Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.

Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.

Nanoscopic lipid domains determined by microscopy and neutron scattering.

Biological membranes are highly complex supramolecular assemblies, which play central roles in biology. However, their complexity makes them challenging to study their nanoscale structures. To overcome this challenge, model membranes assembled using reduced sets of membrane-associated biomolecules have been found to be both excellent and tractable proxies for biological membranes. Due to their relative simplicity, they have been studied using a range of biophysical characterization techniques. In this review article, we will briefly detail the use of fluorescence and electron microscopies, and X-ray and neutron scattering techniques used over the past few decades to study the nanostructure of biological membranes.

19F solid-state NMR approaches to probe antimicrobial peptide interactions with membranes in whole cells.

To address the global problem of bacterial antibiotic resistance, antimicrobial peptides (AMPs) are considered promising therapeutic candidates due to their broad-spectrum and membrane-lytic activity. As preferential interactions with bacteria are crucial, it is equally important to investigate and understand their impact on eukaryotic cells. In this study, we employed 19F solid-state nuclear magnetic resonance (ssNMR) as a novel approach to examine the interaction of AMPs with whole red blood cells (RBCs). We used RBC ghosts (devoid of hemoglobin) and developed a protocol to label their lipid membranes with palmitic acid (PA) monofluorinated at carbon positions 4, 8, or 14 on the acyl chain, allowing us to probe different locations in model and intact RBC ghost membranes. Our work revealed that changes in the 19F chemical shift anisotropy, monitored through a CF bond order parameter (SCF), can provide insights into lipid bilayer dynamics. This information was also obtained using magic-angle spinning 19F ssNMR spectra with and without 1H decoupling, by studying alterations in the second spectral moment (M2) as well as the 19F isotropic chemical shift, linewidth, T1, and T2 relaxation times. The appearance of an additional isotropic peak with a smaller chemical shift anisotropy, a narrower linewidth, and a shorter T1, induced by the AMP caerin 1.1, supports the presence of high-curvature regions in RBCs indicative of pore formation, analogous to its antimicrobial mechanism. In summary, the straightforward incorporation of monofluorinated FAs and rapid signal acquisition offer promising avenues for the study of whole cells using 19F ssNMR.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Study on the effect of blackcurrant extract - based preservative on model membranes and pathogenic bacteria.

In this work the cosmetic preservative based on a Ribes Nigrum (blackcurrant) plant extract (PhytoCide Black Currant Powder abbr. BCE) was investigated to evaluate its antibacterial effect and to gain an insight into its mechanism of action. The influence of this commercially available formulation on model Escherichia coli and Staphylococcus aureus lipid membranes was studied to analyze its interactions with membrane lipids at a molecular level. The mixed lipid monolayers and one component bacteria lipid films were used to investigate the effect of BCE on condensation and morphology of model systems and to study the ability of BCE components to penetrate into the lipid environment. The in vitro tests were also done on different bacteria species (E. coli, Enterococcus faecalis, S. aureus, Salmonella enterica, Pseudomonas aeruginosa) to compare antimicrobial potency of the studied formulation. As evidenced the in vitro studies BCE formulation exerts very similar antibacterial activity against E. coli and S. aureus. Moreover, based on the collected data it is impossible to indicate which bacteria: Gram-positive or Gram-negative are more susceptible to this formulation. Model membrane experiments evidenced that the studied preservative affects organization of both E. coli and S. aureus model system by decreasing their condensation and altering their morphology. BCE components are able to penetrate into the lipid systems. However, all these effects depend on the lipid composition and monolayer organization. The collected results were analyzed from the point of view of the mechanism of action of blackcurrant extract and the factors, which may determine the activity of this formulation.

Nucleoside Analog Reverse-Transcriptase Inhibitors in Membrane Environment: Molecular Dynamics Simulations.

The behavior of four drugs from the family of nucleoside analog reverse-transcriptase inhibitors (zalcitabine, stavudine, didanosine, and apricitabine) in a membrane environment was traced using molecular dynamics simulations. The simulation models included bilayers and monolayers composed of POPC and POPG phospholipids. It was demonstrated that the drugs have a higher affinity towards POPG membranes than POPC membranes due to attractive long-range electrostatic interactions. The results obtained for monolayers were consistent with those obtained for bilayers. The drugs accumulated in the phospholipid polar headgroup region. Two adsorption modes were distinguished. They differed in the degree of penetration of the hydrophilic headgroup region. Hydrogen bonds between drug molecules and phospholipid heads were responsible for adsorption. It was shown that apricitabine penetrated the hydrophilic part of the POPC and POPG membranes more effectively than the other drugs. Van der Waals interactions between S atoms and lipids were responsible for this.

