that the most favorable structure could function as a gated ion channel conducting H⁺ ions. Combining pocket estimation and docking with water, we determined that GLU 8 and ASN 15 in the N-terminal region were in close proximity to form H-bonds which was further validated by insertion of the E protein in an ERGIC-mimic membrane. Additionally, two distinct “core” structures were visible, the hydrophobic core and the central core, which may regulate the opening/closing of the channel. We propose this as a mechanism of viral ion channeling activity which plays a critical role in viral infection and pathogenesis. In addition, it provides a structural basis and additional avenues for vaccine development and generating therapeutic interventions against the virus.
A simple, insoluble monolayer of fatty acid is shown to induce 3D nanotexturing of the air–water interface. This advance has been achieved through the study of monolayers of a methyl-branched long chain fatty acid, analogous to those found on the surface of hair and wool, directly at the air–water interface. Specular neutron reflectometry combined with AFM probing of deposited monolayers shows pronounced 3D surface domains, which are absent for unbranched analogues and which are attributed to hydrocarbon packing constraints. The resulting surface topographies of the water far exceed the height perturbation that can be explained by the presence of capillary waves of a free liquid surface. These have hitherto been considered the only source of perturbation of the flatness of a planar water interface under gravity in the absence of topographical features from the presence of extended, globular or particulate matter. This amounts to a paradigm shift in the study of interfacial films and opens the possibility of 3D texturing of the air–water interface.
Biological membranes are key elements for the maintenance of cell architecture and physiology. Beyond a pure barrier separating the inner space of the cell from the outer, the plasma membrane is a scaffold and player in cell-to-cell communication and the initiation of intracellular signals among other functions. Critical to this function is the plasma membrane compartmentalization in lipid microdomains that control the localization and productive interactions of proteins involved in cell signal propagation. In addition, cells are divided into compartments limited by other membranes whose integrity and homeostasis are finely controlled, and which determine the identity and function of the different organelles. Here, we review current knowledge on membrane lipid composition in the plasma membrane and endomembrane compartments, emphasizing its role in sustaining organelle structure and function. The correct composition and structure of cell membranes define key pathophysiological aspects of cells. Therefore, we explore the therapeutic potential of manipulating membrane lipid composition with approaches like membrane lipid therapy, aiming to normalize cell functions through the modification of membrane lipid bilayers.
Lung surfactant is a complex mixture of lipids and proteins which plays a major role in the respiratory cycle. This makes necessary to understand the effects of different external factors or agents, for example, inhaled particles, as a potential source of alteration of the normal physiological response of lung surfactant. However, in most cases, in vivo studies are difficult to perform, and preliminary studies based in model systems are required. Films of lipids or mixtures of lipids and proteins at the water–vapor interface are accounted as one of the most useful methodologies for initial assessments of the potential toxicity of inhaled particles. Thus, the study of the modifications induced by the incorporation of colloidal particles in the interfacial properties of layers mimicking some of the physicochemical features of lung surfactant might provide a first evaluation of the risks and hazards associated with the inhalation of particulate matter. Considering the importance of particles in technology and industry, it is mandatory to develop strategies providing information about toxicological aspects of these widespread materials. This review focuses its interest on the recent advancements on the application of studied bases on monolayers at the fluid interface as preliminary assay for deepening on a complex situation with biological interest.
The calculation of neutron reflectivity from raw time-of-flight data including instrumental corrections as well as improved resolution calculation is presented. The theoretical calculations are compared to experimental data measured on the vertical sample plane reflectometer D17 and the horizontal sample plane reflectometer FIGARO at the Institut Laue-Langevin, Grenoble, France (ILL). This article comprises the mathematical body of the time-of-flight reflectivity data reduction software COSMOS which is used on D17 and FIGARO.
is a free software application based on MATLAB scripts designed for the graphical analysis, inspection and simulation of neutron reflectivity data. Its architecture, combined with graphics and other advantages of the MATLAB environment, should allow continued development of this software and inclusion of new features and analysis methods. The development of the software was driven by the necessity for a non-commercial open-source application for the analysis of neutron reflectivity data.
provides a robust and reliable method for evaluation of parameter uncertainty, a feature almost absent in similar software applications. In the present paper the main functionalities of the software are presented, together with a comprehensive description of the modeling approaches available at the moment. The code is released under a Creative Commons Attribution Non-Commercial License V2.0. The software application can be downloaded at http://aurorenr.sourceforge.net/.
