Asymmetry of lipid bilayers induced by monovalent salt: atomistic molecular-dynamics study

Interactions between salt ions and lipid components of biological membranes are essential for the structure, stability, and functions of the membranes. The specific ionic composition of aqueous buffers inside and outside of the cell is known to differ considerably. To model such a situation we perform atomistic molecular-dynamics (MD) simulations of a single-component phosphatidylcholine lipid bilayer which separates two aqueous reservoirs with and without NaCl salt. To implement the difference in electrolyte composition near two membrane sides, a double bilayer setup (i.e., two bilayers in a simulation box) is employed. It turns out that monovalent salt, being in contact with one leaflet only, induces a pronounced asymmetry in the structural, electrostatic, and dynamical properties of bilayer leaflets after 50 ns of MD simulations. Binding of sodium ions to the carbonyl region of the leaflet which is in contact with salt results in the formation of "Na-lipids" complexes and, correspondingly, reduces mobility of lipids of this leaflet. In turn, attractive interactions of chloride ions (mainly located in the aqueous phase close to the water-lipid interface) with choline lipid groups lead to a substantial (more vertical) reorientation of postphatidylcholine headgroups of the leaflet adjoined to salt. The difference in headgroup orientation on two sides of a bilayer, being coupled with salt-induced reorientation of water dipoles, leads to a notable asymmetry in the charge-density profiles and electrostatic potentials of bilayer constitutes of the two leaflets. Although the overall charge density of the bilayer is found to be almost insensitive to the presence of salt, a slight asymmetry in the charge distribution between the two bilayer leaflets results in a nonzero potential difference of about 85 mV between the two water phases. Thus, a transmembrane potential of the order of the membrane potential in a cell can arise without ionic charge imbalance between two aqueous compartments.     

Interleaflet interaction and asymmetry in phase separated lipid bilayers: molecular dynamics simulations

In order to investigate experimentally inaccessible, molecular-level detail regarding interleaflet interaction in membranes, we have run an extensive series of coarse-grained molecular dynamics simulations of phase separated lipid bilayers. The simulations are motivated by differences in lipid and cholesterol composition in the inner and outer leaflets of biological membranes. Over the past several years, this phenomenon has inspired a series of experiments in model membrane systems which have explored the effects of lipid compositional asymmetry in the two leaflets. The simulations are directed at understanding one potential consequence of compositional asymmetry, that being regions of bilayers where liquid-ordered (L(o)) domains in one leaflet are opposite liquid-disordered (L(d)) domains in the other leaflet (phase asymmetry). The simulated bilayers are of two sorts: 1) Compositionally symmetric leaflets where each of the two leaflets contains an identical, phase separated (L(o)/L(d)) mixture of cholesterol, saturated and unsaturated phospholipid; and 2) Compositionally asymmetric leaflets, where one leaflet contains a phase separated (L(o)/L(d)) mixture while the other contains only unsaturated lipid, which on its own would be in the L(d) phase. In addition, we have run simulations where the lengths of the saturated lipid chains as well as the mole ratios of the three lipid components are varied. Collectively, we report on three types of interleaflet coupling within a bilayer. First, we show the effects of compositional asymmetry on acyl chain tilt and order, lipid rotational dynamics, and lateral diffusion in regions of leaflets that are opposite L(o) domains. Second, we show substantial effects of compositional asymmetry on local bilayer curvature, with the conclusion that phase separated leaflets resist curvature, while inducing large degrees of curvature in an opposing L(d) leaflet. Finally, in compositionally symmetric, phase separated bilayers, we find phase asymmetry (domain antiregistration) between the two leaflets occurs as a consequence of mismatched acyl chain-lengths in the saturated and unsaturated lipids.     

