Dynamic and Energetic Aspects of Carotenoids In-and-Around Model Lipid Membranes Revealed in Molecular Modelling.

In contrast to plants, humans are unable to synthesise carotenoids and have to obtain them from diet. Carotenoids fulfil several crucial biological functions in the organism; however, due to poor solubility in water, their bioavailability from plant-based food is low. The processes of carotenoid absorption and availability in the human body have been intensively studied. The recent experimental findings concerning these processes are briefly presented in the introductory part of this review, together with a summary of such topics as carotenoid carriers, body transport and tissue delivery, to finally report on molecular-level studies of carotenoid binding by membrane receptors. The main message of the review is contained in the section describing computational investigations of carotenoid intercalation and dynamic behaviour in lipid bilayers. The relevance of these computational studies lies in showing the direct link between the microscopic behaviour of molecules and the characteristics of their macroscopic ensembles. Furthermore, studying the interactions between carotenoids and lipid bilayers, and certainly proteins, on the molecular- and atomic-level using computational methods facilitates the interpretation and explanation of their macroscopic properties and, hopefully, helps to better understand the biological functions of carotenoids.

Protecting the Eye Lens from Oxidative Stress through Oxygen Regulation.

Molecular oxygen is a primary oxidant that is involved in the formation of active oxygen species and in the oxidation of lipids and proteins. Thus, controlling oxygen partial pressure (concentration) in the human organism, tissues, and organs can be the first step in protecting them against oxidative stress. However, it is not an easy task because oxygen is necessary for ATP synthesis by mitochondria and in many biochemical reactions taking place in all cells in the human body. Moreover, the blood circulatory system delivers oxygen to all parts of the body. The eye lens seems to be the only organ that is protected from the oxidative stress through the regulation of oxygen partial pressure. The basic mechanism that developed during evolution to protect the eye lens against oxidative damage is based on the maintenance of a very low concentration of oxygen within the lens. This antioxidant mechanism is supported by the resistance of both the lipid components of the lens membrane and cytosolic proteins to oxidation. Any disturbance, continuous or acute, in the working of this mechanism increases the oxygen concentration, in effect causing cataract development. Here, we describe the biophysical basis of the mechanism and its correlation with lens transparency.

Stacks of monogalactolipid bilayers can transform into a lattice of water channels.

The lipid matrix of thylakoid membranes is a lamellar bilayer, but under a certain condition it can convert locally into a nonlamellar structure. This is possible because one of the main membrane lipids, MGDG, promotes the formation of an inverse hexagonal phase. Here, the spontaneous transformation of aligned hydrated MGDG bilayers into nonlamellar structures is investigated using all-atom molecular dynamics simulation. Previous studies have demonstrated that MGDG polar head groups connect vertically across the interface. In this study, the evolution of the system's initial structure into a lattice of water channels and contacted surfaces created by numerous vertical MGDG connections depended on the width of the hydrating water layers. These widths controlled the bilayers' ability to bend, which was a prerequisite for channel formation. Locally, an intensive exchange of MGDG molecules between apposing bilayer leaflets occurred, although a stable semi-toroidal stalk did not develop.

Lipid/water interface of galactolipid bilayers in different lyotropic liquid-crystalline phases.

In this study, carried out using computational methods, the organisation of the lipid/water interface of bilayers composed of galactolipids with both α-linolenoyl acyl chains is analysed and compared in three different lyotropic liquid-crystalline phases. These systems include the monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG) bilayers in the lamellar phase, the MGDG double bilayer during stalk phase formation and the inverse hexagonal MGDG phase. For each system, lipid-water and direct and water-mediated lipid-lipid interactions between the lipids of one bilayer leaflet and those of two apposing leaflets at the onset of new phase (stalk) formation, are identified. A network of interactions between DGDG molecules and its topological properties are derived and compared to those for the MGDG bilayer.

Molecular oxygen as a probe molecule in EPR spin-labeling studies of membrane structure and dynamics.