The influence of amphiphilic carbosilane dendrons on lipid model membranes.

Amphiphilic dendrons represent a relatively novel class of molecules which may show many unique properties suitable for applications in a field of molecular biology and nanomedicine. They were frequently studied as platforms suitable for drug delivery systems as were, e.g. polymersomes or hybrid lipid-polymer nanoparticles. Recently, natural extracellular lipid vesicles (EVs), called exosomes (EXs), were shown to be a promising candidate in drug delivery applications. Formation of hybrid exosome-dendron nanovesicles could bring benefits in their simple conjugation with selective targeting moieties. Unfortunately, the complex architecture of biological membranes, EXs included, makes obstacles in elucidating the important parameters and mechanisms of interaction with the artificial amphiphilic structures. The aim of the presented work was to study the interaction of two types of amphiphilic carbosilane dendritic structures (denoted as DDN-1 and DDN-2) suitable for further modification with streptavidin (DDN-1) or using click-chemistry approach (DDN-2), with selected neutral and negatively charged lipid model membranes, partially mimicking the basic properties of natural EXs biomembranes. To meet the goal, a number of biophysical methods were used for determination of the degree and mechanisms of the interaction. The results showed that the strength of interactions of amphiphilic dendrons with liposomes was related with surface charge of liposomes. Several steps of interactions were disclosed. The initialization step was mainly coupled with amphiphilic dendrons - liposomes surface interaction resulting in destabilization of large self-assembled amphiphilic dendrons structures. Such destabilization was more significant with liposomes of higher negative charge. With increasing concentration of amphiphilic dendrons in a solution the interactions were taking place also in the hydrophobic part of bilayer. Further increase of nanoparticle concentration resulted in a gradual dendritic cluster formation in a lipid bilayer structure. Due to high affinity of amphiphilic dendrons to model lipid bilayers the conclusion can be drawn that they represent promising platforms also for decoration of exosomes or other kinds of natural lipid vehicles. Such organized hybrid dendron-lipid biomembranes may be advantageous for their subsequent post-functionalization with small molecules, large biomacromolecules or polymers suitable for targeted drug-delivery or theranostic applications.

Effect of γ-Oryzanol on the LE-LC Phase Coexistence Region of DPPC Langmuir Monolayer.

We have studied the effect of relative composition of γ-Oryzanol (γ-Or) on the liquid expanded-liquid condensed phase coexistence region in the mixed Langmuir monolayer of γ-Or and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) molecules at air-water interface. The surface manometry studies at a fixed temperature show that the mixture of γ-Or and DPPC forms a stable monolayer at air-water interface. As the relative composition of γ-Or increases the range of area per molecule over which the coexistence of liquid expanded (LE)-liquid condensed (LC) phases exists reduces. Although the LE-LC phase coexistence corresponds to the first-order phase transition, the slope of the surface pressure-area per molecule isotherm is non-zero. Earlier studies have attributed the non-zero slope in LE-LC phase coexistence region to the influence of the strain between the ordered LC phase and disordered LE phase. The effect of strain on the coexistence of LE-LC phases can be studied in terms of molecular density-strain coupling. Our analysis of the liquid condensed-liquid expanded coexistence region in the isotherms of mixed monolayers of DPPC and γ-Or shows that with the increase in the mole fraction of sterol in the mixed monolayer the molecular lateral density-strain coupling increases. However, at 0.6 mole fraction of γ-Or in the mixed monolayer the coupling decreases. This is corroborated by the observation of minimum Gibb's free energy of the mixed monolayer at this relative composition of γ-Or indicating better packing of molecules.

On a mechanistic impact of transmembrane tetramerization in the pathological activation of RTKs.

Constitutive activation of receptor tyrosine kinases (RTKs) via different mutations has a strong impact on the development of severe human disorders, including cancer. Here we propose a putative activation scenario of RTKs, whereby transmembrane (TM) mutations can also promote higher-order oligomerization of the receptors that leads to the subsequent ligand-free activation. We illustrate this scenario using a computational modelling framework comprising sequence-based structure prediction and all-atom 1 µs molecular dynamics (MD) simulations in a lipid membrane for a previously characterised oncogenic TM mutation V536E in platelet-derived growth factor receptor alpha (PDGFRA). We show that in the course of MD simulations the mutant TM tetramer retains stable and compact configuration strengthened by tight protein-protein interactions, while the wild type TM tetramer demonstrates looser packing and a tendency to dissociate. Moreover, the mutation affects the characteristic motions of mutated TM helical segments by introducing additional non-covalent crosslinks in the middle of the TM tetramer, which operate as mechanical hinges. This leads to dynamic decoupling of the C-termini from the rigidified N-terminal parts and facilitates more pronounced possible displacement between the C-termini of the mutant TM helical regions that can provide more freedom for mutual rearrangement of the kinase domains located downstream. Our results for the V536E mutation in the context of PDGFRA TM tetramer allow for the possibility that the effect of oncogenic TM mutations can go beyond alternating the structure and dynamics of TM dimeric states and might also promote the formation of higher-order oligomers directly contributing to ligand-independent signalling effectuated by PDGFRA and other RTKs.

