Interactions of Malondialdehyde and 4-Hydroxynonenal with Rat Liver Plasma Membranes and their Effect on Binding of Prostaglandin E2 by Specific Receptors

We investigated effect of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydrox-ynonenal (HNE) on prostaglandin (PG) E₂ receptors of liver plasma membranes. The modification of the membranes by MDA diminished PGE₂ binding, decreasing receptor affinity for PGE₂ and receptor density whereas HNE increased PGE₂ binding, enhanced receptor density but did not changed receptor affinity. ESR study showed the decrease of the whole membrane fluidity after modification by MDA whereas HNE lowered membrane fluidity only in the internal zone of lipid bilayer and increased it in the surface area. The possible effects of membrane changes caused by MDA and HNE on PGE₂ receptor parameters are discussed.     

Setting up and optimization of membrane protein simulations

In this paper we describe a method for setting up an atomistic simulation of a membrane protein in a hydrated lipid bilayer and report the effect of differing electrostatic parameters on the drift in the protein structure during the subsequent simulation. The method aims to generate a suitable cavity in the interior of a lipid bilayer, using the solvent-accessible surface of the protein as a template, during the course of a short steered molecular dynamics simulation of a solvated lipid membrane. This is achieved by a two-stage process: firstly, lipid molecules whose headgroups are inside a cylindrical volume equivalent to that defined by the protein surface are removed; then the protein-lipid interface is optimized by applying repulsive forces perpendicular to the protein surface, and of gradually increased magnitude, to the remaining lipid atoms inside the volume occupied by the protein surface until it is emptied. The protein itself may then be inserted. Using the bacterial membrane proteins KcsA and FhuA as test cases, we show how the method achieves the formation of a suitable cavity in the interior of a dimyristoylphosphatidylcholine lipid bilayer without perturbing the configuration of the non-interfacial regions of the previously equilibrated lipid bilayer, even in cases of membrane proteins with irregular geometrical shapes. In addition, we compare subsequent simulations in which the long-range electrostatic interactions are treated via either a cut-off or a particle-mesh Ewald method. The results show that the drift from the initial structure is less in the latter case, especially for KcsA and for the non-core secondary structural elements (i.e. surface loops) of both proteins.     

New EPR Method for Cellular Surface Characterization

An electron paramagnetic resonance (EPR)-based membrane surface characterization method is presented to detect the properties of the carbohydrate-rich part of membrane surfaces as well as carbohydrate interaction with other membrane constituents and water-soluble molecules. The proposed method relies on the spin-labeling and spectral decomposition based on spectral simulation and optimization with EPRSIM software. In order to increase the sensitivity of characterization to the carbohydrate-rich part of the membrane surface, the sucrose-contrasting approach is introduced. With this method, which was established on model membranes with glycolipids and tested on erythrocyte membrane, we were able to characterize the surface and lipid bilayer lateral heterogeneity. Additionally, some properties of the interaction between glycocalyx and lipid bilayer as well as between glycocalyx and sucrose molecules were determined. The experiments also provided some information about the anchoring and aggregation of the glycosylated molecules. According to the results, some functions of the glycosylated surface are discussed.     

Membrane Instability, Plasmalogen Content, and Alzheimer's Disease

Abstract: The normal stability of the cell membrane bilayer depends on its lipid composition being appropriate to the ambient (physiological) temperature, T_p. Membrane lipid composition may be altered by disease such that the bilayer is only stable at a new critical temperature, T^⋆, which may differ from T_p. In Alzheimer's disease (AD) temporal cortex, a defect of lipid composition has previously been identified, namely, a decrease in the ratio of plasmalogen to nonplasmalogen ethanolamine glycerophospholipids. Furthermore, for AD temporal cortex neural membranes, T^⋆≪ T_p, a finding confirmed in the present study in a larger series than previously, using a new method for obtaining T^⋆. This inequality between T^⋆ and T_p has been proposed as a putative contributory pathogenetic mechanism leading to membrane destabilisation in AD brain. The plasmalogen deficiency could account for the change in T^⋆ in AD, as shown by experiments where T^⋆ was measured for artificial lipid mixtures simulating brain membranes with varying plasmalogen/nonplasmalogen ratios. The critical temperature was found to be very sensitive to small alterations in plasmalogen content.     