Molecular oxygen (O2) is the perfect probe molecule for membrane studies carried out using the saturation recovery EPR technique. O2 is a small, paramagnetic, hydrophobic enough molecule that easily partitions into a membrane's different phases and domains. In membrane studies, the saturation recovery EPR method requires two paramagnetic probes: a lipid-analog nitroxide spin label and an oxygen molecule. The experimentally derived parameters of this method are the spin-lattice relaxation times (T 1s) of spin labels and rates of bimolecular collisions between O2 and the nitroxide fragment. Thanks to the long T 1 of lipid spin labels (from 1 to 10 µs), the approach is very sensitive to changes of the local (around the nitroxide fragment) O2 diffusion-concentration product. Small variations in the lipid packing affect O2 solubility and O2 diffusion, which can be detected by the shortening of T 1 of spin labels. Using O2 as a probe molecule and a different lipid spin label inserted into specific phases of the membrane and membrane domains allows data about the lateral arrangement of lipid membranes to be obtained. Moreover, using a lipid spin label with the nitroxide fragment attached to its head group or a hydrocarbon chain at different positions also enables data about molecular dynamics and structure at different membrane depths to be obtained. Thus, the method can be used to investigate not only the lateral organization of the membrane (i.e., the presence of membrane domains and phases), but also the depth-dependent membrane structure and dynamics, and, hence, the membrane properties in three dimensions.

Lutein and Zeaxanthin in the Lipid Bilayer-Similarities and Differences Revealed by Computational Studies.

Lutein and zeaxanthin are two similar carotenoids of the xanthophyll subgroup. Carotenoids are synthesized almost entirely by plants but are also present in significant amounts in animals. They are essential components of the lipid matrix of biomembranes, and one of their functions is to protect cells from light radiation, free radicals and oxidative stress. Carotenoids, depending on their chemical structure, can locate at various positions and in different orientations in the bilayer. Xanthophylls (XAN) are polar and in the bilayer are positionally restricted. In the case of lutein and zeaxanthin, whose both ionone rings are hydroxy-substituted and as such are anchored in the lipid bilayer interfaces, the position is generally transmembrane. However, both experimental and computer modelling studies indicate that lutein can also locate horizontally below the bilayer interface. This location has never been observed for zeaxanthin. To find a molecular-level explanation for the difference in the orientations of the XAN molecules in the bilayer, a number of phosphatidylcholine-XAN bilayers were constructed and molecular dynamics (MD) simulated for 1.1 µs each. The all-trans XAN molecules were initially placed either parallel or perpendicular to the bilayer surface. With the exception of one lutein, the horizontally placed molecules adopted the transmembrane orientation within 100-600 ns. On the basis of detailed analyses of the XAN orientations and the numbers and lifetimes of their interactions in the bilayer, a plausible explanation is offered as to why a lutein molecule may remain in the horizontal orientation while zeaxanthin does not. Contrary to common believe, lutein horizontal orientation is not related to the ε-ring rotation around the C6'-C7' bond.

Data for molecular dynamic simulations in the OPLSAA force field: Partial charges of cholesterol, C7-hydroxycholesterol and C7-hydroperoxycholesterol, torsional parameters for the hydroperoxy group of C7-hydroperoxycholesterol.

This data article contains partial charges generated for cholesterol, C7-hydroxycholesterol and C7-hydroperoxycholesterol and torsional parameters for hydroperoxy of C7-hydroperoxycholesterol for molecular dynamics simulations in the OPLSAA force field [1] using the package Gromacs [2]. The hydroperoxy group remained unparameterized in the OPLSAA force field and the parameters obtained have the potential for re-use in similar simulations. The atom-centred point charges on each sterol molecule were derived using the restrained electrostatic potential (RESP) approach [3]. The parameters for the C7-OET-OH-HO and C8-C7-OET-OH torsion angles were derived by fitting the parameters of the torsional term (Ryckaert-Bellemans function) of the OPLSAA potential energy function to the quantum mechanical rotational energy profile calculated at CCSD(T)/cc-pVQZ level of theory. This article presents data used in the research article "Chirality affects cholesterol-oxysterol association in water, a computational study" [4].

Chirality affects cholesterol-oxysterol association in water, a computational study.

Cholesterol (Chol) is the most prevalent sterol in the animal kingdom and an indispensable component of mammalian cell membranes. Chol content in the membrane is strictly controlled, although the oxidation of phospholipids may change the relative content of membrane Chol. An excess of it results in the formation of pure Chol microdomains in the membrane. It is likely that some Chol molecules detach from the domains and self-assemble in the aqueous environment. This may promote Chol microcrystallisation, which initiates the development of gallstones and atherosclerotic plaque. In this study, the molecular dynamics, free energy perturbation, umbrella sampling and Voronoi diagram methods are used to reveal the details of self-association of Chol and its oxidised forms (oxChol), namely 7α,ß-hydroxycholesterol and 7α,ß-hydroperoxycholesterol, in water. In the first part of the study the interactions between a sterol monomer and water over a short and longer timescale as well as the energy of hydration of each sterol are analysed. This helps one to understand Chol-Chol and Chol-OxChol with different chirality self-association in water better, which is analysed in the second part of the study. The Voronoi diagram approach is used to determine the relative arrangement of molecules in the dimer and, most importantly, to analyse the dehydration of the contacting surfaces of the assembling molecules. Free energy calculations indicate that Chol and 7ß-hydroxycholesterol associate into the most stable dimer and that Chol-Chol is the next most stable of the five dimers studied. Employing different computational methods enables us to obtain an adequate picture of Chol-sterol self-association in water, which includes dynamic, energetic and temporal aspects of the process.

