Nanodisc films are a promising approach to study the equilibrium conformation of membrane bound proteins in native-like environment. Here we compare nanodisc formation for NADPH-dependent cytochrome P450 oxidoreductase (POR) using two different scaffold proteins, MSP1D1 and MSP1E3D1. Despite the increased stability of POR loaded MSP1E3D1 based nanodiscs in comparison to MSP1D1 based nanodiscs, neutron reflection at the silicon–solution interface showed that POR loaded MSP1E3D1 based nanodisc films had poor surface coverage. This was the case, even when incubation was carried out under conditions that typically gave high coverage for empty nanodiscs. The low surface coverage affects the embedded POR coverage in the nanodisc film and limits the structural information that can be extracted from membrane bound proteins within them. Thus, nanodisc reconstitution on the smaller scaffold proteins is necessary for structural studies of membrane bound proteins in nanodisc films.

The characterization of materials at the atomic-, nano-, and microscales is of crucial importance in understanding and then tailoring their macroscale properties and function for end-use applications and for effective modern cradle-to-reuse materials cycling. Synchrotron light, as well as the complementary neutron beams, offer exquisite microscopy probes to look into the heart of materials. This chapter presents some examples of pharma-oriented nanoparticle characterization highlighting the possibilities of synchrotron light and neutron beams. Small-angle X-ray scattering (SAXS) is a well-established technique to probe nanoscale structures. SAXS can also deliver valuable information on the structure of self-assembled nanovectors, such as liposomes, which are recognized as efficient platforms for drug delivery. Future developments for neutron characterization will be driven in parallel with instrumental developments at existing sources and future facilities such as the European Spallation Source (ESS) being built in Sweden.  

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.

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.

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.

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.

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.

The interactions of the bile salt sodium taurocholate (TC) in 50 mM Trizma–HCl buffer and 150 mM NaCl (pH 9) at 37 °C with membranes composed of sphingomyelin (SM) were studied by dynamic light scattering, cryogenic transmission electron microscopy (cryo-TEM) and turbidity measurements. Small unilamellar SM vesicles were prepared by extrusion. Below the CMC of TC, taurocholate addition leads to vesicle growth due to incorporation of the taurocholate molecules into the vesicle bilayer. At around half the CMC of the bile salt, the SM vesicles are transformed into SM/TC mixed worm-like micelles, which are visualized by cryo-TEM for the first time. Further increase in the taurocholate concentration leads to the rupture of these structures into small spherical micelles. Interestingly, large non-spherical micelles were also identified for pure taurocholate solutions. Similar threadlike structures have been reported earlier for the bile salt sodium taurodeoxycholate [Rich, A., Blow, D., 1958. Nature 182, 1777; Blow, D.M., Rich, A., 1960. J. Am. Chem. Soc. 82, 3566–3571; Galantini, L., Giglio, E., La Mesa, C., Viorel-Pavel, N., Punzo, F., 2002. Langmuir 18, 2812] and for mixtures of taurocholate and phosphatidylcholate [Ulmius, J., Lindblom, G., Wennerström, H., Johansson, L.B.-Å., Fontel, K., Söderman, O., Ardvisson, G., 1982. Biochemistry 21, 1553; Hjelm, R.P., Thiyagarajan, P., Alkan-Onyuksel, H., 1992. J. Phys. Chem. 96, 8653] as determined by various scattering methods.

Films formed from saliva on surfaces are important for maintenance of oral health and integrity by protection against chemical and/or biological agents. The aim of the present study was to investigate adsorbed amounts, thickness and the structure of films formed from human whole saliva on alumina surfaces by means of in situ ellipsometry, neutron reflectivity and atomic force microscopy. Alumina (Al2O3, synthetic sapphire) is a relevant and interesting substrate for saliva adsorption studies as it has an isoelectric point close to that of tooth enamel. The results showed that saliva adsorbs rapidly on alumina. The film could be modelled in two layers: an inner and dense thin region which forms a uniform layer, and an outer, more diffuse and thicker region that protrudes towards the bulk of the solution. The film morphology described a uniformly covering dense layer and a second outer layer containing polydisperse adsorbed macromolecules or aggregates.

The contribution of nonspecific interactions to the overall interactions of thiol-ssDNA and dsDNA macromolecules with gold nanoparticles was investigated. A systematic investigation utilizing dynamic light scattering and cryogenic transmission electron microscopy has been performed to directly measure and visualize the changes in particle size and appearance during functionalization of gold nanoparticles with thiol-ssDNA and nonthiolated dsDNA. The results show that both thiol-ssDNA and dsDNA do stabilize gold nanoparticle dispersions, but possible nonspecific interactions between the hydrophobic DNA bases and the gold surface promote interparticle interactions and cause aggregation within rather a short period of time. We also discuss the adsorption mechanisms of dsDNA and thiol-ssDNA to gold particles.

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.