The lipid composition of cellular organelles is tailored to suit their specialized tasks. A fundamental transition in the lipid landscape divides the secretory pathway in early and late membrane territories, allowing an adaptation from biogenic to barrier functions. Defending the contrasting features of these territories against erosion by vesicular traffic poses a major logistical problem. To this end, cells evolved a network of lipid composition sensors and pipelines along which lipids are moved by non-vesicular mechanisms. We review recent insights into the molecular basis of this regulatory network and consider examples in which malfunction of its components leads to system failure and disease.
Fluid Interfaces Grazing Angles ReflectOmeter (FIGARO) is the new horizontal neutron reflectometer at the Institut Laue-Langevin,
Grenoble, France. It is a versatile, high-flux time-of-flight instrument with features suitable for a range of studies in
soft condensed matter, chemistry, physics and biology both at free air/liquid interfaces and buried liquid/liquid and solid/liquid
interfaces. Most of the experiments exploit isotopic contrast variation to determine the structure and composition of surface
layers. Multiple chopper discs allow variable wavelength resolution, with the loose-resolution options increasing the available
neutron flux for fast kinetic studies of thin films and improving the data acquisition efficiency from complex samples requiring
multiple contrast measurements. It is possible to approach the interface with neutrons from below or above the horizon. The
instrument is equipped with a range of sample environments including free liquid adsorption troughs, a Langmuir trough, an
overflowing cylinder and a range of solid/liquid sample cells. FIGARO was launched as a user instrument in April 2009 and
its success has been steadily increasing since. This article includes an introduction to the scientific case, detailed sections
on the instrument components and performance, and descriptions of standard sample environments developed to date as well as
some selected early scientific highlights.
A microscope employing the characteristics of the reflection at the Brewster angle has been built for the study of first‐order phase transitions in monolayers and the growth of two‐dimensional domains without adding fluorescent impurities. It takes about 2.4 s to constitute an image.
The composition of Langmuir monolayers used as cell membrane models is an essential factor for the interaction with biologically-relevant molecules, including pharmaceutical drugs. In this paper, we report the modulation of effects from the antineoplastic drug paclitaxel by the relative concentration of cholesterol in the Langmuir monolayers of ternary mixtures of dipalmitoylphosphatidylcholine, sphingomyelin, and cholesterol. Since the dependence on cholesterol concentration for these monolayers simulating lipid rafts is non-monotonic, we analyzed the surface pressure and compressibility modulus data with the multidimensional projection technique referred to as interactive document mapping (IDMAP). The maximum expansion induced by paclitaxel in surface pressure isotherms was observed for 27% cholesterol, while the compressibility modulus decreased most strongly for the monolayer with 48% cholesterol. Therefore, the physiological action of paclitaxel may vary depending on whether it is associated with penetration in the membrane or with changes in the membrane elasticity.
The pulmonary surfactant (PS) is a complex mixture of lipids and proteins dispersed in the aqueous lining layer of the alveolar surface. Such a layer plays a key role in maintaining the proper lung functionality. It acts as a barrier against inhaled particles and pathogens, including viruses, and may represent an important entry point for drugs delivered via aerosols. Understanding the physico-chemical properties of PS is therefore of importance for the comprehension of pathophysiological mechanisms affecting the respiratory system. That can be of particular relevance for supporting the development of novel therapeutic interventions against COVID-19-induced acute respiratory distress syndrome. Due to the complexity of the in vivo alveolar lining layer, several in vitro methodologies have been developed to investigate the functional and structural properties of PS films or interfacial films made by major constituents of the natural PS. As breathing is a highly dynamic interfacial process, most applied methodologies for studying pulmonary surfactants need to be capable of dynamic measurements, including the study of interfacial dilational rheology. We provide here a review of the most frequently and successfully applied methodologies that have proven to be excellent tools for understanding the biophysics of the PS and of its role in the respiratory mechanics. This overview also discusses recent findings on the dynamics of pulmonary surfactant layers and on the impact of inhalable particles or pathogens, such as the novel coronavirus, on its functionality.
