High-density lipoproteins (HDLs) are responsible for removing cholesterol from arterial walls, through a process known as reverse cholesterol transport. The main protein in HDL, apolipoprotein A-I (ApoA-I), is essential to this process, and changes in its sequence significantly alter HDL structure and functions. ApoA-I amyloidogenic variants, associated with a particular hereditary degenerative disease, are particularly effective at facilitating cholesterol removal, thus protecting carriers from cardiovascular disease. Thus, it is conceivable that reconstituted HDL (rHDL) formulations containing ApoA-I proteins with functional/structural features similar to those of amyloidogenic variants hold potential as a promising therapeutic approach. Here we explored the effect of protein cargo and lipid composition on the function of rHDL containing one of the ApoA-I amyloidogenic variants G26R or L174S by Fourier transformed infrared spectroscopy and neutron reflectometry. Moreover, small-angle x-ray scattering uncovered the structural and functional differences between rHDL particles, which could help to comprehend higher cholesterol efflux activity and apparent lower phospholipid (PL) affinity. Our findings indicate distinct trends in lipid exchange (removal vs. deposition) capacities of various rHDL particles, with the rHDL containing the ApoA-I amyloidogenic variants showing a markedly lower ability to remove lipids from artificial membranes compared to the rHDL containing the native protein. This effect strongly depends on the level of PL unsaturation and on the particles' ultrastructure. The study highlights the importance of the protein cargo, along with lipid composition, in shaping rHDL structure, contributing to our understanding of lipid-protein interactions and their behavior.

Neutrons are characterized by a low absorption in many engineering materials. At the same time the scattering cross section of light elements, such as hydrogen and deuterium, may be large. These properties make neutron scattering experiments performed under grazing incidence geometry an excellent tool for the study of solid–liquid interfaces. In this review we describe the basic concepts of neutron reflection and grazing incidence scattering experiments as well as experimental procedures and sample cells. The full power of the method is exemplified on a range of science areas, including polymers, bio- and ionic liquid lubricants, electrolytes as well as bio-membranes or magnetic liquids.

There is a close relationship between the SARS-CoV-2 virus and lipoproteins, in particular high-density lipoprotein (HDL). The severity of the coronavirus disease 2019 (COVID-19) is inversely correlated with HDL plasma levels. It is known that the SARS-CoV-2 spike (S) protein binds the HDL particle, probably depleting it of lipids and altering HDL function. Based on neutron reflectometry (NR) and the ability of HDL to efflux cholesterol from macrophages, we confirm these observations and further identify the preference of the S protein for specific lipids and the consequent effects on HDL function on lipid exchange ability. Moreover, the effect of the S protein on HDL function differs depending on the individuals lipid serum profile. Contrasting trends were observed for individuals presenting low triglycerides/high cholesterol serum levels (LTHC) compared to high triglycerides/high cholesterol (HTHC) or low triglycerides/low cholesterol serum levels (LTLC). Collectively, these results suggest that the S protein interacts with the HDL particle and, depending on the lipid profile of the infected individual, it impairs its function during COVID-19 infection, causing an imbalance in lipid metabolism.

Lipid nanoparticles (LNPs) are the most versatile and successful gene delivery systems, notably highlighted by their use in vaccines against COVID-19. LNPs have a well-defined core-shell structure, each region with its own distinctive compositions, suited for a wide range of in vivo delivery applications. Here, we discuss how a detailed knowledge of LNP structure can guide LNP formulation to improve the efficiency of delivery of their nucleic acid payload. Perspectives are detailed on how LNP structural design can guide more efficient nucleic acid transfection. Views on key physical characterization techniques needed for such developments are outlined including opinions on biophysical approaches both correlating structure with functionality in biological fluids and improving their ability to escape the endosome and deliver they payload.

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.

Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both alpha-helical and beta-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with alpha-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.

Bicelles are disk-shaped models of cellular membranes used to study lipid–protein interactions, as well as for structural and functional studies on transmembrane proteins. One challenge for the incorporation of transmembrane proteins in bicelles is the limited range of detergent and lipid combinations available for the successful reconstitution of proteins in model membranes. This is important, as the function and stability of transmembrane proteins are very closely linked to the detergents used for their purification and to the lipids that the proteins are embedded in. Here, we expand the toolkit of lipid and detergent combinations that allow the formation of stable bicelles. We use a combination of dynamic light scattering, small-angle X-ray scattering and cryogenic electron microscopy to perform a systematic sample characterization, thus providing a set of conditions under which bicelles can be successfully formed.