This paper describes a combined investigation of the salivary and MUC5B films structure and topography in conditions similar to those found in the oral cavity in terms of ionic strength, pH, and protein concentration. AFM and ellipsometry were successfully used to give a detailed picture of the film structure and topography both on hydrophilic and on hydrophobic substrata. Regardless of the substrata, the salivary film can be described as having a two sublayer structure in which an inner dense layer is decorated by large aggregates. However, the shape and height of these larger aggregates largely depend on the type of substrata used. Additionally, we show that the adsorption of MUC5B is controlled by the type of substrata and the MUC5B film topography is similar to that of the larger aggregates present in the salivary films, especially on hydrophobic substrates. Therefore, we conclude that MUC5B is a major component in the salivary film when formed on hydrophobic substrates. Furthermore, we studied how resistant the salivary and MUC5B films are against elutability by buffer rinsing and addition of SDS solution. We conclude that the adsorbed proteins contain fractions with varying binding strengths to the two types of surfaces. Specifically, we have shown that the large MUC5B biomacromolecules on the hydrophobic substrates are especially resistant to both elution with buffer solution and SDS. Therefore, these large mucins can be responsible for the increased resistance of HWS films on hydrophobic substrates and can protect the intraoral surfaces against surface-active components present in oral health care products.

Hydroxyapatite is a very interesting material given that it is the main component in tooth enamel and because of its uses in bone implant applications. Therefore, not only the characterization of its surface is of high relevance but also designing reliable methods to study the interfacial properties of films adsorbed onto it. In this paper we apply the colloidal probe atomic force microscopy method to investigate the surface properties of commercially available hydroxyapatite surfaces (both microscopic particles and macroscopic discs) in terms of interfacial and frictional forces. In this way, we find that hydroxyapatite surfaces at physiological relevant conditions are slightly negatively charged. The surfaces were then exposed to human whole saliva, and the surface properties were re-evaluated. A thick film was formed that was very resistant to mechanical stress. The frictional measurements demonstrated that the film was indeed highly lubricating, supporting the argument that this system may prove to be a relevant model for evaluating dental and implant systems.

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.

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.

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.

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.

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.

Lipidation of proteins is used in the pharma- ceutical field to increase the therapeutic efficacy of proteins. In this study, we investigate the effect of a 14-carbon fatty acid modification on the adsorption behavior of human insulin to a hydrophobic solid surface and the subsequent fibrillation development under highly acidic conditions and elevated temperature by comparing to the fibrillation of human insulin. At these stressed conditions, the lipid modification accelerates the rate of fibrillation in bulk solution. With the use of several complementary surface-sensitive techniques, including quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and neutron reflectivity (NR), we show that there are two levels of structurally different protein organization at a hydrophobic surface for both human insulin and the lipidated analogue: a dense protein layer formed within minutes on the surface and a diffuse outer layer of fibrillar structures which took hours to form. The two layers may only be weakly connected, and proteins from both layers are able to desorb from the surface. The lipid modification increases the protein surface coverage and the thickness of both layer organizations. Upon lipidation not only the fibrillation extent but also the morphology of the fibrillar structures changes from fibril clusters on the surface to a more homogeneous network of fibrils covering the entire hydrophobic surface.

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.

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.

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.

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.

Hydrophobic resin acids (RAs) are synthesized by conifer trees as part of their defense mechanisms. One of the functions of RAs in plant defense is suggested to be the perturbation of the cellular membrane. However, there is a vast diversity of chemical structures within this class of molecules, and there are no clear correlations to the molecular mechanisms behind the RA's toxicity. In this study we unravel the molecular interactions of the three closely related RAs dehydroabietic acid, neoabietic acid, and the synthetic analogue dichlorodehydroabietic acid with dipalmitoylphosphatidylcholine (DPPC) model membranes and the polar lipid extract of soybeans. The complementarity of the biophysical techniques used (NMR, DLS, NR, DSC, Cryo-TEM) allowed correlating changes at the vesicle level with changes at the molecular level and the co-localization of RAs within DPPC monolayer. Effects on DPPC membranes are correlated with the physical chemical properties of the RA and their toxicity.

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.

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.

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.

Two monoclonal antibodies from the IgG subclasses one and two were compared in their adsorption behavior with hydrophobic surfaces upon dilution to 10 mg/mL with 0.9% NaCl. These conditions simulate handling of the compounds at hospital pharmacies and surfaces encountered after preparation, such as infusion bags and i.v. lines. Total internal reflection fluorescence and quartz crystal microbalance with dissipation monitoring were used to follow and quantify this. Furthermore, the influence of the nonionic surfactant polysorbate 80 (PS80) on the adsorption process of these two antibodies was investigated. Despite belonging to two different IgG subclasses, both antibodies displayed comparable adsorption behavior. Both antibodies readily adsorbed in the absence of PS80, whereas adsorption was reduced in the presence of 30 mg/L surfactant. The sequence of exposure of the surfactant and protein to the surface was found to have a major influence on the extent of protein adsorption. Although only a fraction of adsorbed protein could be removed by rinsing with 30 mg/L surfactant solution, adsorption was entirely prevented when surfaces were pre-exposed to PS80

In this review, the authors discuss the challenges of studying supported lipid bilayers (SLBs) deposited by vesicle fusion in terms of (1) evaluating SLB formation and quality using quartz crystal microbalance with dissipation and (2) analyzing the composition and asymmetry of SLBs composed by lipid mixtures using complementary surface sensitive techniques. An overview of the literature is presented and the inconsistencies on this topic are discussed with the objective to expand beyond simple lipid compositions and set the basis for forming and analyzing SLBs of complex natural lipid extracts formed via the vesicle fusion method. The authors conclude by providing some guidelines to successfully form SLBs of complex lipid mixtures including natural extracts.