Effect of NaCl and KCl on phosphatidylcholine and phosphatidylethanolamine lipid membranes: insight from atomic-scale simulations for understanding salt-induced effects in the plasma membrane

To gain a better understanding of how monovalent salt under physiological conditions affects plasma membranes, we have performed 200 ns atomic-scale molecular dynamics simulations of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipid bilayers. These two systems provide representative models for the outer and inner leaflets of the plasma membrane, respectively. The implications of cation-lipid interactions in these lipid systems have been considered in two different aqueous salt solutions, namely NaCl and KCl, and the sensitivity of the results on the details of interactions used for ions is determined by repeating the simulations with two distinctly different force fields. We demonstrate that the main effect of monovalent salt on a phospholipid membrane is determined by cations binding to the carbonyl region of a membrane, while chloride anions mostly stay in the water phase. It turns out that the strength and character of the cation-lipid interactions are quite different for different types of lipids and cations. PC membranes and Na+ ions demonstrate strongest interactions, leading to notable membrane compression. This finding was confirmed by both force fields (Gromacs and Charmm) employed for the ions. The binding of potassium ions to PC membranes (and the overall effect of KCl), in turn, was found to be much weaker mainly due to the larger size of a K+ ion compared to Na+. Furthermore, the effect of KCl on PC membranes was found to be force-field sensitive: The binding of a potassium ion was not observed at all in simulations performed with the Gromacs force-field, which seems to exaggerate the size of a K+ ion. As far as PE lipid bilayers are concerned, they are found to be influenced by monovalent salt to a significantly lesser extent compared to PC bilayers, which is a direct consequence of the ability of PE lipids to form both intra- and intermolecular hydrogen bonds and hence to adopt a more densely packed bilayer structure. Whereas for NaCl we observed weak binding of Na+ cations to the PE lipid-water interface, in the case of KCl we witnessed almost complete lack of cation binding. Overall, our findings indicate that monovalent salt ions affect lipids in the inner and outer leaflets of plasma cell membranes in substantially different ways.     

Influence of lipid composition on membrane activity of antimicrobial phenylene ethynylene oligomers

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1-3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.     

Local anesthetics facilitate ion transport across lipid planar bilayer membranes under an electric field: dependence on type of lipid bilayer

In order to elucidate the role of structural change of lipid membrane bilayer in the mode of action of local anesthetic, we studied the effects of local anesthetics, charged tetracaine and uncharged benzocaine, on ion permeability across various lipid planar bilayers (PC, mixed PC/PS (4/1, mol/mol); mixed PC/PE (1/1, mol/mol); mixed PC/SM (4/1, mol/mol)) under a constant applied voltage. The membrane conductances increased in the order of PC  PC/PS ≤ PC/SM  PC/PE. When the constant voltage of −100 or −70 mV was applied through the lipid bilayer membranes in the presence of positively charged tetracaine, the fluctuating current pulses with the large amplitude generated, but not appeared in the absence of tetracaine. The addition of uncharged benzocaine generated the fluctuating currents with the small amplitude. Both charged tetracaine and uncharged benzocaine facilitated electrophoretically the transport of small ions such as KCl in the buffer solution through the fluctuating pores in the lipid bilayer membranes formed by interaction with the local anesthetic under the negative applied membrane potential. The current pulses also contained actual transport of charged tetracaine together with the transport of the small ions. The amplitude and the duration time of the electrical current generated by adding the local anesthetics were dependent on the type of the lipid, the applied voltage and its voltage polarity.     

Electrostatic properties of membrane lipids coupled to metarhodopsin II formation in visual transduction

Changes in lipid composition have recently been shown to exert appreciable influences on the activities of membrane-bound proteins and peptides. We tested the hypothesis that the conformational states of rhodopsin linked to visual signal transduction are related to biophysical properties of the membrane lipid bilayer. For bovine rhodopsin, the meta I-meta II conformational transition was studied in egg phosphatidylcholine (PC) recombinants versus the native rod outer segment (ROS) membranes by means of flash photolysis. Formation of metarhodopsin II was observed by the change in absorbance at 478 nm after a single actinic flash was delivered to the sample. The meta I/meta II ratio was investigated as a function of both temperature and pH. The data clearly demonstrated thermodynamic reversibility of the transition for both the egg PC recombinants and the native ROS membranes. A significant shift of the apparent pK(a) for the acid-base equilibrium to lower values was evident in the egg PC recombinant, with little meta II produced under physiological conditions. Calculations of the membrane surface pH using a Poisson-Boltzmann model suggested the free energies of the meta I and meta II states were significantly affected by electrostatic properties of the bilayer lipids. In the ROS membranes, phosphatidylserine (PS) is needed for full formation of meta II, in combination with phosphatidylethanolamine (PE) and polyunsaturated docosahexaenoic acid (DHA; 22:6omega3) chains. We propose that the PS surface potential leads to an accumulation of hydronium ions, H(3)O(+), in the electrical double layer, which drive the reaction together with the large negative spontaneous curvature (H(0)) conferred by PE plus DHA chains. The elastic stress/strain of the bilayer arises from an interplay of the approximately zero H(0) from PS and the negative H(0) due to the PE headgroups and polyunsaturated chains. The lipid influences are further explained in terms of matching of the bilayer spontaneous curvature to the curvature at the lipid/rhodopsin interface, as formulated by the Helfrich bending energy. These new findings guide current ideas as to how bilayer properties govern the conformational energetics of integral membrane proteins. Moreover, they yield knowledge of how membrane lipid-protein interactions involving acidic phospholipids such as PS and neutral polyunsaturated DHA chains are implicated in key biological functions such as vision.     