This review focuses on the description of the structure and composition of a variety of Langmuir monolayers (LMs) deposited at the air/water interface by using ellipsometry, Brewster Angle microscopy and scattering techniques, mainly neutron and X-ray reflectometry. Since the first experiment done by Angels Pockels with a homemade trough in her home kitchen until today, LMs of different materials have been extensively studied providing not only relevant model systems in biology, physics and chemistry but also precursors of novel materials via their deposition on solid substrates. There is a vast amount of surface-active materials that can form LMs and, therefore, far from a revision of the state-of-the-art, we will emphasize here: (i) some fundamental aspects to understand the physics behind the molecular deposition at the air/water interface; (ii) the advantages in using in situ techniques, such as reflectometry or ellipsometry, to resolve the interfacial architecture and conformation of molecular films; and, finally, (iii) a summary of several systems that have certain interest from the experimental or conceptual point of view. Concretely, we will report here advances in polymers confined to interfaces and surfactants, from fatty acids and phospholipids monolayers to more unconventional ones such as graphene oxide.
Antimicrobial peptides (AMPs) kill microorganisms by causing structural damage to bacterial membranes. Different microorganisms often require a different type and concentration of an AMP to achieve full microbial killing. We hypothesise that the difference is caused by different membrane structure and composition.
Given the complexities of bacterial membranes, we have used the binary DPPG/TMCL to mimic the cytoplasmic membrane of Gram-positive bacteria and the binary DPPG/DPPE to mimic the cytoplasmic membrane of Gram-negative bacteria, where DPPG, TMCL and DPPE stand for 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1',3'-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-sn-glycerol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, respectively. A Langmuir trough was specially designed to control the spread lipid monolayers and facilitate neutron reflectivity measurements.
Surface pressure-area isotherm analysis revealed that all binary lipid systems mix non-ideally, but mixing is thermodynamically favoured. An increase in the surface pressure encourages demixing, resulting in phase separation and formation of clusters. Neutron reflectivity measurements were undertaken to study the binding of an antimicrobial peptide G(IIKK)4-I-NH2 (G4) to the binary DPPG/TMCL and DPPG/DPPE monolayer mixtures at the molar ratios of 6/4 and 3/7, respectively. The results revealed stronger binding and penetration of G4 to the DPPG/TMCL monolayer, indicating greater affinity of the antimicrobial peptide due to the electrostatic interaction and more extensive penetration into the more loosely packed lipid film. This work helps explain how AMPs attack different bacterial membranes, and the results are discussed in the context of other lipid models and antibacterial studies.
The time-averaged lateral organization of the lipids and proteins that make up mammalian cell membranes continues to be the subject of intense interest and debate. Since the introduction of the fluid mosaic model almost 50 years ago, the "lipid raft hypothesis" has emerged as a popular concept that has captured the imagination of a large segment of the biomembrane community. In particular, the notion that lipid rafts play a pivotal role in cellular processes such as signal transduction and membrane protein trafficking is now favored by many investigators. Despite the attractiveness of lipid rafts, their composition, size, lifetime, biological function, and even the very existence remain controversial. The central tenet that underlies this hypothesis is that cholesterol and high-melting lipids have favorable interactions (i.e., they pull together), which lead to transient domains. Recent nearest-neighbor recognition (NNR) studies have expanded the lipid raft hypothesis to include the influence that low-melting lipids have on the organization of lipid membranes. Specifically, it has been found that mimics of cholesterol and high-melting lipids are repelled (i.e., pushed away) by low-melting lipids in fluid bilayers. The picture that has emerged from our NNR studies is that lipid mixing is governed by a balance of these "push and pull" forces, which maximizes the number of hydrocarbon contacts and attractive van der Waals interactions within the membrane. The power of the NNR methodology is that it allows one to probe these push/pull interaction energies that are measured in tens of calories per mole.