Tissue factor (TF) is a membrane protein involved in blood coagulation. TF initiates a cascade of proteolytic reactions, ultimately leading to the formation of a blood clot. The first reaction consists of the binding of the coagulation factor VII and its conversion to the activated form, FVIIa. Here, we combined experimental, i.e. quartz crystal microbalance with dissipation monitoring and neutron reflectometry, and computational, i.e. molecular dynamics (MD) simulation, methods to derive a complete structural model of TF and TF/FVIIa complex in a lipid bilayer. This model shows that the TF transmembrane domain (TMD), and the flexible linker connecting the TMD to the extracellular domain (ECD), define the location of the ECD on the membrane surface. The average orientation of the ECD relative to the bilayer surface is slightly tilted towards the lipid headgroups, a conformation that we suggest is promoted by phosphatidylserine lipids, and favours the binding of FVIIa. On the other hand, the formation of the TF/FVIIa complex induces minor changes in the TF structure, and reduces the conformational freedom of both TF and FVIIA. Altogether we describe the protein-protein and protein-lipid interactions favouring blood coagulation, but also instrumental to the development of new drugs.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Emerging therapeutic treatments based on the production of proteins by delivering mRNA have become increasingly important in recent times. While lipid nanoparticles (LNPs) are approved vehicles for small interfering RNA delivery, there are still challenges to use this formulation for mRNA delivery. LNPs are typically a mixture of a cationic lipid, distearoylphosphatidylcholine (DSPC), cholesterol, and a PEG-lipid. The structural characterization of mRNA-containing LNPs (mRNA-LNPs) is crucial for a full understanding of the way in which they function, but this information alone is not enough to predict their fate upon entering the bloodstream. The biodistribution and cellular uptake of LNPs are affected by their surface composition as well as by the extracellular proteins present at the site of LNP administration, e.g., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP’s plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape. 

Continuous and automated bacteria detection is pivotal for a myriad of biomedical, food safety and envi-ronmental applications. This work presents the fabrication of a prototype of a passive (battery-free) radio frequency sensor for wireless detection of bacteria. The sensing mechanism is based on the bacterial-induced (proteases and peptidases) degradation of glutaraldehyde (GTA) cross-linked gelatin-caprylic acid (CA) composite film. Proteolytic degradation of the film resulted in a decrease of its resistivity, a quan-tity that could be wirelessly monitored by coupling the film to a radio-frequency antenna (an inductor-capacitor resonator) and monitoring the frequency for which the transferred power between this antenna and another antenna connected to a Vector Network Analyzer (VNA) was maximized. We experimen-tally proved this concept by monitoring E.coli bacteria in aqueous medium and detected at 18.0 +/- 2.8 h, 23.5 +/- 0.7 h, 27.0 +/- 2.8 h, 40.5 +/- 3.5 h, 45.5 +/- 0.7 h for the initial E.coli concentration of 3.2 +/- 10(8) , 6.8 +/- 10(7) , 2.3 +/- 10(6) , 4.3 +/- 10(5) , and 3.6 +/- 10(4) CFU/mL, respectively. Further, the E.coli induced degrada-tion of the composite film was investigated by evaluating the thickness of the film by optical microscopy as well as morphology by scanning electron microscopy techniques. (C) 2021 Published by Elsevier Ltd.

Supported lipid bilayers (SLBs) are commonly used to investigate the structure and dynamics of biological membranes. Vesicle fusion is a widely exploited method to produce SLBs. However, this process becomes less favoured when the vesicles contain complex lipid mixtures, e.g. natural lipid extracts. In these cases, it is often necessary to change experimental parameters, such as temperature, to unphysiological values to trigger the SLB formation. This may induce lipid degradation and is also not compatible with including membrane proteins or other biomolecules into the bilayers. Here, we show that the peptide discs, ~10 nm discoidal lipid bilayers stabilized in solution by a self-assembled 18A peptide belt, can be used as precursors for SLBs. The characterizations by means of neutron reflectometry and attenuated total reflectance-FTIR spectroscopy show that SLBs were successfully formed both from synthetic lipid mixtures (surface coverage 90-95%) and from natural lipid mixtures (surface coverage ~85%). Traces of 18A peptide (below 0.02 M ratio) left at the support surface after the bilayer formation do not affect the SLB structure. Altogether, we demonstrate that peptide disc formation of SLBs is much faster than the SLB formation by vesicle fusion and without the need of altering any experimental variable from physiologically relevant values.

Cholesterol has been shown to affect the extent of coronavirus binding and fusion to cellular membranes. The severity of Covid-19 infection is also known to be correlated with lipid disorders. Furthermore, the levels of both serum cholesterol and high-density lipoprotein (HDL) decrease with Covid-19 severity, with normal levels resuming once the infection has passed. Here we demonstrate that the SARS-CoV-2 spike (S) protein interferes with the function of lipoproteins, and that this is dependent on cholesterol. In particular, the ability of HDL to exchange lipids from model cellular membranes is altered when co-incubated with the spike protein. Additionally, the S protein removes lipids and cholesterol from model membranes. We propose that the S protein affects HDL function by removing lipids from it and remodelling its composition/structure.

