Model Membrane Systems Research Articles

Nonconverged Constraints Cause Artificial Temperature Gradients in Lipid Bilayer Simulations.

Molecular dynamics (MD) simulations have become an indispensable tool to investigate phase separation in model membrane systems. In particular, simulations based on coarse-grained (CG) models have found widespread use due to their increased computational efficiency, allowing for simulations of multicomponent lipid bilayers undergoing phase separation into liquid-ordered and liquid-disordered domains. Here, we show that a significant temperature difference between molecule types can artificially arise in CG MD membrane simulations with the standard Martini simulation parameters in GROMACS. In particular, the linear constraint solver (LINCS) algorithm does not converge with its default settings, resulting in serious temperature differences between molecules in a time step-dependent manner. We demonstrate that the underlying reason for this behavior is the presence of highly constrained moieties, such as cholesterol. Their presence can critically impact numerous structural and dynamic membrane properties obtained from such simulations. Furthermore, any preference of these molecules toward a certain membrane phase can lead to spatial temperature gradients, which can amplify the degree of phase separation or even induce it in compositions that would otherwise mix well. We systematically investigated the effect of the integration time step and LINCS settings on membrane properties. Our data show that for cholesterol-containing membranes, a time step of 20 fs should be combined with at least lincs_iter = 2 and lincs_order = 12, while using a time step of 30 fs requires at least lincs_iter = 3 and lincs_order = 12 to bring the temperature differences to a level where they do not perturb central membrane properties. Moreover, we show that in cases where stricter LINCS settings are computationally too demanding, coupling the lipids in multiple groups to the temperature bath offers a practical workaround to the problem, although the validity of this approach should be further verified. Finally, we show that similar temperature gradients can also emerge in atomistic simulations using the CHARMM force field in combination with settings that allow for a 5 fs integration step.

ESCRT-III induces phase separation in model membranes prior to budding and causes invagination of the liquid-ordered phase

Membrane fission triggered by the endosomal sorting complex required for transport (ESCRT) is an important process observed in several pathogenic and non-pathogenic cellular events. From a synthetic-biology viewpoint, ESCRT proteins represent an interesting machinery for the construction of cell mimetic sub-compartments produced by fission. Since their discovery, the studies on ESCRT-III-mediated action, have mainly focused on protein dynamics, ignoring the role of lipid organization and membrane phase state. Recently, it has been suggested that membrane buds formed by the action of ESCRT-III are generated from transient microdomains in endosomal membranes. However, the interplay between membrane domain formation and ESCRT remodeling pathways has not been investigated. Here, giant unilamellar vesicles made of ternary lipid mixtures, either homogeneous in phase or exhibiting liquid-ordered/liquid-disordered phase coexistence, were employed as a model membrane system. These vesicles were incubated with purified recombinant ESCRT-III proteins from the parasite Entamoeba histolytica. In homogeneous membranes, we observe that EhVps32 can trigger domain formation while EhVps20 preferentially co-localizes in the liquid disordered phase. The addition of EhVps24 appears to induce the formation of intraluminal vesicles produced from the liquid-ordered phase. In phase separated membranes, the intraluminal vesicles are also generated from the liquid-ordered phase and presumably emerge from the phase boundary region. Our findings reinforce the hypothesis that ESCRT-mediated remodeling depends on the membrane phase state. Furthermore, the obtained results point to a potential synthetic biology approach for establishing eukaryotic mimics of artificial cells with microcompartments of specific membrane composition, which can also differ from that of the mother vesicle.

Unraveling the mechanism of octenidine and chlorhexidine on membranes: Does electrostatics matter?