Cellular membranes are complex structures and simplified analogues in the form of model membranes or biomembranes are used as platforms to understand fundamental properties of the membrane itself as well as interactions with various biomolecules such as drugs, peptides and proteins. Model membranes at the air-liquid and solid-liquid interfaces can be studied using a range of complementary surface-sensitive techniques to give a detailed picture of both the structure and physicochemical properties of the membrane and its resulting interactions. In this review, we will present the main planar model membranes used in the field to date with a focus on monolayers at the air-liquid interface, supported lipid bilayers at the solid-liquid interface and advanced membrane models such as tethered and floating membranes. We will then briefly present the principles as well as the main type of information on molecular interactions at model membranes accessible using a Langmuir trough, quartz crystal microbalance with dissipation monitoring, ellipsometry, atomic force microscopy, Brewster angle microscopy, Infrared spectroscopy, and neutron and X-ray reflectometry. A consistent example for following biomolecular interactions at model membranes is used across many of the techniques in terms of the well-studied antimicrobial peptide Melittin. The overall objective is to establish an understanding of the information accessible from each technique, their respective advantages and limitations, and their complementarity.
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Cell membrane models are useful to obtain molecular-level information on the interaction of biologically-re-levant molecules such as pesticides whose activity is believed to depend on its effects on the membrane. In thisstudy, we investigated the interaction between the widely used pesticide picloram with Langmuir monolayers ofbinary and ternary mixtures comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM)and cholesterol (Chol), which could be taken as representative of ocular membranes in humans. Picloram ex-panded the molecular area of DOPC/SM and DOPC/SM/Chol monolayers as the pesticide penetrated the hy-drophobic region of the mixtures. A clear correlation was also found between the compressibility modulus(Cs−1) and the presence of cholesterol in the ternary monolayer. Data from polarization-modulated infraredreflection absorption spectroscopy (PM-IRRAS) confirmed that picloram interacts with both the acyl chains andheadgroups. Spectral shifts and band broadening were induced by picloram, particularly for the phosphate andcholine groups, probably owing to its H-bonding ability. The effects reported here on the lipid monolayers maybe evidence of the possible activity of picloram on mammalian cell membranes, which highlights the importanceof strict control of the level of exposure of humans dealing with pesticid
The aim of the present study was to determine the interfacial behavior of model biomembrane in the presence of mucin. The Langmuir monolayer technique was used to form a mixed protein–phospholipid film at the air–water interface. Using the surface pressure-area isotherms, the relaxation curves and adsorption experiment, the nature of interactions between mucin and dipalmitoylphosphatidylcholine (DPPC) components of the monolayers under physiological conditions, (characteristic of the vagina and oral cavity), was investigated. It has been shown that at pH 4.2 mucin molecules reveal a strong affinity to the model biomembrane, composed of zwitterionic DPPC, and probably interact with it through the hydrophobic interactions. At pH 6.8 very weak electrostatic interactions are possible between the positively charged quaternary ammonium groups present in the DPPC molecules and the negatively charged mucin molecules. Moreover, from the relaxation experiment we concluded that beneath the DPPC layer there is a layer made of mucin particles due to electrostatic attraction. This layer of mucin beneath the DPPC particles stabilizes the entire system, especially at physiologically significant surface pressure, i.e. at π = 30 mN/m.
Research on lipid/drug interactions at the nanoscale underpins the emergence of synergistic mechanisms for topical drug administration. The structural understanding of bio-mimetic systems employing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) as a lung surfactant model mixed with antibiotics, as well as their biophysical properties, is of critical importance to modulate the effectiveness of therapeutic agents released directly to the airways. In this paper, we investigate the structural details of the interaction between Levofloxacin, 'a respiratory quinolone', and the macrolide Clarithromycin, with DPPC monolayers at the air-water interface, using a combination of Brewster angle microscopy, polarization modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS), surface pressure isotherms and neutron reflectometry (NR) to describe the structural details of this interaction. The results allowed association of changes in the π-A isotherm profile with changes in the molecular organization and the co-localization of the antibiotics within the lipid monolayer by NR measurements. Overall, both antibiotics are able to increase the thickness of the acyl tails in DPPC monolayers with a corresponding reduction in tail tilt as well as to interact with the phospholipid headgroups as shown by PM-IRRAS experiments. The effects on the DPPC monolayers are correlated with the physical-chemical properties of each antibiotic and dependent on its concentration.