Lipid nanoparticles (LNPs) are promising drug and gene carriers. Upon intravenous administration, LNPs' experience different degree of cellular uptake depending on their formulation. Currently, in vitro and in vivo studies are the gold standard for assessing the fate of nano carriers once administered, but they are time consuming and expensive. In this work, we propose a time and cost-effective method to screen a wide range of LNP formulations and select the most promising candidates for in vitro and in vivo studies. Two different approaches were explored to investigate the binding affinity between LNPs and serum proteins using sensor functionalisation with either protein specific antibody or PEG specific antibody. The first approach allowed to identify the presence of a specific protein in the protein corona of lipid particles (reconstituted and native high-density lipoproteins (rHDL and HDL), and low-density lipoproteins LDL); while the second one provided a versatile platform for the immobilisation of pegylated-particles in order to follow the interaction with serum proteins and hence predict the composition of LNP protein corona. Sensing was done using Quartz Crystal Microbalance with Dissipation (QCM-D) but the approach is extendable to other surface sensing techniques such as Surface Plasmon Resonance (SPR) or ellipsometry.

Atherosclerosis, the largest killer in the western world, arises from build-up of plaques at the artery walls and can result in cardiovascular disease. Low- and high-density lipoproteins are involved in the disease development by depositing and removing lipids to and from macrophages at the artery wall. These processes are complex and not fully understood. Thus, determining the specific roles of the different lipoprotein fractions involved is of fundamental importance for the treatment of the disease. In this chapter, we present the state of the art in lipoprotein structure with focus on the comparison between normolipidemic and hypertriglyceridemic individuals. Then we discuss lipid transfer between lipoproteins and receptor-free cellular membranes. Although these models lack any receptor, key clinical observations are mirrored by these, including increased ability of HDL to remove lipids, in contrast to the ability of LDL to deposit them. Also effects of saturated and unsaturated lipids in the presence and absence of cholesterol are revised. These models can then be used to understand the difference in functionality of lipoproteins from individuals showing different lipid profiles and have the potential to be used also for the development of new HDL therapies. 

Atherosclerosis is the main killer in the west and therefore a major health challenge today. Total serum cholesterol and lipoprotein concentrations, used as clinical markers, fail to predict the majority of cases, especially between the risk scale extremes, due to the high complexity in lipoprotein structure and composition. In particular, low-density lipoprotein (LDL) plays a key role in atherosclerosis development, with LDL size being a parameter considered for determining the risk for cardiovascular diseases. Determining LDL size and structural parameters is challenging to address experimentally under physiological-like conditions. This article describes the biochemistry and ultrastructure of normolipidemic and hypertriglyceridemic LDL fractions and subfractions using small-angle X-ray scattering. Our results conclude that LDL particles of hypertriglyceridemic compared to healthy individuals 1) have lower LDL core melting temperature, 2) have lower cholesteryl ester ordering in their core, 3) are smaller, rounder and more spherical below melting temperature, and 4) their protein-containing shell is thinner above melting temperature.

We have performed grazing incidence neutron small angle scattering using a fan shaped incident beam focused along one dimension. This allows significantly reduced counting times for measurements of lateral correlations parallel to an interface or in a thin film where limited depth resolution is required. We resolve the structure factor of iron inclusions in aluminium oxide and show that the ordering of silica particles deposited on a silicon substrate depends on their size. We report hexagonal packing for 50 nm but not for 200 nm silica spheres deposited by a modified Langmuir-Schaefer method on a silicon substrate. For the 200 nm particles we extract the particles shape from the form factor. Moreover, we report dense packing of the particles spread on a free water surface. We name this method π-GISANS to highlight that it differs from GISANS as it gives lateral information while averaging the in-depth structure.

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.

Supramolecular assembly and PEGylation (attachment of a polyethylene glycol polymer chain) of peptides can be an effective strategy to develop antimicrobial peptides with increased stability, antimicrobial efficacy and hemocompatibility. However, how the self-assembly properties and PEGylation affect their lipid membrane interaction is still an unanswered question. In this work, we use state-of-the-art small angle X-ray and neutron scattering (SAXS/SANS) together with neutron reflectometry (NR) to study the membrane interaction of a series of multidomain peptides, with and without PEGylation, known to self-assemble into nanofibers. Our approach allows us to study both how the structure of the peptide and the membrane are affected by the peptide–lipid interactions. When comparing self-assembled peptides with monomeric peptides that are not able to undergo assembly due to shorter chain length, we found that the nanofibers interact more strongly with the membrane. They were found to insert into the core of the membrane as well as to absorb as intact fibres on the surface. Based on the presented results, PEGylation of the multidomain peptides leads to a slight net decrease in the membrane interaction, while the distribution of the peptide at the interface is similar to the non-PEGylated peptides. Based on the structural information, we showed that nanofibers were partially disrupted upon interaction with phospholipid membranes. This is in contrast with the considerable physical stability of the peptide in solution, which is desirable for an extended in vivo circulation time.

Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as 'good' and 'bad' cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as for an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.

In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with similar to 0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.

The structural integrity of living plant cells heavily relies on the plant cell wall containing a nanofibrous cellulose skeleton. Hence, if synthetic plant cells consist of such a cell wall, they would allow for manipulation into more complex synthetic plant structures. Herein, we have overcome the fundamental difficulties associated with assembling lipid vesicles with cellulosic nanofibers (CNFs). We prepare plantosomes with an outer shell of CNF and pectin, and beneath this, a thin layer of lipids (oleic acid and phospholipids) that surrounds a water core. By exploiting the phase behavior of the lipids, regulated by pH and Mg²⁺ ions, we form vesicle-crowded interiors that change the outer dimension of the plantosomes, mimicking the expansion in real plant cells during, e.g., growth. The internal pressure enables growth of lipid tubules through the plantosome cell wall, which paves the way to the development of hierarchical plant structures and advanced synthetic plant cell mimics.

HYPOTHESIS: Permeation of macromolecular drugs across biological plasma membranes is a major challenge in drug delivery. Cationic cell-penetrating peptides (CPPs) are attractive functional excipient candidates for the delivery of macromolecules across membrane barriers, due to their membrane translocating ability. The properties of CPPs can be tailored by lipidation, a promising approach to facilitate enhanced membrane insertion, potentially promoting increased translocation of the CPP and cargo.

EXPERIMENTS: To explore the impact that site and degree of lipidation have on the membrane interaction of a cationic CPP, we designed and investigated CPP conjugates with one or two fatty acid chains.

FINDINGS: Compared to the parent CPP and the single-lipidated conjugates, the double-lipidated conjugate exhibited the most pronounced membrane perturbation effects, as measured by several biophysical techniques. The experimental findings were supported by molecular dynamics (MD) simulations, demonstrating that all CPP conjugates interacted with the membrane by insertion of the lipid chain(s) into the core of the bilayer. Moreover, membrane-thinning effects and induced membrane curvature were displayed upon CPP interaction. Our results demonstrate that the impact exerted by the CPP on the membrane is notably affected by positioning and especially the degree of lipidation, which might influence the properties of CPPs as functional excipients.

Dynamic interactions of proteins with lipid membranes are essential regulatory events in biology, but remain rudimentarily understood and particularly overlooked in membrane proteins. The ubiquitously expressed membrane protein Na+/H+-exchanger 1 (NHE1) regulates intracellular pH (pHi) with dysregulation linked to e.g. cancer and cardiovascular diseases. NHE1 has a long, regulatory cytosolic domain carrying a membrane-proximal region described as a lipid-interacting domain (LID), yet, the LID structure and underlying molecular mechanisms are unknown. Here we decompose these, combining structural and biophysical methods, molecular dynamics simulations, cellular biotinylation- and immunofluorescence analysis and exchanger activity assays. We find that the NHE1-LID is intrinsically disordered and, in presence of membrane mimetics, forms a helical αα-hairpin co-structure with the membrane, anchoring the regulatory domain vis-a-vis the transport domain. This co-structure is fundamental for NHE1 activity, as its disintegration reduced steady-state pHi and the rate of pHi recovery after acid loading. We propose that regulatory lipid-protein co-structures may play equally important roles in other membrane proteins.

The naturally occurring peptide indolicidin from bovine neutrophils exhibits strong biological activity against a broad spectrum of microorganisms. This is believed to arise from selective interactions with the negatively charged cytoplasmic lipid membrane found in bacteria. We have investigated the peptide interaction with supported lipid model membranes using a combination of complementary surface sensitive techniques: neutron reflectometry (NR), atomic force microscopy (AFM), and quartz crystal microbalance with dissipation monitoring (QCM-D). The data are compared with small-angle X-ray scattering (SAXS) results obtained with lipid vesicle/peptide solutions. The peptide membrane interaction is shown to be significantly concentration dependent. At low concentrations, the peptide inserts at the outer leaflet in the interface between the headgroup and tail core. Insertion of the peptide results in a slight decrease in the lipid packing order of the bilayer, although not sufficient to cause membrane thinning. By increasing the indolicidin concentration well above the physiologically relevant conditions, a deeper penetration of the peptide into the bilayer and subsequent lipid removal take place, resulting in a slight membrane thinning. The results suggest that indolicidin induces lipid removal and that mixed indolicidin-lipid patches form on top of the supported lipid bilayers. Based on the work presented using model membranes, indolicidin seems to act through the interfacial activity model rather than through the formation of stable pores.