The increasing problem of antibiotic resistance in bacteria requires the development of new antimicrobial candidates. There are several well-known substances with commercial use, but their molecular mode of action is not fully understood. In this work, we focus on two commonly used antimicrobial agents from the detergent family—octenidine dichloride (OCT) and chlorhexidine digluconate (CHX). Both of them are reported to be agents selectively attacking the cell membrane through interaction inducing membrane disruption by emulsification. They are believed to present electrostatic selectivity toward charged lipids. In this study, we tested this hypothesis and revised previously proposed molecular mechanisms of action. Employing a variety of techniques such as molecular dynamics, ζ potential with dynamic light scattering, vesicle fluctuation spectroscopy, carboxyfluorescein leakage measurement, and fluorescence trimethylammonium-diphenylhexatriene- and diphenylhexatriene-based studies for determination of OCT and CHX membrane location, we performed experimental studies using two model membrane systems—zwitterionic PC and negatively charged PG (18:1/18:1):PC (16:0/18:1) 3:7, respectively. These studies were extended by molecular dynamics simulations performed on a three-component bacterial membrane model system to further test interactions with another negatively charged lipid, cardiolipin. In summary, our study demonstrated that detergent selectivity is far more complicated than supposed simple electrostatic interactions. Although OCT does disrupt the membrane, our results suggest that its primary selectivity was more linked to mechanical properties of the membrane. On the other hand, CHX did not disrupt membranes as a primary activity, nor did it show any sign of electrostatic selectivity toward negatively charged membranes at any stage of interactions, which suggests membrane disruption by influencing more discrete membrane properties.

Influence of different polymer belts on lipid properties in nanodiscs characterized by CW EPR spectroscopy

With this study we aim at comparing the well-known lipid membrane model system of liposomes and polymer-encapsulated nanodiscs regarding their lipid properties. Using differential scanning calorimetry (DSC) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we characterize the temperature-dependent lipid behavior within 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes and nanodiscs made from such liposomes by application of various polymers based on styrene-co-maleic acid (SMA), diisobutylene-alt-maleic acid (DIBMA), and styrene-co-maleic amide sulfobetaine (SMA-SB), a new SMA-derived copolymer containing sulfobetaine side chains. By incorporating a spin label doxyl moiety into the lipid bilayer in position 16 or 5 we were able to study the micropolarity as well as rotational restrictions onto the lipids in the apolar bilayer center and the chain region adjacent to the carbonyl groups, respectively. Our results suggest that all polymers broaden the main melting transition of DMPC, change the water accessibility within the lipid bilayer, and exhibit additional constraints onto the lipids. Independent of the used polymer, the rotational mobility of both spin-labeled lipids decreased with DIBMA exerting less restraints probably due to its aliphatic side chains. Our findings imply that the choice of the solubilizing polymer has to be considered an important step to form lipid nanodiscs which should be included into research of lipid membranes and membrane proteins in the future.

New short cationic antibacterial peptides. Synthesis, biological activity and mechanism of action

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Prediction of Permeate Flux in Ultrafiltration Processes: A Review of Modeling Approaches.

In any membrane filtration, the prediction of permeate flux is critical to calculate the membrane surface required, which is an essential parameter for scaling-up, equipment sizing, and cost determination. For this reason, several models based on phenomenological or theoretical derivation (such as gel-polarization, osmotic pressure, resistance-in-series, and fouling models) and non-phenomenological models have been developed and widely used to describe the limiting phenomena as well as to predict the permeate flux. In general, the development of models or their modifications is done for a particular synthetic model solution and membrane system that shows a good capacity of prediction. However, in more complex matrices, such as fruit juices, those models might not have the same performance. In this context, the present work shows a review of different phenomenological and non-phenomenological models for permeate flux prediction in UF, and a comparison, between selected models, of the permeate flux predictive capacity. Selected models were tested with data from our previous work reported for three fruit juices (bergamot, kiwi, and pomegranate) processed in a cross-flow system for 10 h. The validation of each selected model’s capacity of prediction was performed through a robust statistical examination, including a residual analysis. The results obtained, within the statistically validated models, showed that phenomenological models present a high variability of prediction (values of R-square in the range of 75.91–99.78%), Mean Absolute Percentage Error (MAPE) in the range of 3.14–51.69, and Root Mean Square Error (RMSE) in the range of 0.22–2.01 among the investigated juices. The non-phenomenological models showed a great capacity to predict permeate flux with R-squares higher than 97% and lower MAPE (0.25–2.03) and RMSE (3.74–28.91). Even though the estimated parameters have no physical meaning and do not shed light into the fundamental mechanistic principles that govern these processes, these results suggest that non-phenomenological models are a useful tool from a practical point of view to predict the permeate flux, under defined operating conditions, in membrane separation processes. However, the phenomenological models are still a proper tool for scaling-up and for an understanding the UF process.