In this study, the interaction between Lycium barbarum polysaccharide (LBP) and unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or saturated 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was explored using the Langmuir films technique and atomic force microscopy (AFM). Comparing the pure lipid monolayer with the mixed monolayers, the π-A isotherms of the mixed monolayers shifted to larger molecular areas when LBP was added to the subphase. The compression modulus showed that the compressibility of the monolayer films decreased with the addition of LBP. Adsorption curves revealed that the variation in the surface pressure of LBP with POPC was larger than that with DPPC. This phenomenon was verified by the AFM images and the number of each lipid molecule combining with polysaccharide molecules in the mixed monolayer (A p value), indicating that hydrophobic interactions between LBP and POPC are stronger than those of DPPC. These findings lay the foundation for exploring the pharmacological mechanism of LBP as an in vivo therapeutic.
Specular neutron reflectometry is a powerful technique to resolve interfacial compositions and structures in soft matter. Surprisingly however, even after several decades, a universal modeling approach for the treatment of data of surfactant and phospholipid monolayers at the air/water interface has not yet been established. To address this shortcoming, first a systematic evaluation of the suitability of different models is presented. The result is a comprehensive validation of an optimum model, which is evidently much needed in the field, and which we recommend as a starting point for future data treatment. While its limitations are openly discussed, consequences of failing to take into account various key aspects are critically examined and the systematic errors quantified. On the basis of this physical framework, we go on to show for the first time that neutron reflectometry can be used to quantify directly in situ at the air/water interface the extent of acyl chain compaction of phospholipid monolayers with respect to their phase. The achieved precision of this novel quantification is ∼10%. These advances together enhance significantly the potential for exploitation in future studies data from a broad range of systems including those involving synthetic polymers, proteins, DNA, nanoparticles and drugs.
The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that the tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.
The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures.
Coronaviruses are a major infectious disease threat, and include the pathogenic human pathogens of zoonotic origin: SARS-CoV and MERS-CoV. Entry of coronaviruses into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the coronavirus fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2' cleavage site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous ESR spectroscopic studies, the membrane ordering effect of influenza virus, HIV and Dengue virus FPs have been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium-dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca(2+)-dependency membrane ordering was not observed with influenza FP, indicating that the coronavirus FP exhibits a mechanistically different behavior. Membrane ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform".
In recent years, several studies based on the interaction of self-assembling short peptides derived from Viroporins with model membranes, have improved our understanding of the molecular mechanism of Corona virus (CoV) infection under physiological conditions. In this study, we have characterized the mechanism of membrane interaction of a short, 9-residue peptide TK9 (T(55)VYVYSRVK(63)) that had been derived from the carboxyl terminal of the Severe Acute Respiratory Syndrome (SARS) Corona virus (SARS CoV) Envelope (E) protein. The peptide has been studied for its physical changes in the presence of both zwitterionic DPC and negatively charged SDS model membrane micelles, respectively, with the help of a battery of biophysical techniques including two-dimensional solution state NMR spectroscopy. Interestingly, in both micelle environments, TK9 adopted an alpha helical conformation; however, the helical propensities were much higher in the case of DPC compared to those of SDS micelle, suggesting that TK9 has more specificity towards eukaryotic cell membrane than the bacterial cell membrane. The orientation of the peptide TK9 also varies in the different micellar environment. The peptide's affinity was further manifested by its pronounced membrane disruption ability towards the mammalian compared to the bacterial membrane mimic. Collectively, the in-depth structural information on the interaction of TK9 with different membrane environment explains the host specificity and membrane orientation owing to subsequent membrane disruption implicated in the viral pathogenesis.