Antimicrobial drug resistance is a major human health threat. Among the many attempts to tackle this problem, the synthesis of antimicrobial compounds that mimic natural antimicrobial peptides appears as a promising approach. Peptide-based dendrimers can be designed to have higher potency than natural antimicrobial peptides and at the same time they can evade the bacterial defense system. Novel dendrimers with similar chemical structure but varying potency in terms of minimum inhibitory concentration were designed. The dependency between dendrimer structure and antibacterial activity as well as their capacity to attack model cell membranes was studied. The data suggests that supramolecular structure in terms of charge distribution and amphiphilicity, rather than net charge, is the main driver for disruption of cellular membranes and this correlates well with dendrimer hemolytic activity.

We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main phase-transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid bilayers formed below or above Tm by neutron reflection and investigated the effect of subsequent cooling to below the Tm. Our results clearly show that a continuous supported bilayer can be formed with high surface coverage below the lipid Tm. We also demonstrate that the high dissipation responses observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported.

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.

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

The interaction mechanism of a novel amphiphilic antimicrobial peptide dendrimer, BALY, with model lipid bilayers was explored through a combination of neutron reflection and molecular dynamics simulations. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phos-phocholine (DPPC) lipid bilayers were examined at room temperature to extract information on the interaction of BALY with fluid and gel phases, respectively. Furthermore, a 1:4 mixture of POPC and DPPC was used as a model of a phase-separated membrane. Upon interaction with fluid membranes, BALY inserted in the distal leaflet and caused thinning and disordering of the headgroups. Membrane thinning and expansion of the lipid cross-sectional area were observed for gel phase membranes, also with limited insertion to the distal leaflet. However, dendrimer insertion through the entire lipid tail region was observed upon crossing the lipid phase transition temperature of DPPC and in phase separated membranes. The results show clear differences in the interaction mechanism of the dendrimer depending on the lipid membrane fluidity, and suggest a role for lipid phase separation in promoting its antimicrobial activity.

Films formed from saliva on surfaces are important for maintenance of oral health and integrity by serving as barriers against chemical and/or biological agents. OBJECTIVES: The aim of the present study was to investigate the structure of films formed from human whole saliva onto alumina surfaces. METHODS: In situ ellipsometry was used to investigate adsorbed amounts and thickness of the adsorbed layers. Neutron reflectivity and atomic force microscopy (AFM) were used to study the density profile within the adsorbed layer and morphology, respectively, of the adsorbed salivary film onto alumina (Al2O3). RESULTS: The results show that saliva adsorbed rapidly on alumina. First a thin and dense layer covered the surfaces. With time, a thicker and diffuser layer was developed. The film morphology described a uniformly covering dense layer and a second outer layer containing polydisperse adsorbed macromolecules or aggregates. CONCLUSION: The film was found to be composed of two layers: an inner and dense thin region, that forms a uniform layer, and an outer, more diffuse and thicker region that protrudes towards the bulk of the solution. This study was supported by research grants from the Knowledge Foundation, Malmö University, The Swedish Dental Society, The Swedish Patent Revenue Fund for Research in Preventive Dentistry, The Swedish Research Council and Uppsala University. We are grateful also to the Institute Laue Langevin, Grenoble for an allocation of neutron beam time.

Abstract In situ ellipsometry was used to study layer-by-layer film formation on hydrophilic and hydrophobized silica surfaces by alternating sequential adsorption of human mucin MUC5B and cationic proteins lysozyme, lactoferrin, lactoperoxidase or histatin 5, respectively. The stability of the multilayers was investigated by addition of sodium dodecyl sulfate solution (SDS). Atomic force microscopy was employed to investigate morphological structures on the surfaces during the layer-by-layer film build-up. It was clearly shown that, on both hydrophilic and hydrophobized silica, only MUC5B and lactoperoxidase showed the ability for multilayer formation, resulting in an approximately linear increase in adsorbed amount and film thickness with each deposition cycle. The net increase in amounts per cycle was larger on the hydrophilic silica. Further, MUC5B needs to be adsorbed first on the hydrophilic substrates to obtain this fast build-up behavior. Generally, addition of SDS solution showed that a large fraction of the adsorbed film could be desorbed. However, films on the hydrophobized silica were more resistant to surfactant elution. In conclusion, MUC5B–cationic protein multilayers can be formed on hydrophilic and hydrophobized silica, depending on the choice of the cationic protein as well as in which order the build-up is started on hydrophilic silica. Additionally, SDS disrupts the layer-by-layer film formed by MUC5B and lactoperoxidase.

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.

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.

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.

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.

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.

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.