Interfacial tension of phosphatidylcholine-phosphatidylserine system in bilayer lipid membrane

The effect of pH of electrolyte solution on the interfacial tension of lipid membrane formed of phosphatidylcholine (PC, lecithin)–phosphatidylserine (PS) system was studied. In this article, three models describing the H⁺ and OH⁻ ions adsorption in the bilayer lipid surface are presented. In Model I and Model II, the surface is continuous with uniformly distributed functional groups constituting the centres of H⁺ and OH⁻ ions adsorption while in the other the surface is built of lipid molecules, free or with attached H⁺ and OH⁻ ions. In these models contribution of the individual lipid molecule forms to interfacial tension of the bilayer were assumed to be additive. In Model III the adsorption of the H⁺ and OH⁻ ions at the PC–PS bilayer surface was described in terms of the Gibbs isotherm. Theoretical equations are derived to describe this dependence in the whole pH range.     

Lipid-protein interactions

Intrinsic membrane proteins are solvated by a shell of lipid molecules interacting with the membrane-penetrating surface of the protein; these lipid molecules are referred to as annular lipids. Lipid molecules are also found bound between transmembrane α-helices; these are referred to as non-annular lipids. Annular lipid binding constants depend on fatty acyl chain length, but the dependence is less than expected from models based on distortion of the lipid bilayer alone. This suggests that hydrophobic matching between a membrane protein and the surrounding lipid bilayer involves some distortion of the transmembrane α-helical bundle found in most membrane proteins, explaining the importance of bilayer thickness for membrane protein function. Annular lipid binding constants also depend on the structure of the polar headgroup region of the lipid, and hotspots for binding anionic lipids have been detected on some membrane proteins; binding of anionic lipid molecules to these hotspots can be functionally important. Binding of anionic lipids to non-annular sites on membrane proteins such as the potassium channel KcsA can also be important for function. It is argued that the packing preferences of the membrane-spanning α-helices in a membrane protein result in a structure that matches nicely with that of the surrounding lipid bilayer, so that lipid and protein can meet without either having to change very much.     

The influence of proteins and peptides on the phase properties of lipids

This paper reviews model membrane studies on the modulation of the macroscopic structure of lipids by lipid-protein interactions, with particular emphasis on the gramicidin molecule. This hydrophobic peptide has three main effects on lipid polymorphism: (1) in lysophosphatidylcholine it triggers a micellar to bilayer transition, (2) in phosphatidylethanolamine it lowers the bilayer to hexagonal H_II phase transition temperature and (3) in phosphatidylcholine and other bilayer preferring lipids it is able to induce the formation of an H_II phase. From experiments in which the gramicidin molecule was chemically modified it can be concluded that the tryptophan residues play a determining role in the peptide-induced changes in polymorphism. The experimental data lead to the proposal that gramicidin molecules have a tendency to self-associate, possibly mediated by tryptophan-tryptophan interactions and organize into tubular structures such as found in the H_II phase.     

Different effects of lipid chain length on the two sides of a membrane and the lipid annulus of MscL

Quenching of the fluorescence of Trp residues in a membrane protein by lipids with bromine-containing fatty acyl chains provides a powerful technique for measuring lipid-protein binding constants. Single Trp residues have been placed on the periplasmic and cytoplasmic sides of the mechanosensitive channel of large conductance MscL from Mycobacterium tuberculosis to measure, separately, lipid binding constants on the two faces of MscL. The chain-length dependence of lipid binding was found to be different on the two sides of MscL, the chain-length dependence being more marked on the cytoplasmic than on the periplasmic side. To determine if lipid binding constants are affected by the properties of the lipid molecules not in direct contact with MscL (the bulk lipid), the amount of bulk lipid present in the system was varied. The binding constant of the short-chain phospholipid didodecylphosphatidylcholine was found to be independent of the molar ratio of lipid/MscL pentamer over the range 500:1-50:1, suggesting that lipid binding constants are determined largely by the properties of the lipid molecules interacting directly with MscL. These results point to a model in which lipid molecules located on the transmembrane surface of a membrane protein (the annular lipid molecules), by playing a dominant role in the interaction between a membrane protein and the surrounding lipid bilayer, could effectively buffer the membrane protein from changes in the properties of the bulk lipid bilayer.     

