Interaction of MRI Contrast Agent [Gd(DOTA)]- with Lipid Membranes: A Molecular Dynamics Study.

Contrast agents are important imaging probes in clinical MRI, allowing the identification of anatomic changes that otherwise would not be possible. Intensive research on the development of new contrast agents is being made to image specific pathological markers or sense local biochemical changes. The most widely used MRI contrast agents are based on gadolinium(III) complexes. Due to their very high charge density, they have low permeability through tight biological barriers such as the blood-brain barrier, hampering their application in the diagnosis of neurological disorders. In this study, we explore the interaction between the widely used contrast agent [Gd(DOTA)]- (Dotarem) and POPC lipid bilayers by means of molecular dynamics simulations. This metal complex is a standard reference where several chemical modifications have been introduced to improve key properties such as bioavailability and targeting. The simulations unveil detailed insights into the agent's interaction with the lipid bilayer, offering perspectives beyond experimental methods. Various properties, including the impact on global and local bilayer properties, were analyzed. As expected, the results indicate a low partition coefficient (KP) and high permeation barrier for this reference compound. Nevertheless, favorable interactions are established with the membrane leading to moderately long residence times. While coordination of one inner-sphere water molecule is maintained for the membrane-associated chelate, the physical-chemical attributes of [Gd(DOTA)]- as a MRI contrast agent are affected. Namely, increases in the rotational correlation times and in the residence time of the inner-sphere water are observed, with the former expected to significantly increase the water proton relaxivity. This work establishes a reference framework for the use of simulations to guide the rational design of new contrast agents with improved relaxivity and bioavailability and for the development of liposome-based formulations for use as imaging probes or theranostic agents.

State-of-the-Art Macromolecules in Portugal.

This Special Issue presents five contributions covering various topics, as it would be expected for an area as comprehensive and multidisciplinary as Macromolecules [...].

Nutrient-efficient catfish-based aquaponics for producing lamb's lettuce at two light intensities.

BACKGROUND: Aquaponic systems are sustainable processes of managing water and nutrients for food production. An innovate nutrient-efficient catfish-based (Clarias gariepinus) aquaponics system was implemented for producing two cultivars of two leafy vegetables largely consumed worldwide: lamb's lettuce (Valerianella locusta var. Favor and Valerianella locusta var. de Hollande) and arugula (Eruca vesicaria var. sativa and Eruca sativa). Different growing treatments (4 × 2 factorial design) were applied to plants of each cultivar, grown at two light intensities (120 and 400 µmol m-2 s-1). During growth, several morphological characteristics (root length, plant height, leaf number, foliage diameter and biggest leaf length) were measured. At harvest, plants were weighed and examined qualitatively in terms of greenness and health status. Additionally, leaf extracts were obtained and used to determine total phenolic contents, antioxidant capacities, and levels of cytotoxicity to Caco-2 intestinal model cells. RESULTS: After a 5-week growth period, both lamb's lettuce cultivars presented high levels of greenness and health status, at both light intensities, particularly the var. de Hollande that also showed higher average performance in terms of plant morphology. In turn, arugula cultivars showed lower levels of greenness and health status, especially the cultivar E. vesicaria var. sativa submitted to direct sunlight during growth. In addition, plant specimens submitted to higher levels of light intensity showed higher contents in antioxidants/polyphenols. Cultivars with a higher content in antioxidants/polyphenols led to higher Caco-2 cell viability. CONCLUSION: For successful industrial implementation of the aquaponics technology, different and optimized acclimatizing conditions must be applied to different plant species and cultivars. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Ligand's Partition to the Lipid Bilayer Should Be Accounted for When Estimating Their Affinity to Proteins.

Ligand-protein interactions are usually studied in complex media that also contain lipids. This is particularly relevant for membrane proteins that are always associated with lipid bilayers, but also for water-soluble proteins studied in in vivo conditions. This work addresses the following two questions: (i) How does the neglect of the lipid bilayer influence the apparent ligand-protein affinity? (ii) How can the intrinsic ligand-protein affinity be obtained? Here we present a framework to quantitatively characterize ligand-protein interactions in complex media for proteins with a single binding site. The apparent affinity obtained when following some often-used approximations is also explored, to establish these approximations' validity limits and to allow the estimation of the true affinities from data reported in literature. It is found that an increase in the ligand lipophilicity or in the volume of the lipid bilayer always leads to a decrease in the apparent ligand-protein affinity, both for water-soluble and for membrane proteins. The only exceptions are very polar ligands (excluded from the lipid bilayer) and ligands whose binding affinity to the protein increases supralinearly with ligand lipophilicity. Finally, this work discusses which are the most relevant parameters to consider when exploring the specificity of membrane proteins.