Counterion-mediated pattern formation in membranes containing anionic lipids

Most lipid components of cell membranes are either neutral, like cholesterol, or zwitterionic, like phosphatidylcholine and sphingomyelin. Very few lipids, such as sphingosine, are cationic at physiological pH. These generally interact only transiently with the lipid bilayer, and their synthetic analogs are often designed to destabilize the membrane for drug or DNA delivery. However, anionic lipids are common in both eukaryotic and prokaryotic cell membranes. The net charge per anionic phospholipid ranges from − 1 for the most abundant anionic lipids such as phosphatidylserine, to near − 7 for phosphatidylinositol 3,4,5 trisphosphate, although the effective charge depends on many environmental factors. Anionic phospholipids and other negatively charged lipids such as lipopolysaccharides are not randomly distributed in the lipid bilayer, but are highly restricted to specific leaflets of the bilayer and to regions near transmembrane proteins or other organized structures within the plane of the membrane. This review highlights some recent evidence that counterions, in the form of monovalent or divalent metal ions, polyamines, or cationic protein domains, have a large influence on the lateral distribution of anionic lipids within the membrane, and that lateral demixing of anionic lipids has effects on membrane curvature and protein function that are important for biological control.     

Membrane lipid influences on the energetics of the metarhodopsin I and metarhodopsin II conformational states of rhodopsin probed by flash photolysis.

We have investigated the relationship between rhodopsin photochemical function and the retinal rod outer segment (ROS) disk membrane lipid composition using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form recombinant membrane vesicles, in which the lipid acyl chain composition was maintained at that of egg phosphatidylcholine (PC), while the nature of the headgroups was varied. The ratio of metarhodopsin II (MII)/metarhodopsin I (MI) in these recombinants produced by an actinic flash was investigated as a function of pH, and compared with the photochemical activity observed for rhodopsin in native ROS membranes and dimyristoylphosphatidylcholine recombinants. In recombinants made with lipids derived from egg PC, as well as in native ROS membranes, MI and MII were found to be present in a pH-dependent, acid-base equilibrium on the millisecond timescale. The recombinants made with phospholipids containing unsaturated acyl chains were capable of full native-like MII production, but each demonstrated a titration curve with a different pK. In addition, some of the recombinants exhibited apparent deviations from the Henderson-Hasselbalch curve shape. The presence of either phosphatidylethanolamine (PE) or phosphatidylserine (PS) headgroups appeared to increase the amount of MII produced. This may result from alteration of the curvature free energy, in the case of PE, and from the influence of the membrane surface potential in the case of PS. An investigation of the effects of temperature on the MI-MII transition in native ROS membranes and the recombinants was also carried out.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrophobic/superhydrophobic oxidized metal surfaces showing negligible contact angle hysteresis

The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ～6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems.     

Microscopic Characterization of GRP1 PH Domain Interaction with Anionic Membranes