Comparative molecular dynamics study of lipid membranes containing cholesterol and ergosterol

Sterol molecules are essential for maintaining the proper structure and function of eukaryotic cell membranes. The influence of cholesterol (the principal sterol of higher animals) on the lipid bilayer properties was extensively studied by both experimental and simulation methods. In contrast, the effect of ergosterol (the principal fungal sterol) on the membrane structure and dynamics is much less recognized. This work presents the results of comparative molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine bilayer containing approximately 25 mol % of cholesterol or ergosterol. A detailed analysis of the molecular properties (e.g., bilayer thickness, lipid order, diffusion, intermolecular interactions, etc.) of both sterol-induced liquid-ordered membrane phases is presented. Presence of sterols in the membrane significantly changes its property, especially fluidity and molecular packing. Moreover, in accordance with the experiments, our calculations show that, compared to cholesterol, ergosterol has higher ordering effect on the phospholipid acyl chains. This different influence on the properties of the lipid bilayer stems from differences in conformational freedom of sterol side chains. Additionally, obtained models of lipid membranes containing human and fungal sterols, constituting the result of our work, can be also utilized in other chemotherapeutic studies on interaction of selected ligands (e.g., antifungal compounds) with membranes.     

A molecular toolkit for highly insulating tethered bilayer lipid membranes on various substrates

Tethered bilayer lipid membranes (tBLMs) are promising model architectures that mimic the structure and function of natural biomembranes. They provide a fluid, stable, and electrically sealing platform for the study of membrane related processes, specifically, the function of incorporated membrane proteins. This paper presents a generic approach toward the synthesis of functional tBLMs adapted for application to various surfaces. The central element of a tethered membrane consists of a lipid bilayer. Its proximal layer is covalently attached via a spacer unit to a solid support, either gold or silicon oxide. The membranes are characterized optically by using surface plasmon resonance spectroscopy (SPR) or ellipsometry and electrically by using electrochemical impedance spectroscopy (EIS). The bilayer membranes obtained show high electrical barrier properties and can be used to incorporate and study small membrane proteins in a functional form.     

The protein-tethered lipid bilayer: a novel mimic of the biological membrane

A new concept of solid-supported tethered bilayer lipid membrane (tBLM) for the functional incorporation of membrane proteins is introduced. The incorporated protein itself acts as the tethering molecule resulting in a versatile system in which the protein determines the characteristics of the submembraneous space. This architecture is achieved through a metal chelating surface, to which histidine-tagged (His-tagged) membrane proteins are able to bind in a reversible manner. The tethered bilayer lipid membrane is generated by substitution of protein-bound detergent molecules with lipids using in-situ dialysis or adsorption. The system is characterized by surface plasmon resonance, quartz crystal microbalance, and electrochemical impedance spectroscopy. His-tagged cytochrome c oxidase (CcO) is used as a model protein in this study. However, the new system should be applicable to all recombinant membrane proteins bearing a terminal His-tag. In particular, combination of surface immobilization and membrane reconstitution opens new prospects for the investigation of functional membrane proteins by various surface-sensitive techniques under a defined electric field.     

The relationship between compositional phase separation and vesicle morphology: Implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A₂ (PLA₂) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA₂. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Effect of Gold Nanoparticle on Structure and Fluidity of Lipid Membrane

This paper deals with the effect of different size gold nanoparticles on the fluidity of lipid membrane at different regions of the bilayer. To investigate this, we have considered significantly large bilayer leaflets and incorporated only one nanoparticle each time, which was subjected to all atomistic molecular dynamics simulations. We have observed that, lipid molecules located near to the gold nanoparticle interact directly with it, which results in deformation of lipid structure and slower dynamics of lipid molecules. However, lipid molecules far away from the interaction site of the nanoparticle get perturbed, which gives rise to increase in local ordering of the lipid domains and decrease in fluidity. The bilayer thickness and area per head group in this region also get altered. Similar trend, but with different magnitude is also observed when different size nanoparticle interact with the bilayer.     

The cryoprotectant trehalose destabilises the bilayer organisation of Escherichia coli-derived membrane systems at elevated temperatures as determined by H and P-NMR