Hypothetical Pathway for Formation of Cholesterol Microcrystals Initiating the Atherosclerotic Process.

Major factors leading to the development of atherosclerosis are a high cholesterol (Chol) level in the blood and oxidative stress. Both promote the formation of Chol microcrystals in blood vessel walls. Deposition of Chol microcrystals in arterial intima causes inflammation, which initiates and accompanies the atherosclerotic process in all its phases. One of the possible sources of Chol in the blood vessel walls is oxidized low-density lipoproteins-this atherosclerotic plaque formation pathway has already been described in the literature. Here, we hypothesize that initiation of the atherosclerotic process may involve Chol domains in the plasma membranes of arterial cells. Increased Chol content and the presence of polyunsaturated phospholipids in these membranes together with oxidative stress (phospholipid peroxidation) may lead to the formation of pure Chol bilayer domains that, with further peroxidation and increased Chol content, may collapse in the form of Chol seed crystals. Independent of their origin, Chol microcrystals activate inflammasomes, thereby stimulate immune responses, and initiate inflammation that may lead to the development of atherosclerosis. This new, hypothetical pathway has not yet been investigated in depth; however, data from the literature and our own results support its feasibility.

Formation of cholesterol Bilayer Domains Precedes Formation of Cholesterol Crystals in Membranes Made of the Major Phospholipids of Human Eye Lens Fiber Cell Plasma Membranes.

Purpose/Aim: The goal of this study is to reveal how age-related changes in phospholipid (PL) composition in the fiber cell plasma membranes of the human eye lens affect the cholesterol (Chol) content at which Chol bilayer domains (CBDs) and Chol crystals start to form.Materials and Methods: Saturation-recovery electron paramagnetic resonance with spin-labeled cholesterol analogs and differential scanning calorimetry were used to determine the Chol contents at which CBDs and cholesterol crystals, respectively, start to form in in membranes made of the major PL constituents of the plasma membrane of the human eye lens fiber cells. To preserve compositional homogeneity throughout the membrane suspension, the lipid multilamellar dispersions investigated in this work were prepared using a rapid solvent exchange method. The cholesterol content changed from 0 to 75 mol%.Results: The saturation recovery electron paramagnetic resonance results show that CBDs start to form at 33, 50, 46, and 48 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively. The differential scanning calorimetry results show that Chol crystals start to form at 50, 66, 70, and 66 mol% Chol in the phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and sphingomyelin bilayers, respectively.Conclusions: These results, as well those of our previous studies, indicate that the formation of CBDs precedes the formation of Chol crystals in all of the studied systems, and the appearance of each depends on the type of PL forming the bilayer. These findings contribute to a better understanding of the molecular mechanisms involved in the regulation of Chol-dependent processes in eye lens fiber cell membranes.

Asymmetric Spontaneous Intercalation of Lutein into a Phospholipid Bilayer, a Computational Study.

Lutein, a hydroxylated carotenoid, is a pigment synthesised by plants and bacteria. Animals are unable to synthesise lutein, nevertheless, it is present in animal tissues, where its only source is dietary intake. Both in plants and animals, carotenoids are associated mainly with membranes where they carry out important physiological functions. Their trafficking to and insertion into membranes are not well recognised due to experimental difficulties. In this paper, a computational approach is used to elucidate details of the dynamics and energetics of lutein intercalation from the water to the phospholipid bilayer phase. The dynamics is studied using molecular dynamics simulation, and the energetics using umbrella sampling. Lutein spontaneous insertion into the bilayer and translocation across it proceed via formation of hydrogen bonds between its hydroxyl groups and water and/or phospholipid oxygen atoms as well as desolvation of its polyene chain. As lutein molecule is asymmetric, its bilayer intercalation is also asymmetric. The course of events and timescale of the intercalation are different from those of helical peptides. The time of full lutein intercalation ranges from 20 to 100 ns and its final orientation is predominately vertical. Nevertheless, some lutein molecules are in the final horizontal position and some aggregate in the water phase and remain there for the whole simulation time. The highest energy barrier for the intercalation process is ~2.2 kcal/mol and the energy gain is ~18 kcal/mol. The results obtained for lutein can be applied to other xanthophylls and molecules of a similar structure.