New Meloxicam Derivatives-Synthesis and Interaction with Phospholipid Bilayers Measured by Differential Scanning Calorimetry and Fluorescence Spectroscopy.

The purpose of the present paper was to assess the ability of five newly designed and synthesized meloxicam analogues to interact with phospholipid bilayers. Calorimetric and fluorescence spectroscopic measurements revealed that, depending on the details of the chemical structure, the studied compounds penetrated bilayers and affected mainly their polar/apolar regions, closer to the surface of the model membrane. The influence of meloxicam analogues on the thermotropic properties of DPPC bilayers was clearly visible because these compounds reduced the temperature and cooperativity of the main phospholipid phase transition. Additionally, the studied compounds quenched the fluorescence of prodan to a higher extent than laurdan, what pointed to a more pronounced interaction with membrane segments close to its surface. We presume that a more pronounced intercalation of the studied compounds into the phospholipid bilayer may be related to the presence of the molecule of a two-carbon aliphatic linker with a carbonyl group and fluorine substituent/trifluoromethyl group (compounds PR25 and PR49) or the three-carbon linker together with the trifluoromethyl group (PR50). Moreover, computational investigations of the ADMET properties have shown that the new meloxicam analogues are characterized by beneficial expected physicochemical parameters, so we may presume that they will have a good bioavailability after an oral administration.

Biophysical Chemistry in Groningen - A Personal Note.

This paper describes the start of the Biophysical Chemistry group of prof. Herman J.C. Berendsen (1934-2019) in Groningen, The Netherlands in the years from 1964 to 1974.

Impact of Pollutant Ozone on the Biophysical Properties of Tear Film Lipid Layer Model Membranes.

Ozone exposure from environmental smog has been implicated as a risk factor for developing dry eye disease (DED). The tear film lipid layer (TFLL), which is the outermost layer of the tear film and responsible for surface tension reduction while blinking, is in direct contact with the environment and serves as the first line of defense against external aggressors such as environmental pollution. The impact of exposure to ozone on the biophysical properties of three TFLL model membranes was investigated. These model membranes include a binary mixture of cholesteryl oleate (CO) and L-α-phosphatidylcholine (egg PC), a ternary mixture of CO, glyceryl trioleate (GT) and PC, as well as a quaternary mixture of CO, GT, a mixture of free fatty acids palmitic acid and stearic acid (FFAs) and PC. Biophysical impacts were evaluated as changes to the surface activity, respreadability, morphology and viscoelastic properties of the films. Expansion to higher molecular areas was observed in all the TFLL model membrane films which is attributable to the accommodation of the cleaved chains in the film. Significant morphological changes were observed, namely fluidization and the disruption of the phase transition behaviour of GT, and multilayer formation of CO. This fluidization reduces the hysteresis loops for the model membranes. On the other hand, the viscoelastic properties of the films exhibited differential impacts from ozone exposure as a function of composition. These findings are correlated to chemical changes to the lipids determined using ESI-MS.

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

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

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

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

Effects of Natural Rhamnolipid Mixture on Dioleoylphosphatidylcholine Model Membrane Depending on Method of Preparation and Sterol Content.

Rhamnolipids as biosurfactants have a potentially wide range of applications, for example, as "green" surfactants or components of drug delivery systems, which is associated with the features of their interaction with cell membranes. However, as noted in the literature, those kind of features have not been sufficiently studied now. This paper presents a study of the interaction of a natural mixture of rhamnolipids produced by bacteria of the rhizosphere zone of plants Pseudomonas aeruginosa with model membranes-liposomes based on dioleoylphosphatidylcholine (DOPC), depending on the method of their preparation and the content of sterols-ergosterol, cholesterol, lanosterol. Liposomes with rhamnolipids were prepared by two protocols: with film method from a mixture of DOPC and rhamnolipids; with film method from DOPC and injection of water solution of rhamnolipids. Joint analysis of the data of 31P NMR spectroscopy and ATR-FTIR spectroscopy showed that in the presence of rhamnolipids, the mobility of the head group of the DOPC phospholipid increases, the conformational disorder of the hydrophobic tail increases, and the degree of hydration of the C=O and P=O groups of the phospholipid decreases. It can be assumed that, when prepared from a mixture, rhamnolipids are incorporated into the membrane in the form of clusters and are located closer to the middle of the bilayer; while when prepared by injection, rhamnolipid molecules migrate into the membrane in the form of individual molecules and are located closer to the head part of phospholipids. The sterol composition of the model membrane also affects the interaction of rhamnolipids with the membrane. Here it is worth noting the possible presence of type of interaction between rhamnolipids and ergosterol differ from other investigated sterols, due to which rhamnolipid molecules are embedded in the area where ergosterol is located.