Modeling Gd3+ Complexes for Molecular Dynamics Simulations: Toward a Rational Optimization of MRI Contrast Agents.

The correct parametrization of lanthanide complexes is of the utmost importance for their characterization using computational tools such as molecular dynamics simulations. This allows the optimization of their properties for a wide range of applications, including medical imaging. Here we present a systematic study to establish the best strategies for the correct parametrization of lanthanide complexes using [Gd(DOTA)]- as a reference, which is used as a contrast agent in MRI. We chose the bonded model to parametrize the lanthanide complexes, which is especially important when considering the study of the complex as a whole (e.g., for the study of the dynamics of its interaction with proteins or membranes). We followed two strategies: a so-called heuristic approach employing strategies already published by other authors and another based on the more recent MCPB.py tool. Adjustment of the Lennard-Jones parameters of the metal was required. The final topologies obtained with both strategies were able to reproduce the experimental ion to oxygen distance, vibrational frequencies, and other structural properties. We report a new strategy to adjust the Lennard-Jones parameters of the metal ion in order to capture dynamic properties such as the residence time of the capping water (τm). For the first time, the correct assessment of the τm value for Gd-based complexes was possible by recording the dissociative events over up to 10 µs all-atom simulations. The MCPB.py tool allowed the accurate parametrization of [Gd(DOTA)]- in a simpler procedure, and in this case, the dynamics of the water molecules in the outer hydration sphere was also characterized. This sphere was divided into the first hydration layer, an intermediate region, and an outer hydration layer, with a residence time of 18, 10 and 19 ps, respectively, independent of the nonbonded parameters chosen for Gd3+. The Lennard-Jones parameters of Gd3+ obtained here for [Gd(DOTA)]- may be used with similarly structured gadolinium MRI contrast agents. This allows the use of molecular dynamics simulations to characterize and optimize the contrast agent properties. The characterization of their interaction with membranes and proteins will permit the design of new targeted contrast agents with improved pharmacokinetics.

Analysis of the Equilibrium Distribution of Ligands in Heterogeneous Media-Approaches and Pitfalls.

The equilibrium distribution of small molecules (ligands) between binding agents in heterogeneous media is an important property that determines their activity. Heterogeneous systems containing proteins and lipid membranes are particularly relevant due to their prevalence in biological systems, and their importance to ligand distribution, which, in turn, is crucial to ligand's availability and biological activity. In this work, we review several approaches and formalisms for the analysis of the equilibrium distribution of ligands in the presence of proteins, lipid membranes, or both. Special attention is given to common pitfalls in the analysis, with the establishment of the validity limits for the distinct approaches. Due to its widespread use, special attention is given to the characterization of ligand binding through the analysis of Stern-Volmer plots of protein fluorescence quenching. Systems of increasing complexity are considered, from proteins with single to multiple binding sites, from ligands interacting with proteins only to biomembranes containing lipid bilayers and membrane proteins. A new formalism is proposed, in which ligand binding is treated as a partition process, while considering the saturation of protein binding sites. This formalism is particularly useful for the characterization of interaction with membrane proteins.

Interactions between Rhodamine Dyes and Model Membrane Systems-Insights from Molecular Dynamics Simulations.

BACKGROUND: rhodamines are dyes widely used as fluorescent tags in cell imaging, probing of mitochondrial membrane potential, and as P-glycoprotein model substrates. In all these applications, detailed understanding of the interaction between rhodamines and biomembranes is fundamental. METHODS: we combined atomistic molecular dynamics (MD) simulations and fluorescence spectroscopy to characterize the interaction between rhodamines 123 and B (Rh123 and RhB, respectively) and POPC bilayers. RESULTS: while the xanthene moiety orients roughly parallel to the membrane plane in unrestrained MD simulations, variations on the relative position of the benzoic ring (below the xanthene for Rh123, above it for RhB) were observed, and related to the structure of the two dyes and their interactions with water and lipids. Subtle distinctions were found among different ionization forms of the probes. Experimentally, RhB displayed a lipid/water partition coefficient more than two orders of magnitude higher than Rh123, in agreement with free energy profiles obtained from umbrella sampling MD. CONCLUSIONS: this work provided detailed insights on the similarities and differences in the behavior of bilayer-inserted Rh123 and RhB, related to the structure of the probes. The much higher affinity of RhB for the membranes increases the local concentration and explains its higher apparent affinity for P-glycoprotein reconstituted in model membranes.