The pleckstrin homology (PH) domain of general receptor for phosphoionositides 1 (GRP1-PHD) binds specifically to phosphatidylinositol (3,4,5)-triphosphate (PIP₃), and acts as a second messenger. Using an extensive array of molecular dynamics (MD) simulations employing highly mobile membrane mimetic (HMMM) model as well as complementary full membrane simulations, we capture differentiable binding and dynamics of GRP1-PHD in the presence of membranes containing PC, PS, and PIP₃ lipids in varying compositions. While GRP1-PHD forms only transient interactions with pure PC membranes, incorporation of anionic lipids resulted in stable membrane-bound configurations. We report the first observation of two distinct PIP₃ binding modes on GRP1-PHD, involving PIP₃ interactions at a “canonical” and at an “alternate” site, suggesting the possibility of simultaneous binding of multiple anionic lipids. The full membrane simulations confirmed the stability of the membrane bound pose of GRP1-PHD as captured from our HMMM membrane binding simulations. By performing additional steered membrane unbinding simulations and calculating nonequilibrium work associated with the process, as well as metadynamics simulations, on the protein bound to full membranes, allowing for more quantitative examination of the binding strength of the GRP1-PHD to the membrane, we demonstrate that along with the bound PIP₃, surrounding anionic PS lipids increase the energetic cost of unbinding of GRP1-PHD from the canonical mode, causing them to dissociate more slowly than the alternate mode. Our results demonstrate that concurrent binding of multiple anionic lipids by GRP1-PHD contributes to its membrane affinity, which in turn control its signaling activity. © 2019 Wiley Periodicals, Inc.     

Profiling and quantification of Drosophila melanogaster lipids using liquid chromatography/mass spectrometry

We present here the findings of global profiling of Drosophila lipids using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on an LTQ-Orbitrap instrument. In addition, we present a multiple reaction monitoring (LC-MRM) method for the absolute quantification of the major phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids of Drosophila. Using both normal- and reversed-phase LC followed by accurate mass analysis and MS/MS on an LTQ-Orbitrap instrument, we evaluated the lipid composition of the fruit fly Drosophila melanogaster. A total of 74 lipid species were identified consisting of glycerphospholipids belonging to the PE, PC, phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) classes including several plasmanyl PE species, as well as triacylglycerides, cardiolipins, ceramides, and PE ceramides. Individual PE and PC phospholipids were then quantified using an LC-MRM approach. Reversed-phase chromatography followed by monitoring on a QTrap 4000 instrument of 21 MRM transitions combined with calibration curves constructed using internal standards enabled the absolute quantification of 28 PE and PC lipid species with limits of quantification of 3 and 5 pg/μL, respectively. Internal standards accounted for the differences in ionization efficiencies of PE and PC phospholipids, facilitating more accurate lipid abundance measurements. The method presented here builds on previous Drosophila work by making the quantification of absolute lipid abundance possible and will be of interest to scientists who study variation and changes in the degree of unsaturation, fatty acid carbon length, and head-group concentration among individuals of different genotypes in response to environmental, genetic, or physiological perturbation in small insects. It will also be particularly useful to biologists interested in adaptation and acclimation of cellular membranes in response to thermal heterogeneity.     

Two components of boundary potentials at the lipid membrane surface: electrokinetic and complementary methods studies

Bilayer lipid membranes (BLM) are commonly used as models for cell membranes to study their interactions with inorganic ions and molecules of biological importance. In this work the principal electrostatic effects at the BLM surface are demonstrated by two methods: by the inner membrane field compensation (IFC) which is applied to planar BLM and sensitive to changes in the total boundary potential; and by electrokinetic measurements in liposome suspensions, sensitive to diffuse (surface) component of this potential. The difference in these two potentials allows us to conclude on changes in the dipole component of the boundary potential caused by structural changes at the membrane–water interface. No difference in the experimental data of both methods was observed for Be²⁺ and other divalent cation adsorption to unchanged phosphatidyl choline (PC) membranes. These data are in a good agreement with the Gouy–Champan–Stern (GCS) theory of diffuse double layer. This theory gives the value of binding constants for Be²⁺ about 400 M⁻¹ and 10⁴ M⁻¹ for DPPC liposomes in the liquid and solid states of the lipids, respectively. Clear isotope effects for normal and heavy water solutions of Be²⁺ were observed both by the electrostatic measurements and by differential scanning calorimetry. In contrast to PC, the electrostatic potentials induced by Be²⁺ and Gd³⁺ adsorption to membranes from phosphatidyiserine (PS) show the difference between the data of mentioned methods — total boundary potential changes are much higher in comparison to the surface potential. Dipole potential changes (about 150 mV) caused by changes in PS head group orientation may be more important in this specific case.     