This review addresses the advances made with specular neutron reflectometry in studies of aqueous mixtures of polymers and surfactants at fluid interfaces during the last decade. The increase in neutron flux due to improvements in instrumentation has led to routine measurements at the air/water interface that are both faster and involve samples with low isotopic contrast. One can now resolve the surface excess of a single deuterated component in one second and the composition of a mixture on the minute time scale, and information about adsorption processes and dynamic rheology can also be accessed. Research areas addressed include the types of formed equilibrium surface structures, the link to the foam film stability and the range of non-equilibrium effects that dominate the behavior of oppositely charged polyelectrolyte/surfactant mixtures, macroscopic film formation in like-charged polymer/surfactant mixtures, and the properties of mixtures of bio-polymers with surfactants and phospholipids.
Brewster angle microscopy (BAM) is a powerful technique that allows for real-time visualization of Langmuir monolayers. The lateral organization of these films can be investigated, including phase separation and the formation of domains, which may be of different sizes and shapes depending on the properties of the monolayer. Different molecules or small changes within a molecule such as the molecule's length or presence of a double bond can alter the monolayer's lateral organization that is usually undetected using surface pressure-area isotherms. The effect of such changes can be clearly observed using BAM in real-time, under full hydration, which is an experimental advantage in many cases. While previous BAM reviews focused more on selected compounds or compared the impact of structural variations on the lateral domain formation, this review provided a broader overview of BAM application using biological materials and systems including the visualization of amphiphilic molecules, proteins, drugs, extracts, DNA, and nanoparticles at the air-water interface.
The interactions between three triterpene saponins: α-hederin, hederacoside C and ammonium glycyrrhizate with model lipids: cholesterol and dipalmitoylphosphatidylcholine (DPPC) are described. The oleanolic acid-type saponins (α-hederin and hederacoside C) were shown to form 1:1 complexes with lipids in bulk, characterized by stability constants in the range (4.0±0.2)·10(3)-(5.0±0.4)·10(4)M(-1). The complexes with cholesterol are generally stronger than those with DPPC. On the contrary, ammonium glycyrrhizate does not form complexes with any of the lipids in solution. The saponin-lipid interactions were also studied in a confined environment of Langmuir monolayers of DPPC and DPPC/cholesterol with the saponins present in the subphase. A combined monolayer relaxation, surface dilational rheology, fluorescence microscopy and neutron reflectivity (NR) study showed that all three saponins are able to penetrate pure DPPC and mixed DPPC/cholesterol monolayers. Overall, the effect of the saponins on the model lipid monolayers does not fully correlate with the lipid-saponin complex formation in the homogeneous solution. The best correlation was found for α-hederin, for which even the preference for cholesterol over DPPC observed in bulk is well reflected in the monolayer studies and the literature data on its membranolytic activity. Similarly, the lack of interaction of ammonium glycyrrhizate with both lipids is evident equally in bulk and monolayer experiments, as well as in its weak membranolytic activity. The combined bulk and monolayer results are discussed in view of the role of confinement in modulating the saponin-lipid interactions and possible mechanism of membranolytic activity of saponins.
Lipid monolayers are often considered as model membranes, but they are also the physiologic lipid part of the peripheral envelope of lipoproteins and cytosolic lipid bodies. However, their structural organization is still rather elusive, in particular in case of simultaneous presence of cholesterol and sphingomyelin. To investigate such structural organization of hemi-membranes, we measured, using alternative current voltammetry, the differential capacitance of condensed phosphatidylcholine-based monolayers as a function of applied potential, which is sensitive to their lipid composition and molecular arrangement. In particular, monolayers containing both sphingomyelin and cholesterol, at 15 % w/w, presented specific characteristics for the differential capacitance vs potential curves recorded, which was indicative of specific interactions between these two lipid components. We then compared the behavior of two cholesterol derivatives (at 15 % w/w), 21-methylpyrenyl-cholesterol (Pyr-met-Chol) and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol), with that of cholesterol when present in model monolayers, because these two probes have been previously reported to present opposite behaviors within bilayer membranes regarding their interaction with ordered lipids, only Pyr-met-Chol well mimicking cholesterol. Remarkably, in monolayers containing sphingomyelin or not, Pyr-met-Chol and NBD-Chol presented contrasting behaviors, and Pyr-met-Chol mimicked cholesterol only in the presence of sphingomyelin. These two observations (i.e. optimal amounts of sphingomyelin and cholesterol, and ability to discriminate between Pyr-met-Chol and NBD-Chol) can be interpreted by the existence of heterogeneities including ordered patches in sphingomyelin- and cholesterol-containing monolayers. Since such monolayer lipid arrangement shares some properties with the raft-type lipid microdomains well-described in sphingomyelin- and cholesterol-containing bilayer membranes, these data thus fairly suggest the existence of compact and ordered microdomains in model lipid monolayers.
Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer–Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form “lipid rafts” that float in a “sea” of low-melting lipids.
The detailed organization of cellular membranes remains rather elusive. Based on large-scale molecular dynamics simulations, we provide a high-resolution view of the lipid organization of a plasma membrane at an unprecedented level of complexity. Our plasma membrane model consists of 63 different lipid species, combining 14 types of headgroups and 11 types of tails asymmetrically distributed across the two leaflets, closely mimicking an idealized mammalian plasma membrane. We observe an enrichment of cholesterol in the outer leaflet and a general non-ideal lateral mixing of the different lipid species. Transient domains with liquid-ordered character form and disappear on the microsecond time scale. These domains are coupled across the two membrane leaflets. In the outer leaflet, distinct nanodomains consisting of gangliosides are observed. Phosphoinositides show preferential clustering in the inner leaflet. Our data provide a key view on the lateral organization of lipids in one of life's fundamental structures, the cell membrane.
Analysis of the shape of the curve of reflected x-ray intensity vs glancing angle in the region of total reflection provides a new method of studying certain structural properties of the mirror surface about 10 to several hundred angstroms deep. Dispersion theory, extended to treat any (small) number of stratified homogeneous media, is used as a basis of interpretation.Curves for evaporated copper on glass at room temperature are studied as an example. These curves may be explained by assuming that the copper (exposed to atmospheric air at room temperature) has completely oxidized about 150A deep. If oxidation is less deep, there probably exists some general reduction of density (e.g., porosity) and an electron density minimum just below an internal oxide seal. This seal, about 25A below the nominal surface plane, arrests further oxidation of more deeply-lying loose-packed copper crystallites.All measurements to date have been carried out under laboratory atmospheric conditions which do not allow satisfactory separation or control of the physical and chemical variables involved in the surface peculiarities. The method, under more controlled conditions of preparation and treatment of the surface, promises to be useful.
The contrast-variation technique is employed in multiple-contrast neutron/X-ray reflectometry experiments to highlight scattering from different structural components that are present at a surface or interface. The advantage of this technique is that the structural model used to describe the interfacial scattering length density profile must apply to all the contrasts measured. A new reflectivity analysis package, MOTOFIT, which runs in the IGOR Pro environment (http://www.wavemetrics.com), has been created to aid the simultaneous fitting (with the same structural model) of these multiple-contrast data, using an intuitive graphical user interface, which most co-refinement packages do not possess. MOTOFIT uses a slab-model approach with the Abeles matrix method, and extensions for surface roughness to perform non-linear least-squares regression on the experimental reflectivity curves. Other features, such as the ability to create complicated interparameter constraints or analyse reflectivity from multilayers, simulated annealing, etc., make MOTOFIT a powerful reflectometry analysis package
Langmuir monolayers of Cholesterol (Chol) and a mixture of Chol with 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), at a ratio of 17:83 in weight, spread on pure water and on silica nanoparticle dispersions, have been investigated measuring the compression isotherms as well as the surface pressure response to harmonic area variation of the monolayer. Aim of this study was to evaluate the effects of the interaction of silica nanoparticles with Chol and the conditions for the incorporation in the monolayer. In previous works on different kind of lipid monolayers, it has been shown that hydrophilic silica nanoparticles dispersed in the sub-phase may transfer into the monolayer, driven by the interaction with the lipid molecules that make them partially hydrophobic. The results here obtained indicate that also for Chol and Chol-DPPC mixtures the presence of silica nanoparticles may have important effects on the phase behavior and structural properties of the monolayer. As confirmed by complementary structural characterisations, BAM, AFM and ellipsometry, the principal effect of the nanoparticle incorporation is the disruption of the monolayer packing, owing to the alteration of the cohesive interactions of lipid components.