The influence of ergosterol on the action of the hop oil and its major terpenes on model fungi membranes. Towards understanding the mechanism of action of phytocompounds for food and plant protection

The aim of this work was to find the correlation between the content of ergosterol in fungi membrane and the action of the hop essential oil, myrcene and humulene on its properties. To reach this goal, the monolayers and bilayers composed of phosphatidylcholine, phosphatidyethanol amine and ergosterol, differing in the concentration of sterol, were used as model membrane systems. The impact of the essential oil and its major terpenes on one component ergosterol film was also investigated.It was found that pure isolated terpenes, in contrast to the hop oil being the mixture of them, do not incorporate into pure ergosterol membrane, however, they cause the loss of monolayer material from the interface. These results are in contrast to the effect of these terpenes on phospholipid films reported previously and they may suggest a strong effect of ergosterol on the behavior of terpenes in the mixed systems. Surprisingly, for model membranes, the effect of myrcene was qualitatively similar to the effect of the hop oil and ergosterol was found to regulate the incorporation of both these substances into the film. In contrast, very strong correlation between ergosterol content and the action of humulene was found. Namely, the ability of humulene to change model membrane properties was found to increase with ergosterol concentration. Additionally, the differentiating effect of ergosterol on humulene action in membranes was much more pronounced than for myrcene or the hop oil. Interestingly, at the highest ergosterol level the influence of humulene was even stronger than the effect of the hop oil. This is very important finding suggesting that ergosterol may regulate the sensitivity of particular membrane to the impact of humulene. Summarizing, ergosterol substantially differentiates the effect of the hop oil, myrcene and humulene on the lipid systems and it can be the molecule important for antifungal effect of the essential oil and terpenes.

Unveiling a Hidden Event in Fluorescence Correlative Microscopy by AFM Nanomechanical Analysis.

Fluorescent imaging combined with atomic force microscopy (AFM), namely AFM-fluorescence correlative microscopy, is a popular technology in life science. However, the influence of involved fluorophores on obtained mechanical information is normally underestimated, and such subtle changes are still challenging to detect. Herein, we combined AFM with laser light excitation to perform a mechanical quantitative analysis of a model membrane system labeled with a commonly used fluorophore. Mechanical quantification was additionally validated by finite element simulations. Upon staining, we noticed fluorophores forming a diffuse weakly organized overlayer on phospholipid supported membrane, easily detected by AFM mechanics. The laser was found to cause a degradation of mechanical stability of the membrane synergically with presence of fluorophore. In particular, a 30 min laser irradiation, with intensity similar to that in typical confocal scanning microscopy experiment, was found to result in a ∼40% decrease in the breakthrough force of the stained phospholipid bilayer along with a ∼30% reduction in its apparent elastic modulus. The findings highlight the significance of analytical power provided by AFM, which will allow us to “see” the “unseen” in correlative microscopy, as well as the necessity to consider photothermal effects when using fluorescent dyes to investigate, for example, the deformability and permeability of phospholipid membranes.

How Does Liquid-Liquid Phase Separation in Model Membranes Reflect Cell Membrane Heterogeneity?

Although liquid–liquid phase separation of cytoplasmic or nuclear components in cells has been a major focus in cell biology, it is only recently that the principle of phase separation has been a long-standing concept and extensively studied in biomembranes. Membrane phase separation has been reconstituted in simplified model systems, and its detailed physicochemical principles, including essential phase diagrams, have been extensively explored. These model membrane systems have proven very useful to study the heterogeneity in cellular membranes, however, concerns have been raised about how reliably they can represent native membranes. In this review, we will discuss how phase-separated membrane systems can mimic cellular membranes and where they fail to reflect the native cell membrane heterogeneity. We also include a few humble suggestions on which phase-separated systems should be used for certain applications, and which interpretations should be avoided to prevent unreliable conclusions.