Is the tilt of the lipid head group correlated with the number of intermolecular interactions at the bilayer interface?

Lipid and water molecules comprising the bilayer form an integral entity owing to only weak physical interactions. At the bilayer interface, these interactions chiefly involve hydrogen bonding and charge pairing. Lipid head groups make hydrogen bonds (H-bonds) predominantly with water, whereas interlipid H-bonds and charge pairs are less numerous. Both interlipid H-bonding and charge pairing depend on the distance and relative orientation of the interacting head groups. In this computational paper, correlations are analysed between the orientation of the lipid head group and the number of interlipid interactions at the interface of a bilayer made of galactolipids, forming direct interlipid H-bonds, and of phosphatidylcholines forming interlipid charge pairs. The correlations are not strong, however, in both bilayers they show a similar trend.

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (PM) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the PM across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the PM of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens.

High Cholesterol/Low Cholesterol: Effects in Biological Membranes: A Review.

Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.

Assessing gastric toxicity of xanthone derivatives of anti-inflammatory activity using simulation and experimental approaches.

Xanthones are tricyclic compounds of natural or synthetic origin exhibiting a broad spectrum of therapeutic activities. Three synthetic xanthone derivatives (KS1, KS2, and KS3) with properties typical for nonsteroidal anti-inflammatory drugs (NSAID) were objects of the presented model study. NSAIDs are in common use however; several of them exhibit gastric toxicity predominantly resulting from their direct interactions with the outermost lipid layer of the gastric mucosa that impair its hydrophobic barrier property. Among the studied xanthones, gastric toxicity of only KS2 has been determined in previous pharmacological studies, and it is low. In this study, carried out using X-ray diffraction and computer simulation, a palmitoyloleoylphosphatidylcholine-cholesterol bilayer (POPC-Chol) was used as a model of a hydrophobic layer of lipids protecting gastric mucosa as POPC and Chol are the main lipids in human mucus. X-ray diffraction data were used to validate the computer model. The aim of the study was to assess potential gastric toxicity of the xanthones by analysing their atomic level interactions with lipids, ions, and water in the lipid bilayer and their effect on the bilayer physicochemical properties. The results show that xanthones have small effect on the bilayer properties except for its rigidity whereas their interactions with water, ions, and lipids depend on their protonation state and for a given state, are similar for all the xanthones. As gastric toxicity of KS2 is low, based on MD simulations one can predict that toxicity of KS1 and KS3 is also low.

Fitmunk: improving protein structures by accurate, automatic modeling of side-chain conformations.

Improvements in crystallographic hardware and software have allowed automated structure-solution pipelines to approach a near-`one-click' experience for the initial determination of macromolecular structures. However, in many cases the resulting initial model requires a laborious, iterative process of refinement and validation. A new method has been developed for the automatic modeling of side-chain conformations that takes advantage of rotamer-prediction methods in a crystallographic context. The algorithm, which is based on deterministic dead-end elimination (DEE) theory, uses new dense conformer libraries and a hybrid energy function derived from experimental data and prior information about rotamer frequencies to find the optimal conformation of each side chain. In contrast to existing methods, which incorporate the electron-density term into protein-modeling frameworks, the proposed algorithm is designed to take advantage of the highly discriminatory nature of electron-density maps. This method has been implemented in the program Fitmunk, which uses extensive conformational sampling. This improves the accuracy of the modeling and makes it a versatile tool for crystallographic model building, refinement and validation. Fitmunk was extensively tested on over 115 new structures, as well as a subset of 1100 structures from the PDB. It is demonstrated that the ability of Fitmunk to model more than 95% of side chains accurately is beneficial for improving the quality of crystallographic protein models, especially at medium and low resolutions. Fitmunk can be used for model validation of existing structures and as a tool to assess whether side chains are modeled optimally or could be better fitted into electron density. Fitmunk is available as a web service at http://kniahini.med.virginia.edu/fitmunk/server/ or at http://fitmunk.bitbucket.org/.

Computer modelling studies of the bilayer/water interface.