Uncertainty in protein-ligand binding constants: asymmetric confidence intervals versus standard errors.

Equilibrium binding constants (Kb) between chemical compounds and target proteins or between interacting proteins provide a quantitative understanding of biological interaction mechanisms. Reported uncertainties of measured experimental parameters are critical for decision-making in many scientific areas, e.g., in lead compound discovery processes and in comparing computational predictions with experimental results. Uncertainties in measured Kb values are commonly represented by a symmetric normal distribution, often quoted in terms of the experimental value plus-minus the standard deviation. However, in general, the distributions of measured Kb (and equivalent Kd) values and the corresponding free energy change ΔGb are all asymmetric to varying degree. Here, using a simulation approach, we illustrate the effect of asymmetric Kb distributions within the realm of isothermal titration calorimetry (ITC) experiments. Further we illustrate the known, but perhaps not widely appreciated, fact that when distributions of any of Kb, Kd and ΔGb are transformed into each other, their degree of asymmetry is changed. Consequently, we recommend that a more accurate way of expressing the uncertainties of Kb, Kd, and ΔGb values is to consistently report 95% confidence intervals, in line with other authors' suggestions. The ways to obtain such error ranges are discussed in detail and exemplified for a binding reaction obtained by ITC.

Polysaccharide Structures and Their Hypocholesterolemic Potential.

Several classes of polysaccharides have been described to have hypocholesterolemic potential, namely cholesterol bioaccessibility and bioavailability. This review will highlight the main mechanisms by which polysaccharides are known to affect cholesterol homeostasis at the intestine, namely the effect (i) of polysaccharide viscosity and its influence on cholesterol bioaccessibility; (ii) on bile salt sequestration and its dependence on the structural diversity of polysaccharides; (iii) of bio-transformations of polysaccharides and bile salts by the gut microbiota. Different quantitative structure-hypocholesterolemic activity relationships have been explored depending on the mechanism involved, and these were based on polysaccharide physicochemical properties, such as sugar composition and ramification degree, linkage type, size/molecular weight, and charge. The information gathered will support the rationalization of polysaccharides' effect on cholesterol homeostasis and highlight predictive rules towards the development of customized hypocholesterolemic functional food.

The Secret Lives of Fluorescent Membrane Probes as Revealed by Molecular Dynamics Simulations.

Fluorescent probes have been employed for more than half a century to study the structure and dynamics of model and biological membranes, using spectroscopic and/or microscopic experimental approaches. While their utilization has led to tremendous progress in our knowledge of membrane biophysics and physiology, in some respects the behavior of bilayer-inserted membrane probes has long remained inscrutable. The location, orientation and interaction of fluorophores with lipid and/or water molecules are often not well known, and they are crucial for understanding what the probe is actually reporting. Moreover, because the probe is an extraneous inclusion, it may perturb the properties of the host membrane system, altering the very properties it is supposed to measure. For these reasons, the need for independent methodologies to assess the behavior of bilayer-inserted fluorescence probes has been recognized for a long time. Because of recent improvements in computational tools, molecular dynamics (MD) simulations have become a popular means of obtaining this important information. The present review addresses MD studies of all major classes of fluorescent membrane probes, focusing in the period between 2011 and 2020, during which such work has undergone a dramatic surge in both the number of studies and the variety of probes and properties accessed.

Rat cardiomyocyte H9c2(2-1)-based sulforhodamine B assay as a promising in vitro method to assess the biological component of effluent toxicity.