Supported lipid bilayers (SLBs) are simple and robust biomimics with controlled lipid composition that are widely used as models of both mammalian and bacterial membranes. However, the lipids typically used for SLB formation poorly resemble those of bacterial cell membranes due to the lack of available protocols to form SLBs using mixtures of lipids relevant for bacteria such as phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). Although a few reports have been published recently on the formation of SLBs from Escherichia coli lipid extracts, a detailed understanding of these systems is challenging due to the complexity of the lipid composition in such natural extracts. Here, we present for the first time a simple and reliable protocol optimized to form high-quality SLBs using mixtures of PE and PG at compositions relevant for Gram-negative membranes. We show using neutron reflection and quartz microbalance not only that Ca2+ ions and temperature are key parameters for successful bilayer deposition but also that mass transfer to the surface is a limiting factor. Continuous flow of the lipid suspension is thus crucial for obtaining full SLB coverage. We furthermore characterize the resulting bilayers and report structural parameters, for the first time for PE and PG mixtures, which are in good agreement with those reported earlier for pure POPE vesicles. With this protocol in place, more suitable and reproducible studies can be conducted to understand biomolecular processes occurring at cell membranes, for example, for testing specificities and to unravel the mechanism of interaction of antimicrobial peptides.

A hybrid coating based on multilayers of proteins and biopolymers was developed to enhance the protection performance of alginate microbeads against acidic conditions for delivery of probiotics (Lactobacillus rhamnosus GG). Zeta potential measurements and quartz crystal microbalance with dissipation confirmed layer-by-layer deposition of protein-polymer layers. The stability of protein-based coatings during simulated gastric fluid (SGF) treatment was monitored by microscopy. Protein-coated microbeads were partially dismantled, whereas polymer-coated microbeads were intact after a sequential treatment in simulated gastric and intestinal fluids. This suggests that hybrid formulation offers an advantage over the coatings based on biopolymer multilayers in terms of better release of bacteria. Uncoated alginate microbeads completely dissolved and could not protect bacteria after SGF treatment whereas microbeads with hybrid coating showed increased physical stability and a modest decrease of culturability of 3.8 log units. Therefore, this work provides a concept for future protein-based hybrid coatings for bacterial delivery systems.

An important consequence of protein-lipid interaction is the effect on stability of the protein in solution as well as on its behavior at interfaces. Here we will discuss key aspects of protein aggregation and unfolding as well as the effects of protein structure (random coil proteins versus globular) that are relevant for our understanding protein-lipid interaction. The main types of emulsifiers are the (1) aqueous soluble, surfactant type and (2) lipids with low aqueous solubility. The monomer concentration as defined by cmc is an important parameter for the soluble lipids. For emulsifiers with low aqueous solubility the emulsifier self-assembly structure and its properties control the interaction with proteins. We will therefore summarize the main features of lipid self-assembly. It also allows us to define different plausible scenarios and principles and models for factors that control the interactions in real food (and Pharmaceutical) systems. For the food applications the fate of the lipid during digestion is important and therefore we will discuss some aspects of enzyme-catalyzed lipolysis in terms of the structural evolution. New products and concepts of using protein/emulsifier interactions will be exemplified by illustrating how food nanotechnology possibly can be used for the delivery of functionality.

The deuteration of biomolecules provides advanced opportunities for neutron scattering studies. For low resolution studies using techniques such as small-angle neutron scattering and neutron reflection, the level of deuteration of a sample can be varied to match the scattering length density of a specific DO/HO solvent mixture. This can be of major value in structural studies where specific regions of a complex system can be highlighted, and others rendered invisible. This is especially useful in analyses of the structure and dynamics of membrane components. In mammalian membranes, the presence of cholesterol is crucial in modulating the properties of lipids and in their interaction with proteins. Here, a protocol is described for the production of partially deuterated cholesterol which has a neutron scattering length density that matches that of 100% DO solvent (hereby named matchout cholesterol). The level of deuteration was determined by mass spectrometry and nuclear magnetic resonance. The cholesterol match-point was verified experimentally using small angle neutron scattering. The matchout cholesterol was used to investigate the incorporation of cholesterol in various phosphatidylcholine supported lipid bilayers by neutron reflectometry. The study included both saturated and unsaturated lipids, as well as lipids with varying chain lengths. It was found that cholesterol is distributed asymmetrically within the bilayer, positioned closer to the headgroups of the lipids than to the middle of the tail core, regardless of the phosphatidylcholine species.

Atherosclerosis is the main killer in the western world. Today's clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, "neutron invisible" phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.