This review summarises high resolution studies on the interface of lamellar lipid bilayers composed of the most typical lipid molecules which constitute the lipid matrix of biomembranes. The presented results were obtained predominantly by computer modelling methods. Whenever possible, the results were compared with experimental results obtained for similar systems. The first and main section of the review is concerned with the bilayer-water interface and is divided into four subsections. The first describes the simplest case, where the interface consists only of lipid head groups and water molecules and focuses on interactions between the lipid heads and water molecules; the second describes the interface containing also mono- and divalent ions and concentrates on lipid-ion interactions; the third describes direct inter-lipid interactions. These three subsections are followed by a discussion on the network of direct and indirect inter-lipid interactions at the bilayer interface. The second section summarises recent computer simulation studies on the interactions of antibacterial membrane active compounds with various models of the bacterial outer membrane. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Cis and trans unsaturated phosphatidylcholine bilayers: A molecular dynamics simulation study.

Trans unsaturated lipids are uncommon in nature. In the human diet, they occur as natural products of ruminal bacteria or from industrial food processing like hydrogenation of vegetable oils. Consumption of trans unsaturated lipids has been shown to have a negative influence on human health; in particular, the risk of cardiovascular disease is higher when the amount of trans unsaturated lipids in the diet is elevated. In this study, we first performed quantum mechanical calculations to specifically and accurately parameterize cis and trans mono-unsaturated lipids and subsequently validated the newly derived parameter set. Then, we carried out molecular dynamics (MD) simulations of lipid bilayers composed of cis or trans unsaturated lipids with and without cholesterol. Our results show that trans mono-unsaturated chains are more flexible than cis mono-unsaturated chains due to lower barriers for rotation around the single bonds next to the trans double bond than those next to the cis double bond. In effect, interactions between cholesterol and trans unsaturated chains are stronger than cis unsaturated chains, which results in a higher ordering effect of cholesterol in trans unsaturated bilayers.

Topologies, structures and parameter files for lipid simulations in GROMACS with the OPLS-aa force field: DPPC, POPC, DOPC, PEPC, and cholesterol.

In this data article we provide topologies and force field parameters files for molecular dynamics simulations of lipids in the OPLS-aa force field using the GROMACS package. This is the first systematic parameterization of lipid molecules in this force field. Topologies are provided for four phosphatidylcholines: saturated DPPC, mono-cis unsaturated POPC and DOPC, and mono-trans unsaturated PEPC. Parameterization of the phosphatidylcholines was achieved in two steps: first, we supplemented the OPLS force field parameters for DPPC with new parameters for torsion angles and van der Waals parameters for the carbon and hydrogen atoms in the acyl chains, as well as new partial atomic charges and parameters for torsion angles in the phosphatidylcholine and glycerol moieties [1]. Next, we derived parameters for the cis and trans double bonds and the neighboring them single bonds [2]. Additionally, we provide GROMACS input files with parameters describing simulation conditions (md.mdp), which are strongly recommended to be used with these lipids models. The data are associated with the research article "Cis and trans unsaturated phosphatidylcholine bilayers: a molecular dynamics simulation study" [2] and provided as supporting materials.

A computer model of a polyunsaturated monogalactolipid bilayer.

1,2-di-O-acyl-3-O-ß-D-galactopyranosyl-sn-glycerol (MGDG) is the main lipid component of thylakoid membranes of higher plants and algae. This monogalactolipid is thought of as a non-bilayer lipid but actually it can form both lamellar and nonlamellar phases. In this study, molecular dynamics (MD) simulations of the fully hydrated di-18:3 MGDG bilayer in the lamellar phase were carried out at 310 and 295 K for 200 and 450 ns, respectively, using the GROMACS 4 software package and OPLS-AA force field. At both temperatures, the lamellar phase of the systems was stable. The pure di-18:3 MGDG bilayer is the first step towards creating a computer model of the lipid matrix of the thylakoid membrane and the main aim of this study was to validate the computer model of di-18:3 MGDG in the bilayer and also to assess the properties of the bilayer. However, only a few of the predicted properties could be compared with those derived experimentally and in other MD simulations because of insufficient amount of such data. Thus, direct validation of the MGDG bilayer proved difficult. Therefore, in the validation process also an indirect approach was used, in which a computer model of the 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) bilayer simulated at the same temperatures using the same force field as the MGDG bilayer was assessed. Successful validation of the DOPC bilayer parameterized in the OPLS-AA force field and similar properties of the MGDG molecules in the pure 18:3 MGDG and in binary 18:3 MGDG-PC bilayers indicate that the computer model of the MDGD molecule is faithful and the MGDG bilayer is representative on the time scales covered in these MD simulations.