In this study, ²H and ³¹P-NMR techniques were used to study the effects of trehalose and glycerol on phase transitions and lipid acyl chain order of membrane systems derived from cells of E. coli unsaturated fatty acid auxotroph strain K1059, which was grown in the presence of [11,11-²H₂]-oleic acid or [11,11-²H₂]-elaidic acid. From an analysis of the temperature dependence of the quadrupolar splitting it could be concluded that neither 1 M trehalose or glycerol generally had any significant effect on the temperature of the lamellar gel to liquid-crystalline phase transition. In the case of the oleate-containing hydrated total lipid extract, glycerol but not trehalose caused a 5°C increase of this transition temperature. In general, both cryoprotectants induced an ordering of the acyl chains in the liquid-crystalline state. Trehalose and glycerol both decrease the bilayer to non-bilayer transition temperature of the hydrated lipid extract of oleate-grown cells by about 5°C, but only trehalose in addition induces an isotropic to hexagonal (H_II) phase transition. In the biological membranes, trehalose and not glycerol destabilised the lipid bilayer, and in the case of the E. coli spheroplasts, part of the induced non-bilayer structures is ascribed to a hexagonal (H_II) phase in analogy with the total lipids. Interestingly, 1 mM Mg²⁺ was a prerequisite for the destabilisation of the lipid bilayer. In the hydrated total lipid extract of E. coli grown on the more ordered elaidic acid, both transition temperatures were shifted about 20°C upwards compared with the oleate-containing lipid, but the effect of trehalose on the lipid phase behaviour was similar. The bilayer destabilising ability of trehalose might have implications for the possible protection of biological systems by (cryo-)protectants during dehydration, in that protection is unlikely to be caused by preventing the occurrence of polymorphic phase transitions.     

The dependence of Fluorescein-PE fluorescence intensity on lipid bilayer state. Evaluating the interaction between the probe and lipid molecules

The degree of dependence of a lipid bilayer's surface properties on its conformational state is still an unresolved question. Surface properties are functions of molecular organization in the complex interfacial region. In the past, they were frequently measured using fluorescence spectroscopy. Since a fluorescent probe provides information on its local environment, there is a need to estimate the effect caused by the probe itself. In this paper, we address this question by calculating how lipid head-group orientation effects the fluorescence intensity of Fluorescein-PE (a probe that is sensitive to surface potential). In the theoretical model assumed the lipid bilayer state and the interactions between the charged fluorescent probe and the surrounding lipid molecules was evaluated. The results of this theoretical analysis were compared with experimentally obtained data. A lipid bilayer formed from DPPC was chosen as the experimental system, since it exhibits all the major conformational states within a narrow temperature range of 30 degrees C-45 degrees C. Fluorescein-PE fluorescence intensity depends on local pH, which in turn is sensitive to local electrostatic potential in the probe's vicinity. This local electrostatic potential is generated by lipid head-group dipole orientation. We have shown that the effect of the probe on lipid bilayer properties is limited when the lipid bilayer is in the gel phase, whereas it is more pronounced when the membrane is liquid-crystalline. This implies that Fluorescein-PE is a good reporter of local electrostatic fields when the lipid bilayer is in the gel phase, and is a poor reporter when the membrane is in the liquid-crystalline state.     

A new interpretation of eutectic behavior for distearoylphosphatidylcholine-cholesterol binary bilayer membrane

We investigated the thermotropic phase behavior of the distearoylphosphatidylcholine (DSPC)–cholesterol binary bilayer membrane as a function of the cholesterol composition (X_ch) by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry (DSC). The fluorescence spectra, each of which has a single maximum, showed that the wavelength at the maximum intensity (λ_max) changed depending on the bilayer state: ca. 440 nm for the lamellar gel (L_β′ or L_β) and the liquid ordered (L_o) phases, ca. 470 nm for the ripple gel (P_β′) phase and ca. 490 nm for the liquid crystalline (L) phase, respectively. The transition temperatures were determined from the temperature dependences of the λ_max and endothermic peaks of the DSC thermograms. Both measurements showed that the pretransition disappears around X_ch = 0.035. The constructed temperature–X_ch phase diagram indicated that the phase behavior of the binary bilayer membrane at X_ch ≤ 0.15 is similar to that of general liquid–solid equilibrium for a binary system where both components are completely miscible in the liquid phase and completely immiscible in the solid phase. It was also revealed that the diagram has two characteristic points: a congruent melting point at X_ch = 0.08 and a peritectic-like point at X_ch = 0.15. The hexagonal lattice model was used for the interpretation of the phase behavior of the binary bilayer membrane. These characteristic compositions well correspond to the bilayer states in each of which cholesterol molecules are regularly distributed in the hexagonal lattice in a different way. That is, each composition of 0.035, 0.08 and 0.15 is nearly equal to that for the binary bilayer membrane which is entirely occupied with units, each composed of a cholesterol and 30 surrounding DSPC molecules within the next-next-next nearest neighbor sites (Unit (1:30): L_β(1:30)), with units, each of a cholesterol and 12 surrounding DSPC molecules within the next nearest sites (Unit (1:12): L_β(1:12)) or with units, each of a cholesterol and 6 surrounding DSPC molecules at the nearest neighbor sites (Unit (1:6): L_β(1:6)), respectively. Therefore, the eutectic behavior observed in the phase diagram was fully explainable in terms of a kind of phase separation between two different types of regions with different types of regular distributions of cholesterol. Further, the L_o phase was found in the higher X_ch-region (X_ch > 0.15). No endothermic peak over the temperature range from 10 to 80 °C at X_ch = 0.50 suggested that the single L_o phase can exist at X_ch > 0.50.     