Phosphoinositide (PIP) lipids are anionic phospholipids playing a fundamental role for the activity of several transmembrane and soluble proteins. Among all, phosphoinositol-3',4',5'-trisphosphate (PIP3) is a secondary signaling messenger that regulates the function of proteins involved in cell growth and gene transcription. The present study aims to reveal the structure of PIP-containing lipid membranes, which so far has been little explored. For this purpose, supported lipid bilayers (SLBs) containing 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-3',4',5'-trisphosphate (DOPIP3) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were used as mimics of biomembranes. Surface sensitive techniques, i.e. Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), Atomic Force Microscopy (AFM) and Neutron Reflectometry (NR), provided detailed information on the formation of the SLB and the location of DOPIP3 in the lipid membrane. Specifically, QCM-D and AFM were used to identify the best condition for lipid deposition and to estimate the total bilayer thickness. On the other hand, NR was used to collect experimental structural data on the DOPIP3 location and orientation within the lipid membrane. The two bilayer leaflets showed the same DOPIP3 concentration, thus suggesting the formation of a symmetric bilayer. The headgroup layer thicknesses of the pure POPC and the mixed POPC/DOPIP3 bilayer suggest that the DOPIP3-headgroups have a preferred orientation, which is not perpendicular to the membrane surface, but instead it is close to the surrounding lipid headgroups. These results support the proposed PIP3 tendency to interact with the other lipid headgroups as PC, so far exclusively suggested by MD simulations.

Lipoproteins play a key role in the onset and development of atherosclerosis, the formation of lipid plaques at blood vessel walls. The plaque formation, as well as subsequent calcification, involves not only endothelial cells but also connective tissue, and is closely related to a wide range of cardiovascular syndromes, that together constitute the number one cause of death in the Western World. High (HDL) and low (LDL) density lipoproteins are of particular interest in relation to atherosclerosis, due to their protective and harmful effects, respectively. In an effort to elucidate the molecular mechanisms underlying this, and to identify factors determining lipid deposition and exchange at lipid membranes, we here employ neutron reflection (NR) and quartz crystal microbalance with dissipation (QCM-D) to study the effect of membrane charge on lipoprotein deposition and lipid exchange. Dimyristoylphosphatidylcholine (DMPC) bilayers containing varying amounts of negatively charged dimyristoylphosphatidylserine (DMPS) were used to vary membrane charge. It was found that the amount of hydrogenous material deposited from either HDL or LDL to the bilayer depends only weakly on membrane charge density. In contrast, increasing membrane charge resulted in an increase in the amount of lipids removed from the supported lipid bilayer, an effect particularly pronounced for LDL. The latter effects are in line with previously reported observations on atherosclerotic plaque prone regions of long-term hyperlipidaemia and type 2 diabetic patients, and may also provide some molecular clues into the relation between oxidative stress and atherosclerosis. (C) 2018 Elsevier B.V. All rights reserved.

The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.

Cholesterol is an essential component of mammalian membranes and is known to induce a series of physicochemical changes in the lipid bilayer. Such changes include the formation of liquid-ordered phases with an increased thickness and a configurational order as compared to liquid-disordered phases. For saturated lipid membranes, cholesterol molecules localize close to the lipid head group-tail interface. However, the presence of polyunsaturated lipids was recently shown to promote relocation of cholesterol toward the inner interface between the two bilayer leaflets. Here, neutron reflection is used to study the location of cholesterol (both non-deuterated and per-deuterated versions are used) within supported lipid bilayers composed of a natural mixture of phosphatidylcholine (PC). The lipids were produced in a genetically modified strain of Escherichia coli and grown under specific deuterated conditions to give an overall neutron scattering length density (which depends on the level of deuteration) of the lipids matching that of D2O. The combination of solvent contrast variation method with specific deuteration shows that cholesterol is located closer to the lipid head group-tail interface in this natural PC extract rather than in the center of the core of the bilayer as seen for very thin or polyunsaturated membranes.

Lipid bilayers, forming the outer barrier of cells, display a wide array of proteins and carbohydrates for modulating interfacial biological interactions. Formed by the spontaneous self-assembly of lipid molecules, these bilayers feature liquid crystalline order, while retaining a high degree of lateral mobility. Studies of these dynamic phenomena have been hampered by the fragility and instability of corresponding biomimetic cell membrane models. Here, we present the construct of a series of oligoethylene glycol-terminated reversible self-assembled monolayers (rSAMs) featuring lipid-bilayer-like fluidity, while retaining air and protein stability and resistance. These robust and ordered layers were prepared by simply immersing a carboxylic acid terminated self-assembled monolayer into 5-50 mu M aqueous omega-(4-ethylene glycol-phenoxy)-alpha-(4-amidinophenoxy)decane solutions. It is anticipated that this new class of robust and fluidic two-dimensional biomimetic surfaces will impact the design of rugged cell surface mimics and high-performance biosensors.

The potential of a lactylate (the sodium caproyl lactylate or C10 lactylate), a typical food grade emulsifier, as an anionic environmental friendly anti-fungal additive was tested in growth medium and formulated in a protective coating for exterior wood. Different laboratory growth tests on the blue stain fungus Aureobasidium pullulans were performed and its interactions on a model fungal cell membrane were studied. Promising short term anti-fungal effects in growth tests were observed, although significant but less dramatic effects took place in coating test on wood panels. Scanning electron microscope analysis shows clear differences in the amount of fungal slime on the mycelium of Aureobasidium pullulans when the fungus was exposed of C10 lactylate. This could indicate an effect on the pullulan and melanin production by the fungus. Moreover, the interaction studies on model fungal cell membranes show that C10 lactylate affects the phospholipid bilayer in a similar manner to other negative charged detergents. (C) 2017 Elsevier Inc. All rights reserved.