Biomembrane Structure and Material Properties Studied With Neutron Scattering.

Cell membranes and their associated structures are dynamical supramolecular structures where different physiological processes take place. Detailed knowledge of their static and dynamic structures is therefore needed, to better understand membrane biology. The structure–function relationship is a basic tenet in biology and has been pursued using a range of different experimental approaches. In this review, we will discuss one approach, namely the use of neutron scattering techniques as applied, primarily, to model membrane systems composed of lipid bilayers. An advantage of neutron scattering, compared to other scattering techniques, is the differential sensitivity of neutrons to isotopes of hydrogen and, as a result, the relative ease of altering sample contrast by substituting protium for deuterium. This property makes neutrons an ideal probe for the study of hydrogen-rich materials, such as biomembranes. In this review article, we describe isotopic labeling studies of model and viable membranes, and discuss novel applications of neutron contrast variation in order to gain unique insights into the structure, dynamics, and molecular interactions of biological membranes. We specifically focus on how small-angle neutron scattering data is modeled using different contrast data and molecular dynamics simulations. We also briefly discuss neutron reflectometry and present a few recent advances that have taken place in neutron spin echo spectroscopy studies and the unique membrane mechanical data that can be derived from them, primarily due to new models used to fit the data.

Unveiling the Dynamics of KRAS4b on Lipid Model Membranes

Small GTPase proteins are ubiquitous and responsible for regulating several processes related to cell growth and differentiation. Mutations that stabilize their active state can lead to uncontrolled cell proliferation and cancer. Although these proteins are well characterized at the cellular scale, the molecular mechanisms governing their functions are still poorly understood. In addition, there is limited information about the regulatory function of the cell membrane which supports their activity. Thus, we have studied the dynamics and conformations of the farnesylated KRAS4b in various membrane model systems, ranging from binary fluid mixtures to heterogeneous raft mimics. Our approach combines long time-scale coarse-grained (CG) simulations and Markov state models to dissect the membrane-supported dynamics of KRAS4b. Our simulations reveal that protein dynamics is mainly modulated by the presence of anionic lipids and to some extent by the nucleotide state (activation) of the protein. In addition, our results suggest that both the farnesyl and the polybasic hypervariable region (HVR) are responsible for its preferential partitioning within the liquid-disordered (Ld) domains in membranes, potentially enhancing the formation of membrane-driven signaling platforms.Graphic

Rhodopsin Channel Activity Can Be Evaluated by Measuring the Photocurrent Voltage Dependence in Planar Bilayer Lipid Membranes.

The studies of the functional properties of retinal-containing proteins often include experiments in model membrane systems, e.g., measurements of electric current through planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one of the membrane surfaces. However, the possibilities of this method have not been fully explored yet. We demonstrated that the voltage dependence of stationary photocurrents for two light-sensitive proteins, bacteriorhodopsin (bR) and channelrhodopsin 2 (ChR2), in the presence of protonophore had very different characteristics. In the case of the bR (proton pump), the photocurrent through the BLM did not change direction when the polarity of the applied voltage was switched. In the case of the photosensitive channel protein ChR2, the photocurrent increased with the increase in voltage and the current polarity changed with the change in the voltage polarity. The protonophore 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole (TTFB) was more efficient in the maximizing stationary photocurrents. In the presence of carbonyl cyanide-m-chlorophenylhydrazone (CCCP), the amplitude of the measured photocurrents for bR significantly decreased, while in the case of ChR2, the photocurrents virtually disappeared. The difference between the effects of TTFB and CCCP was apparently due to the fact that, in contrast to TTFB, CCCP transfers protons across the liposome membranes with a higher rate than through the decane-containing BLM used as a surface for the proteoliposome adsorption.

Structural Characterization of Natural Yeast Phosphatidylcholine and Bacterial Phosphatidylglycerol Lipid Multilayers by Neutron Diffraction.