The treatment of wastewaters is crucial to maintain the ecological status of receiving waters, and thereby guarantee the protection of aquatic life and human health. Wastewater quality evaluation is conventionally based on physicochemical parameters, but increasing attention has been paid to integrate physicochemical and biological data. Nevertheless, the regulatory use of fish in biological testing methods has been subject to various ethical and cost concerns, and in vitro cell-based assays have thus become an important topic of interest. Hence, the present study intends: (a) to evaluate the efficiency of two different sample pre-concentration techniques (lyophilisation and solid phase extraction) to assess the toxicity of municipal effluents on rat cardiomyoblast H9c2(2-1) cells, and (b) maximizing the use of the effluent sample collected, to estimate the environmental condition of the receiving environment. The gathered results demonstrate that the H9c2(2-1) sulforhodamine B-based assay is an appropriate in vitro method to assess biological effluent toxicity, and the best results were attained by lyophilising the sample as pre-treatment. Due to its response, the H9c2(2-1) cell line might be a possible alternative in vitro model for fish lethal testing to assess the toxicity of municipal effluents. The physicochemical status of the sample suggests a high potential for eutrophication, and iron exceeded the permissible level for wastewater discharge, possibly due to the addition of ferric chloride for wastewater treatment. In general, the levels of carbamazepine and sulfamethoxazole are higher than those reported for other countries, and both surpassed the aquatic protective values for long-term exposure.

Quantification of Cholesterol Solubilized in Dietary Micelles: Dependence on Human Bile Salt Variability and the Presence of Dietary Food Ingredients.

The solubility of cholesterol in bile salt (BS) micelles is important to understand the availability of cholesterol for absorption in the intestinal epithelium and to develop strategies to decrease cholesterol intake from the intestinal lumen. This has been the subject of intense investigation, due to the established relation between the development of diseases such as atherosclerosis and high levels of cholesterol in the blood. In this work we quantify the effect of BS variability on the amount of cholesterol solubilized. The effect of some known hypocholesterolemic agents usually found in the diet is also evaluated, as well as some insight regarding the mechanisms involved. The results show that, depending on the bile salt composition, the average value of sterol per micelle is equal to or lower than 1. The amount of cholesterol solubilized in the BS micelles is essentially equal to its total concentration until the solubility limit is reached. Altogether, this indicates that the maximum cholesterol solubility in the BS micellar solution is the result of saturation of the aqueous phase and depends on the partition coefficient of cholesterol between the aqueous phase and the micellar pseudophase. The effect on cholesterol maximum solubility for several food ingredients usually encountered in the diet was characterized using methodology developed recently by us. This method allows the simultaneous quantification of both cholesterol and food ingredient solubilized in the BS micelles even in the presence of larger aggregates, therefore avoiding their physical separation with possible impacts on the overall equilibrium. The phytosterols stigmasterol and stigmastanol significantly decreased cholesterol solubility with a concomitant reduction in the total amount of sterol solubilized, most pronounced for stigmasterol. Those results point toward coprecipitation being the major cause for the decrease in cholesterol solubilization by the BS micelles. The presence of tocopherol and oleic acid leads to a small decrease in the amount of cholesterol solubilized while palmitic acid slightly increases the solubility of cholesterol. Those dietary food ingredients are completely solubilized by the BS micelles, indicating that the effects on cholesterol solubility are due to changes in the properties of the mixed micelles.

Interaction of Bile Salts with Model Membranes Mimicking the Gastrointestinal Epithelium: A Study by Isothermal Titration Calorimetry.

Bile salts (BS) are biosurfactants synthesized in the liver and secreted into the intestinal lumen where they solubilize cholesterol and other hydrophobic compounds facilitating their gastrointestinal absorption. Partition of BS toward biomembranes is an important step in both processes. Depending on the loading of the secreted BS micelles with endogeneous cholesterol and on the amount of cholesterol from diet, this may lead to the excretion or absorption of cholesterol, from cholesterol-saturated membranes in the liver or to gastrointestinal membranes, respectively. The partition of BS toward the gastrointestinal membranes may also affect the barrier properties of those membranes affecting the permeability for hydrophobic and amphiphilic compounds. Two important parameters in the interaction of the distinct BS with biomembranes are their partition coefficient and the rate of diffusion through the membrane. Altogether, they allow the calculation of BS local concentrations in the membrane as well as their asymmetry in both membrane leaflets. The local concentration and, most importantly, its asymmetric distribution in the bilayer are a measure of induced membrane perturbation, which is expected to significantly affect its properties as a cholesterol donor and hydrophobic barrier. In this work we have characterized the partition of several BS, nonconjugated and conjugated with glycine, to large unilamellar vesicles (LUVs) in the liquid-disordered phase and with liquid-ordered/liquid-disordered phase coexistence, using isothermal titration calorimetry (ITC). The partition into the liquid-disordered bilayer was characterized by large partition coefficients and favored by enthalpy, while association with the more ordered membrane was weak and driven only by the hydrophobic effect. The trihydroxy BS partitions less efficiently toward the membranes but shows faster translocation rates, in agreement with a membrane protective effect of those BS. The rate of translocation through the more ordered membrane was faster, indicating accumulation of BS at specific locations in this membrane.