Cellular membranes are densely crowded with a diverse population of integral and membrane associated proteins. In this complex environment, lipid rafts, which are phase-separated membrane domains enriched in cholesterol and saturated lipids, are thought to organize the membrane surface. Specifically, rafts may help to locally concentrate proteins and lipids, enabling cellular processes such as assembly of caveolae, budding of enveloped viruses, and sorting of lipids and proteins in the Golgi. However, the ability of rafts to concentrate protein species has not been quantified experimentally. Here we show that when membrane-bound proteins become densely crowded within liquid-ordered membrane regions, steric pressure arising from collisions between proteins can destabilize lipid phase-separations, resulting in a homogenous distribution of proteins and lipids over the membrane surface. Using a reconstituted system of lipid vesicles and recombinant proteins, we demonstrate that protein-protein steric pressure creates an energetic barrier to the stability of phase-separated membrane domains, which increases in significance as the molecular weight of proteins increases. Comparison with a simple analytical model reveals that do-mains are destabilized when steric pressure exceeds the approximate enthalpy of membrane mixing. These results suggest that a subtle balance of free energies governs the stability of phase-separated cellular membranes, providing a new perspective on the role of lipid rafts as concentrators of membrane proteins.
Thermally induced capillary waves on a monolayer of behenic acid on water have been investigated by x-ray reflectivity. The corresponding roughness is measured as a function of the surface pressure. The experimental data are discussed using a theory of thermal fluctuations. A strong decrease in the roughness is observed at the transition to the solid state. This striking feature is attributed to the rigidity of the monolayer in the solid phase whose value has been determined.
Mixed film studies of the systems cholesterol/tetradecanoic acid and cholesterol/dipalmitoylphosphatidylcholine have been carried out over the entire compositional range at 21°C. When compared on an acyl chain basis the condensing effects were found to be essentially independent of which host-lipid was utilized. The phase change of the host lipid was shifted to higher pressures, then broadened and eliminated. Maximal condensation occurred at just above 42 mol% for the cholesterol/DPPC system. In both systems the two components were initially found to be miscible at all proportions.The results are interpreted in terms of the molecular packing of cholesterol with acyl boundary layers, one significantly, one weakly affected. Maximum condensation is a result of packing that provides maximum cholesterol/acyl chain contact. Consideration is given to both long term stability of such mixed monolayers and the behaviour of the corresponding bilayer states.
A detailed statistical mechanical study has been carried out on a microscopic interaction model of lipid monolayers on the air—water interface. The model is Pink's ten-state model which accounts for the internal conformational states of the lipid acyl chains and their statistics and mutual interactions. Particular attention is paid to the chain-melting phase transition of saturated lecithin monolayers (e.g., DPPC) and the microscopic phenomena which accompany this phase transition. By means of computer-simulation techniques we have calculated the equilibrium and nonequilibrium isotherms and obtained quantitative measures of the lateral density fluctuations in the transition region in terms of the size distribution of lipid domains and the average coherence length of the conformational order of the acyl chains. Indications of the presence of a critical point are obtained. The results are compared with experimental observations of DPPC lipid monolayer phase behavior with special emphasis on an understanding of the origin of the nonhorizontal experimental isotherms.
The phospholipid molecular species of freshwater (pangasius, Nile perch, trout), marine fish fillets (horse mackerel, European hake, common sole, European anchovy, European pilchard, Atlantic mackerel) and the edible muscle foot of bivalves (clam, mussel, oyster) commonly available in the Italian market during spring and summer were characterized by means of normal-phase high performance liquid chromatography coupled online with positive electrospray ionization ion-trap tandem mass spectrometry. From principal component analysis (PCA), it was observed that the total fatty acid profile was not suitable to differentiate among the shellfish genera. The fatty acid molecular combinations of phosphatidylcholine, the main phospholipid class, as well as phosphatidylinositol and phosphatidylethanolamine allowed for the differentiation of shellfish from the bony fishes. Phosphatidylserine and phosphatidylethanolamine plasmalogen profile allowed for the discrimination of each bony fish or shellfish genus since PS and pPE classes included a large number of fatty acid combinations that were specific for a fish genus or group.