Lipid Structure Determines the Differential Impact of Single Metal Additions and Binary Mixtures of Manganese, Calcium and Magnesium on Membrane Fluidity and Liposome Size.

Unilamellar vesicles of the biologically relevant lipids phosphatidic acid (PA) and phosphatidylserine (PS) with fully saturated (DM-) or partly unsaturated (PO-) acyl side chains were exposed to Ca, Mn and Mg in single metal additions; in equimolar mixtures or by sequential additions of one metal at a time. Laurdan generalized polarization measured the membrane fluidity, while dynamic light scattering reported liposome size changes complemented by zeta potential. All metals induced membrane rigidity and increased liposome sizes across all systems. Mn had the strongest effect overall, but Mg was comparable for DMPS. Lipid side chain architecture was important as GP values for binary mixtures were higher than expected from the sum of values for single additions added to POPS but smaller for DMPS. Sequential additions were predominantly different for Ca:Mg mixtures. Mn induced the strongest increase of liposome size in saturated lipids whereas Ca effects dominated unsaturated matrices. Binary additions induced larger sizes than the sum of single additions for POPS, but much lower changes in DMPA. The order of addition was relevant for PS systems. Thus, lipid structure determines metal effects, but their impact is modulated by other ions. Thus, metal effects may differ with the local lipid architecture and metal concentrations within cells.

A biophysical study of tear film lipid layer model membranes.

The tear film lipid layer (TFLL), the final layer of the human tear film is responsible for surface tension reduction while blinking, water evaporation retardation and maintaining the stability of the tear film. The study of the composition-structure-function relationship of TFLL is paramount, as a compromised structure of TFLL leads to the emergence of dry eye disease (DED) which is one the most prevalent ophthalmic surface diseases of the modern world, associated with chronic pain and reduced visual capability. In this model membrane study, a systematic approach is used to study the biophysical properties of TFLL model membranes as a function of composition. Three mixed-lipid model membranes are studied along with their individual components comprising cholesteryl oleate (CO), glyceryl trioleate (GT), L-α-phosphatidylcholine (egg PC) and a free fatty acid mixture. The models become progressively more complex from binary to quaternary mixtures, allowing the role of each individual lipid to be derived. Langmuir balance, Brewster Angle Microscopy (BAM) and Profile Analysis Tensiometer (PAT) are used to study the surface activity and compression-expansion cycles, morphology, and rheological behaviour of the model membranes, respectively. Evidence of multilayering is observed with inclusion of CO and a reversible collapse is associated with the GT phase transition. An initially more coherent film is observed due to the addition of polar PC. Notably, these individual behaviours are retained in the mixed films and suggest a possible role for each physiological component of TFLL.

Expanding the Toolbox for Bicelle-Forming Surfactant-Lipid Mixtures.

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.

Dissecting the mechanisms of environment sensitivity of smart probes for quantitative assessment of membrane properties.

The plasma membrane, as a highly complex cell organelle, serves as a crucial platform for a multitude of cellular processes. Its collective biophysical properties are largely determined by the structural diversity of the different lipid species it accommodates. Therefore, a detailed investigation of biophysical properties of the plasma membrane is of utmost importance for a comprehensive understanding of biological processes occurring therein. During the past two decades, several environment-sensitive probes have been developed and become popular tools to investigate membrane properties. Although these probes are assumed to report on membrane order in similar ways, their individual mechanisms remain to be elucidated. In this study, using model membrane systems, we characterized the probes Pro12A, NR12S and NR12A in depth and examined their sensitivity to parameters with potential biological implications, such as the degree of lipid saturation, double bond position and configuration (cis versus trans), phospholipid headgroup and cholesterol content. Applying spectral imaging together with atomistic molecular dynamics simulations and time-dependent fluorescent shift analyses, we unravelled individual sensitivities of these probes to different biophysical properties, their distinct localizations and specific relaxation processes in membranes. Overall, Pro12A, NR12S and NR12A serve together as a toolbox with a wide range of applications allowing to select the most appropriate probe for each specific research question.