Behaviour of NBD-head group labelled phosphatidylethanolamines in POPC bilayers: a molecular dynamics study.

A complete homologous series of fluorescent phosphatidylethanolamines (diCnPE), labelled at the head group with a 7-nitrobenz-2-oxa-1,3-diazo-4-yl(NBD) fluorophore and inserted in 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, was studied using atomistic molecular dynamics simulations. The longer-chained derivatives of NBD-diCnPE, with n = 14, 16, and 18, are commercially available, and widely used as fluorescent membrane probes. Properties such as location of atomic groups and acyl chain order parameters of both POPC and NBD-diCnPE, fluorophore orientation and hydrogen bonding, membrane electrostatic potential and lateral diffusion were calculated for all derivatives in the series. Most of these probes induce local disordering of POPC acyl chains, which is on the whole counterbalanced by ordering resulting from binding of sodium ions to lipid carbonyl/glycerol oxygen atoms. An exception is found for NBD-diC16PE, which displays optimal matching with POPC acyl chain length and induces a slight local ordering of phospholipid acyl chains. Compared to previously studied fatty amines, acyl chain-labelled phosphatidylcholines, and sterols bearing the same fluorescent tag, the chromophore in NBD-diCnPE locates in a similar region of the membrane (near the glycerol backbone/carbonyl region) but adopts a different orientation (with the NO2 group facing the interior of the bilayer). This modification leads to an inverted orientation of the P-N axis in the labelled lipid, which affects the interface properties, such as the membrane electrostatic potential and hydrogen bonding to lipid head group atoms. The implications of this study for the interpretation of the photophysical properties of NBD-diCnPE (complex fluorescence emission kinetics, differences with other NBD lipid probes) are discussed.

Interaction of NBD-labelled fatty amines with liquid-ordered membranes: a combined molecular dynamics simulation and fluorescence spectroscopy study.

A complete homologous series of fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl-(NBD) labelled fatty amines of varying alkyl chain lengths, NBD-Cn, inserted in 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) or N-palmitoyl sphingomyelin (SpM) bilayers, with 50 mol% and 40 mol% cholesterol (Chol), respectively, was studied using atomistic molecular dynamics simulations. For all amphiphiles in both bilayers, the NBD fluorophore locates at the interface, in a more external position than that previously observed for pure POPC bilayers. This shallower location of the NBD group agrees with the lower fluorescent quantum yield, shorter fluorescence lifetime, and higher ionisation constants (smaller pKa) determined experimentally. The more external location is also consistent with the changes measured in steady-state fluorescence anisotropy from POPC to POPC/Chol (1 : 1) vesicles. Accordingly, the equilibrium location of the NBD group within the various bilayers is mainly dictated by bilayer compositions, and is mostly unaffected by the length of the attached alkyl chain. Similarly to the behaviour observed in POPC bilayers, the longer-chained NBD-Cn amphiphiles show significant mass density near the mixed bilayers' midplanes, and the alkyl chains of the longer derivatives, mainly NBD-C16, penetrate the opposite bilayer leaflet to some extent. However, this effect is quantitatively less pronounced in these ordered bilayers than in POPC. Similarly to POPC bilayers, the effects of these amphiphiles on the structure and dynamics of the host lipid were found to be relatively mild, in comparison with acyl-chain phospholipid analogues.

Homeostasis of free cholesterol in the blood: a preliminary evaluation and modeling of its passive transport.

The rate of noncatalyzed transfer of cholesterol (Chol) among lipoproteins and cells in the blood is of fundamental importance as a baseline to assess the role of active transport mechanisms, but remains unknown. Here we address this gap by characterizing the associa-tion of the Chol analog, ergosta-5,7,9(11),22-tetraen-3ß-ol (DHE), with the lipoproteins VLDL, LDL, HDL2, and HDL3 Combining these results with data for the association of DHE with liposomes, we elaborated a kinetic model for the noncatalyzed exchange of free Chol among blood compartments. The computational results are in good agreement with experimental values. The small deviations are explained by the nonequilibrium distribution of unesterified Chol in vivo, due to esterification and entry of new unesterified Chol, and eventual effects introduced by incubations at low temperatures. The kinetic profile of the homeostasis of unesterified Chol in the blood predicted by the model developed in this work is in good agreement with the observations in vivo, highlighting the importance of passive processes.

Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling.

Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the CLN3 gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3exΔ7/8 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.

Nutritive Value and Bioactivities of a Halophyte Edible Plant: Crithmum maritimum L. (Sea Fennel).

Crithmum maritimum L. (sea fennel), an edible xerophyte of coastal habitats, is considered an emerging cash crop for biosaline agriculture due to its salt-tolerance ability and potential applications in the agri-food sector. Here, the nutritional value and bioactive properties of sea fennel are described. Sea fennel leaves, flowers, and schizocarps are composed of carbohydrates (>65%) followed by ash, proteins, and lipids. Sea fennel's salty, succulent leaves are a source of omega-6 and omega-3 polyunsaturated fatty acids, especially linoleic acid. Extracts obtained from flowers and fruits/schizocarps are rich in antioxidants and polyphenols and show antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermis, Candida albicans, and Candida parapsilosis. Plant material is particularly rich in sodium (Na) but also in other nutritionally relevant minerals, such as calcium (Ca), chlorine (Cl), potassium (K), phosphorus (P), and sulfur (S), beyond presenting a potential prebiotic effect on Lactobacillus bulgaricus and being nontoxic to human intestinal epithelial Caco-2 model cells, up to 1.0% (w/v). Hence, the rational use of sea fennel can bring nutrients, aroma, and flavor to culinary dishes while balancing microbiomes and contributing to expanding the shelf life of food products.

Cholesterol and POPC segmental order parameters in lipid membranes: solid state 1H-13C NMR and MD simulation studies.

The concentration of cholesterol in cell membranes affects membrane fluidity and thickness, and might regulate different processes such as the formation of lipid rafts. Since interpreting experimental data from biological membranes is rather intricate, investigations on simple models with biological relevance are necessary to understand the natural systems. We study the effect of cholesterol on the molecular structure of multi-lamellar vesicles (MLVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a phospholipid ubiquitous in cell membranes, with compositions in the range 0-60 mol% cholesterol. Order parameters, |S(CH)|, are experimentally determined by using (1)H-(13)C solid-state nuclear magnetic resonance (NMR) spectroscopy with segmental detail for all parts of both the cholesterol and POPC molecules, namely the ring system and alkyl chain of the sterol, as well as the glycerol backbone, choline headgroup and the sn-1 and sn-2 acyl chains of POPC. With increasing cholesterol concentration the acyl chains gradually adopt a more extended conformation while the orientation and dynamics of the polar groups are rather unaffected. Additionally, we perform classical molecular dynamics simulations on virtual bilayers mimicking the POPC-cholesterol MLVs investigated by NMR. Good agreement between experiments and simulations is found for the cholesterol alignment in the bilayer and for the |S(CH)| profiles of acyl chains below 15 mol% cholesterol. Deviations occur for the choline headgroup and glycerol backbone parts of POPC, as well as for the phospholipid and cholesterol alkyl chains at higher cholesterol concentrations. The unprecedented detail of the NMR data enables a more complete comparison between simulations and experiments on POPC-cholesterol bilayers and may aid in developing more realistic model descriptions of biological membranes.

Permeation of a Homologous Series of NBD-Labeled Fatty Amines through Lipid Bilayers: A Molecular Dynamics Study.

Permeation through biomembranes is ubiquitous for drugs to reach their active sites. Asymmetry of the cell plasma membrane (PM) has been described as having an important role in this process. Here we describe the interaction of a homologous series of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled amphiphiles (NBD-Cn, n = 4 to 16) with lipid bilayers of different compositions (1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol (1:1) and palmitoylated sphingomyelin (SpM):cholesterol (6:4)), including an asymmetric bilayer. Both unrestrained and umbrella sampling (US) simulations (at varying distances to the bilayer center) were carried out. The free energy profile of NBD-Cn at different depths in the membrane was obtained from the US simulations. The behavior of the amphiphiles during the permeation process was described regarding their orientation, chain elongation, and H-bonding to lipid and water molecules. Permeability coefficients were also calculated for the different amphiphiles of the series, using the inhomogeneous solubility-diffusion model (ISDM). Quantitative agreement with values obtained from kinetic modeling of the permeation process could not be obtained. However, for the longer, and more hydrophobic amphiphiles, the variation trend along the homologous series was qualitatively better matched by the ISDM when the equilibrium location of each amphiphile was taken as reference (ΔG = 0), compared to the usual choice of bulk water.