Eukaryotic and prokaryotic cell membranes are difficult to characterize directly with biophysical methods. Membrane model systems, that include fewer molecular species, are therefore often used to reproduce their fundamental chemical and physical properties. In this context, natural lipid mixtures directly extracted from cells are a valuable resource to produce advanced models of biological membranes for biophysical investigations and for the development of drug testing platforms. In this study we focused on single phospholipid classes, i.e. Pichia pastoris phosphatidylcholine (PC) and Escherichia coli phosphatidylglycerol (PG) lipids. These lipids were characterized by a different distribution of their respective acyl chain lengths and number of unsaturations. We produced both hydrogenous and deuterated lipid mixtures. Neutron diffraction experiments at different relative humidities were performed to characterize multilayers from these lipids and investigate the impact of the acyl chain composition on the structural organization. The novelty of this work resides in the use of natural extracts with a single class head-group and a mixture of chain compositions coming from yeast or bacterial cells. The characterization of the PC and PG multilayers showed that, as a consequence of the heterogeneity of their acyl chain composition, different lamellar phases are formed.

Studies the interactions of ascorbic acid isoforms with a simple model of DPPC monolayer as a biomimetic membrane by Langmuir-Blodgett technique

There is ongoing interest motivated by the desire regarding monolayer to understand the nature of interaction forces within oriented structures. Monolayer of phospholipids dipalmitoylphosphatidylcholine DPPC were examined mainly because they are accepted as membrane model system and can offer a stable frame to investigate the interactions of various biomolecules and biomaterial compounds with the lipid membrane. Identifying the monolayer behavior in the presence of ascorbic acid (AscA) isoforms and if this isoforms could alter packing and organizing the thin film. By Langmuir-Blodgett technique, the DPPC monolayer were studied in absence and presence of (Conc.: 10−3M, 10−2M and 10−1M) L- and D- AscA isoforms in water (subphase) at temperatures 25, 37 and 41°C and fixed pH=7. It’s ascertained in this study that, the subphase with L- and D-AscA addition created a fixed monolayer at 25°C, while at high temperature 41°C causes alteration in DPPC monolayer to somewhat less densely pack especially L-AscA this is for instance disclosed by left-area shifted of the DPPC monolayer curve shape. The Langmuir monolayer studies revealed that AscA isoforms interrupt the DPPC monolayer during its formation, leads to make the variations in such monolayers properties. This work demonstrates that AscA addition in subphase has applied promising significant disturbing in monolayer play an essential role in biomimetic membrane.

Topographically smooth and stable supported lipid bilayer for high-resolution AFM studies.

The cellular membrane has been identified to play a critical role in various biological processes including the assembly of biological systems. Membranes are complex, primarily two-dimensional assemblies with varied lipid compositions depending on the particular region of the cell. Supported lipid bilayers are considered as appropriate models for physio-chemical studies of membranes including numerous single molecule techniques. Atomic force microscopy (AFM) as a topographic technique is a fully appropriate single molecule technique capable of direct observation of molecular processes on membranes. However, reliable experimental AFM studies require the preparation of the bilayer with a sub-nanometer smooth morphology, which remains stable over long-time observation. Here we present the methodology, which allows one to prepare a smooth, stable, structurally homogeneous lipid bilayer without the presence of any trapped vesicles. We described the application of such lipid bilayers to probe time-dependent early stages of aggregation of monomeric amyloid proteins. Importantly, the proposed methodology can be extended to bilayers with various compositions, by incorporating different lipids for on-membrane aggregation study including cholesterol. Furthermore, this methodology development allowed us to monitor the aggregation of amyloid protein at its physiologically relevant low protein concentration. The flexibility of altering the membrane composition allows to identify the specific role of a particular lipid towards the aggregation kinetics, revealing the plausible mechanism of disease development.