Physical and chemical (crosslinked with genipin) hydrogels based on chitosan and dextran sulfate were developed and characterized as novel bio-materials suitable for probiotic encapsulation. The swelling of the hydrogels was dependent on the composition and weakly influenced by the pH of the media. The morphology analysis supports the swelling data showing distinct changes in microstructure depending on the composition. The viability and culturability tests showed approx. 3.6 log CFU/mL decrease of cells (L. acidophilus as model) incorporated into chemical hydrogels when compared to the number of viable native cells. However, the live/dead viability assay evidenced that a considerable amount of viable cells were still entrapped in the hydrogel network and therefore the viability is most likely underestimated. Overall, the developed systems are robust and their structure, rheology and swelling properties can be tuned by changing the blend ratio, thus constituting appealing bio-matrices for cell encapsulation.

Hypothesis: Chitosan and sulfated oat beta-glucan are materials suitable to create a prebiotic coating for targeted delivery to gastrointestinal system, using the layer by layer technology. Experiment: Quartz crystal microbalance with dissipation (QCM-D), spectroscopic ellipsometry (SE) and atomic force microscopy (AFM) were used to assess the multilayer formation capacity and characterize the resulting coatings in terms of morphology and material properties such as structure and rigidity. The coating of colloidal materials was proven, specifically on L acidophilus bacteria as measured by changes in the bacterial suspension zeta potential. Viability of coated cells was shown using plate counting method. The coatings on solid surfaces were examined after exposure to mimics of gastrointestinal fluids and a commercially available beta-glucanase. Findings: Successful build-up of multilayers was confirmed with QCM-D and SE. Zeta potential values proved the coating of cells. There was 2 log CFU/mL decrease after coating cells with four alternating layers of chitosan and sulfated p-glucan when compared to viability of uncoated cells. The coatings were partially degraded after exposure to simulated intestinal fluid and restructured as a result of beta-glucanase treatment, mimicking enzymes present in the microflora of the human gut, but seemed to resist acidic gastric conditions. Therefore, coatings of chitosan and sulfated beta-glucan can potentially be exploited as carriers for probiotics and delicate nutraceuticals. (C) 2016 Elsevier Inc. All rights reserved.

We report the preparation and characterization of aqueous Pickering foams using bio-particles constituted by lactic acid bacteria surface modified by oppositely charged milk proteins. Cell surface modification was shown by zeta potential measurements. Foams stabilized by bacterial Pickering bio-particles showed improved stability compared to purely milk protein stabilized foams. The stability of foams increased with the bacterial concentration whereas the foam volume (foamability) decreased. On the other hand, protein concentration was correlated with foamability but not with the foam stability. Optical and fluorescence microscopy revealed organized cell structures around and in between the air bubbles providing for an internal network that effectively stabilizes the foam. Therefore, entirely food grade stable foams can be produced by using modified health promoting bacterial cells and surface active milk proteins. Such Pickering systems can potentially be utilized in bottom up construction of more complex hierarchical food structures and further improve properties such as foam stability.

High and low density lipoproteins (HDL and LDL) are thought to play vital roles in the onset and development of atherosclerosis; the biggest killer in the western world. Key issues of initial lipoprotein (LP) interactions at cellular membranes need to be addressed including LP deposition and lipid exchange. Here we present a protocol for monitoring the in situ kinetics of lipoprotein deposition and lipid exchange/removal at model cellular membranes using the non-invasive, surface sensitive methods of neutron reflection and quartz crystal microbalance with dissipation. For neutron reflection, lipid exchange and lipid removal can be distinguished thanks to the combined use of hydrogenated and tail-deuterated lipids. Both HDL and LDL remove lipids from the bilayer and deposit hydrogenated material into the lipid bilayer, however, the extent of removal and exchange depends on LP type. These results support the notion of HDL acting as the ‘good’ cholesterol, removing lipid material from lipid-loaded cells, whereas LDL acts as the ‘bad’ cholesterol, depositing lipid material into the vascular wall.