The influence of dioxan on the bilayer and monolayer phase transition of phospholipids

The influence of 1.4.-dioxan on the bilayer phase transition of various phospholipids was studied by differential scanning calorimetry and turbidity measurements. The addition of 1.4.-dioxan to lipid bilayers decreases the transition temperature T_m increases the transition enthalpy of the transition. The cooperativity of the transition is unaffected. The phospholipid monolayer transition from the liquid-condensed to the liquid-expanded phase was measured by recording area versus temperature curves at constant surface pressure (isobars). The monolayer transition temperature at constant surface pressure is increased when 1.4.-dioxan is added to the subphase. The change in molecular area becomes larger. A comparison of monolayer isobars on water and water/dioxan as subphase at constant surface tension rather than surface pressure leads to a decrease of the transition temperature on water/dioxan as subphase. This decrease as well as the larger change in molecular area at the monolayer transition can be correlated to the decrease in T_m and the increase in the transition enthalpy of the corresponding bilayer system. 1.4.-Dioxan seems to accumulate at the lipid head group/water interface, thus lowering the tension of the bilayer membrane. This cyclic ether can be used for altering the characteristics of bilayer membranes without disturbing the lipid chain organization.     

Interaction of low-molecular-weight chitosan with mimic membrane studied by electrochemical methods and surface plasmon resonance

Chitosan has shown its potential as a non-viral gene carrier and an adsorption enhancer for subsequent drug delivery to cells. These results showed that chitosan acted as a membrane perturbant. However, there is currently a lack of direct experimental evidence of this membrane perturbance effect, especially for chitosans with low molecular weight (LMW). In this report, the interaction between a lipid (didodecyl dimethylammonium bromide; DDAB) bilayer and chitosan with molecular weight (MW) of 4200 Da was studied with cyclic voltammetry (CV), electrochemical impedance spectroscopy and surface plasmon resonance (SPR). A lipid bilayer was formed by fusion of oppositely charged lipid vesicles on a mercaptopropionic acid (MPA)-modified gold surface to mimic a cell membrane. The results showed that the LMW chitosan could disrupt the lipid bilayer, and the effect seemed to be in a concentration-dependent manner.     

Phospholipase A2 (PLA2) enzymes in membrane trafficking: mediators of membrane shape and function

Since the mid-1990s, there have been tremendous advances in our understanding of the roles that lipid-modifying enzymes play in various intracellular membrane trafficking events. Phospholipases represent the largest group of lipid-modifying enzymes and accordingly display a wide range of functions. The largest class of phospholipases are the phospholipase A(₂) (PLA₂) enzymes, and these have been most extensively studied for their roles in the generation lipid signaling molecules, e.g. arachidonic acid. In recent years, however, cytoplasmic PLA₂ enzymes have also become increasingly associated with various intracellular trafficking events, such as the formation of membrane tubules from the Golgi complex and endosomes, and membrane fusion events in the secretory and endocytic pathways. Moreover, the ability of cytoplasmic PLA₂ enzymes to directly affect the structure and function of membranes by altering membrane curvature suggests novel functional roles for these enzymes. This review will focus on the role of cytoplasmic PLA₂ enzymes in intracellular membrane trafficking and the mechanisms by which they influence membrane structure and function .     

Impedance spectroscopy of bilayer lipid membranes self-assembled on agar support - interaction with HDL

The electrical properties of the supported lipid bilayer membrane (s-BLM) of egg phosphatidylcholine (PC) self-assembled on agar surface were examined. To characterize the insulating properties of s-BLMs, electrochemical impedance spectroscopy (EIS) was used. The analysis of impedance spectra in terms of an equivalent circuit of agar/electrolyte and agar/s-BLM/electrolyte in the frequency range of 0.1 Hz-10 kHz was performed. The high-density lipoproteins (HDL)/s-BLM interaction in the concentration range from 20 microg/ml to 80 microg/ml of HDL was investigated. It is evident that treatment of s-BLM with HDL resulted in an increase of the lipid film resistance and a decrease of membrane capacitance.