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

Bacterial Mimetic Systems for Studying Bacterial Inactivation and Infection

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins and glycans, which forms a rigid protective layer against the environment. The major component of the outer membrane is the large and amphiphilic molecule lipopolysaccharide (LPS). Understanding the cell wall is important for various fields such as electric field treatments of bacteria and exploration of new antibiotics and alternative treatments (e.g. bacteriophage therapies). Due to the high complexity of the Gram-negative cell wall, there is a great demand for reduced and defined model membrane systems. Giant unilamellar vesicles (GUVs) can provide such a model membrane system. GUVs are versatile synthetic membrane systems that allow the investigation of membrane properties, while controlling parameters such as membrane composition, surrounding media and temperature. Furthermore, effects of external factors like ions, hydrodynamic flows and electromagnetic fields can be studied. In contrast to eukaryotic membranes, models of the Gram-negative outer membrane are lacking. Here, we aim at generating an in vitro model of the Gram-negative bacterial outer membrane. To create such a model membrane system, LPS are integrated in the GUV outer leaflet (LPS-GUVs) using an adapted method based on inverted emulsions. A microfluidic approach is employed to optimize production. The obtained LPS-GUVs will be characterized according to their LPS content, membrane asymmetry and membrane material properties such as bending rigidity and edge tension. Their stability against externally applied electric fields will be compared to that of conventional phospholipid vesicles. Furthermore, the interaction with LPS specific bacteriophages will be studied.

Sphingolipid-Enriched Domains in Yeast: Biophysical Properties and Antifungal Interaction

The presence of sphingolipid-enriched domains (SLED), first identified in the yeast Saccharomyces cerevisiae, is now established in different fungal species. These sterol-depleted fungal membrane regions are in a tightly-packed yet dynamic gel phase. To understand at the molecular level the formation and stability of those domains, we characterized different membrane model systems, composed by POPC, phytoceramide -the yeast sphingolipid backbone- and/or ergosterol. Phytoceramide, but not ergosterol, is essential to obtain rigid domains with an acyl chain packing similar to that found in yeast SLED.

Lipid Nanosystems and Serum Protein as Biomimetic Interfaces: Predicting the Biodistribution of a Caffeic Acid-Based Antioxidant.

PurposeAntiOxCIN3 is a novel mitochondriotropic antioxidant developed to minimize the effects of oxidative stress on neurodegenerative diseases. Prior to an investment in pre-clinical in vivo studies, it is important to apply in silico and biophysical cell-free in vitro studies to predict AntiOxCIN3 biodistribution profile, respecting the need to preserve animal health in accordance with the EU principles (Directive 2010/63/EU). Accordingly, we propose an innovative toolbox of biophysical studies and mimetic models of biological interfaces, such as nanosystems with different compositions mimicking distinct membrane barriers and human serum albumin (HSA).MethodsIntestinal and cell membrane permeation of AntiOxCIN3 was predicted using derivative spectrophotometry. AntiOxCIN3 –HSA binding was evaluated by intrinsic fluorescence quenching, synchronous fluorescence, and dynamic/electrophoretic light scattering. Steady-state and time-resolved fluorescence quenching was used to predict AntiOxCIN3-membrane orientation. Fluorescence anisotropy, synchrotron small- and wide-angle X-ray scattering were used to predict lipid membrane biophysical impairment caused by AntiOxCIN3 distribution.Results and DiscussionWe found that AntiOxCIN3 has the potential to permeate the gastrointestinal tract. However, its biodistribution and elimination from the body might be affected by its affinity to HSA (>90%) and by its steady-state volume of distribution (VDSS=1.89± 0.48 L∙Kg−1). AntiOxCIN3 is expected to locate parallel to the membrane phospholipids, causing a bilayer stiffness effect. AntiOxCIN3 is also predicted to permeate through blood-brain barrier and reach its therapeutic target – the brain.ConclusionDrug interactions with biological interfaces may be evaluated using membrane model systems and serum proteins. This knowledge is important for the characterization of drug partitioning, positioning and orientation of drugs in membranes, their effect on membrane biophysical properties and the study of serum protein binding. The analysis of these interactions makes it possible to collect valuable knowledge on the transport, distribution, accumulation and, eventually, therapeutic impact of drugs which may aid the drug development process.