3-Aminopropylsilane (APS) coupling agent is widely used in industrial, biomaterial, and medical applications to improve adhesion of polymers to inorganic materials. However, during exposure to elevated humidity and temperature, the deposited APS layers can decompose, leading to reduction in coupling efficiency, thus decreasing the product quality and the mechanical strength of the polymer–inorganic material interface. Therefore, a better understanding of the chemical state and stability of APS on inorganic surfaces is needed. In this work, we investigated APS adhesion on silica wafers and compared its properties with those on complex silicate surfaces such as those used by industry (mineral fibers and fiber melt wafers). The APS was deposited from aqueous and organic (toluene) solutions and studied with surface sensitive techniques, including X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), streaming potential, contact angle, and spectroscopic ellipsometry. APS configuration on a model silica surface at a range of coverages was simulated using density functional theory (DFT). We also studied the stability of adsorbed APS during aging at high humidity and elevated temperature. Our results demonstrated that APS layer formation depends on the choice of solvent and substrate used for deposition. On silica surfaces in toluene, APS formed unstable multilayers, while from aqueous solutions, thinner and more stable APS layers were produced. The chemical composition and substrate roughness influence the amount of deposited APS. More APS was deposited and its layers were more stable on fiber melt than on silica wafers. The changes in the amount of adsorbed APS can be successfully monitored by streaming potential. These results will aid in improving industrial- and laboratory-scale APS deposition methods and increasing adhesion and stability, thus increasing the quality and effectiveness of materials where APS is used as a coupling agent.

In this work, the influence of phosphate buffer and proteins on the potentiometric response of a polymeric membrane-based solid-contact Pb2+-selective electrode (Pb2+-ISE) was studied. The effects of bovine serum albumin (BSA) adsorption at the surface of the ion-selective membrane combined with electrode conditioning in phosphate-buffered saline (PBS) solution was elucidated by potentiometry and electrochemical impedance spectroscopy. The adsorbed BSA at the surface of the Pb2+-ISE slightly lowered the detection limit but did not influence the selectivity of the Pb2+-ISE towards the interfering ions studied (Cu2+, Cd2+). Conditioning of the Pb2+-ISE in 0.01 mol dm–3 PBS resulted in a super-Nernstian response which was related to fixation/extraction of Pb2+ in the ion-selective membrane via precipitation of Pb3(PO4)2 by PO43– anions present in PBS. By conditioning of the Pb2+-ISE in 0.01 mol dm–3 PBS + 1 mg/ml BSA it was possible to extend the linear response range of the Pb2+-ISE towards lower analyte concentrations. The utilization of this conditioning procedure was validated by determination of Pb2+ concentrations down to ca 20 ppb in aqueous samples by Pb2+-ISEs and by comparing the results with those obtained by ICP-MS.

Atherosclerosis and its clinical consequences are the leading cause of death in the western hemisphere. While many studies throughout the last decades have aimed at understanding the disease, the clinical markers in use today still fail to accurately predict the risks. The role of the current main clinical indicator, low density lipoprotein (LDL), in depositing fat to the vessel wall is believed to be the onset of the process. However, many subfractions of the LDL, which differ both in structure and composition, are present in the blood and among different individuals. Understanding the relationship between LDL structure and composition is key to unravel the specific role of various LDL components in the development and/or prevention of atherosclerosis. Here, we describe a model for analyzing small-angle X-ray scattering data for rapid and robust structure determination for the LDL. The model not only gives the overall structure but also the particular internal layering of the fats inside the LDL core. Thus, the melting of the LDL can be followed in situ as a function of temperature for samples extracted from healthy human patients and purified using a double protocol based on ultracentrifugation and size-exclusion chromatography. The model provides information on: (i) the particle-specific melting temperature of the core lipids, (ii) the structural organization of the core fats inside the LDL, (iii) the overall shape of the particle, and (iv) the flexibility and overall conformation of the outer protein/hydrophilic layer at a given temperature as governed by the organization of the core. The advantage of this method over other techniques such as cryo-TEM is the possibility of in situ experiments under near-physiological conditions which can be performed relatively fast (minutes at home source, seconds at synchrotron). This approach now allows the monitoring of structural changes in the LDL upon different stresses from the environment, such as changes in temperature, oxidation, or external agents used or currently in development against atherosclerotic plaque build-up and which are targeting the LDL.

Proteins are key components in a multitude of biological processes, of which the functions carried out by transmembrane (membrane-spanning) proteins are especially demanding for investigations. This is because this class of protein needs to be incorporated into a lipid bilayer representing its native environment, and in addition, many experimental conditions also require a solid support for stabilization and analytical purposes. The solid support substrate may, however, limit the protein functionality due to protein–material interactions and a lack of physical space. We have in this work tailored the pore size and pore ordering of a mesoporous silica thin film to match the native cell-membrane arrangement of the transmembrane protein human aquaporin 4 (hAQP4). Using neutron reflectivity (NR), we provide evidence of how substrate pores host the bulky water-soluble domain of hAQP4, which is shown to extend 7.2 nm into the pores of the substrate. Complementary surface analytical tools, including quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy, revealed successful protein-containing supported lipid bilayer (pSLB) formation on mesoporous silica substrates, whereas pSLB formation was hampered on nonporous silica. Additionally, electron microscopy (TEM and SEM), light scattering (DLS and stopped-flow), and small-angle X-ray scattering (SAXS) were employed to provide a comprehensive characterization of this novel hybrid organic–inorganic interface, the tailoring of which is likely to be generally applicable to improve the function and stability of a broad range of membrane proteins containing water-soluble domains.