Electrostatic induction of lipid asymmetry

The asymmetric arrangement of phospholipids between the two leaflets of the plasma membrane of eukaryotic cells is an integral part of cellular function. ATP-dependent translocases capable of selective lipid transport across the membrane are believed to play a role in this lipid asymmetry, but our understanding of this process is incomplete. Here we show the first direct and quantitative experiments demonstrating the induction of phosphatidylserine asymmetry in a membrane by electrostatic association of poly-l-lysine in an attempt to elucidate the complex factors which govern the establishment and maintenance of lipid compositional asymmetry in the plasma membrane on a fundamental level. The attractive electrostatic interactions between the charged surface-associated polylysine and phosphatidylserine are sufficient to both induce and maintain an asymmetric arrangement of phosphatidylserine in a planar supported membrane, as measured by sum-frequency vibrational spectroscopy. These studies provide a glimpse of the physical and chemical underpinnings of lipid asymmetry in the eukaryotic plasma membrane.     

Glycolipid membranes through atomistic simulations: effect of glucose and galactose head groups on lipid bilayer properties

Though glycolipids are involved in a multitude of cellular functions, the understanding of their atom-scale properties in lipid membranes has remained very limited due to the lack of atomistic simulations. In this work, we employ extensive simulations to characterize one-component membranes comprised of glycoglycerolipids, focusing on two common glyco head groups, namely glucose and galactose. The properties of these two glycoglycerolipid bilayers are compared in a systematic manner with membranes consisting of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) lipids, whose structures aside from the head group are identical with those of the two glycolipids. We find that the glycolipid systems are characterized by a substantial number of hydrogen bonds in the head group region, leading to membrane packing that is stronger than in a PC but less significant than that in a PE bilayer. The role played by the glyco head group is especially evident in the electrostatic membrane potential, which is particularly large in the glycolipid membranes. For the same reason, the interfacial forces near glycolipid bilayers are significantly different from those found in PC and PE bilayers, affecting, e.g., the ordering of water close to the membrane. These effects are particularly important for the case of galactose, an important component in thylacoids.     

Lipid-based mechanisms for vesicle fission

Shape transformations and topological changes of lipid vesicles, such as fusion, budding, and fission, have important chemical physical and biological significance. In this paper, we study the fission process of lipid vesicles. Two distinct routes are considered that are both based on an asymmetry of the lipid distribution within the membrane. This asymmetry consists of a nonuniform distribution of two types of lipids. In the first mechanism, the two types of lipids are equally distributed over both leaflets of the membrane. Phase separation of the lipids within both leaflets, however, results in the formation of rafts, which form buds that can split off. In the second mechanism, the asymmetry consists of a difference in composition between the two monolayers of the membrane. This difference in composition yields a spontaneous curvature, reshaping the vesicle into a dumbbell such that it can split. Both pathways are studied with molecular dynamics simulations using a coarse-grained lipid model. For each of the pathways, the conditions required to obtain complete fission are investigated, and it is shown that for the second pathway, much smaller differences between the lipids are needed to obtain fission than for the first pathway. Furthermore, the lipid composition of the resulting split vesicles is shown to be completely different for both pathways, and essential differences between the fission pathway and the pathway of the inverse process, i.e., fusion, are shown to exist.     

The influence of zwitterionic lipids on the electrostatic adsorption of macroions onto mixed lipid membranes

Charged lipid membranes commonly consist of a mixture of charged and zwitterionic lipids. We suggest a model that characterizes the influence of the dipolar nature of the zwitterionic lipid species on the electrostatic adsorption of macroions onto mixed membranes in the fluid state. The model is based on Poisson-Boltzmann theory which we have modified so as to account for the dipolar character of the zwitterionic lipids. In addition the membrane lipids are allowed to adjust their lateral distribution upon macroion adsorption. We consider and compare two experimentally relevant scenarios: cationic macroions adsorbed onto anionic membranes and anionic macroions adsorbed onto cationic membranes. We show that in the former case the adsorption strength is slightly weakened by the presence of the headgroup dipoles of the zwitterionic lipids. Here, macroion-induced lipid demixing is more pronounced and the lipid headgroups tilt away from a cationic macroion upon adsorption. In contrast, for the adsorption of anionic macroions onto a cationic membrane the zwitterionic lipids strongly participate in the electrostatic interaction between membrane and macroion, thus enhancing the adsorption strength significantly (we predict up to 20%). Consistent with that we find less lateral demixing of the charged lipids and a reorientation of the dipoles of the zwitterionic headgroups towards the anionic macroions. Our results may be of importance to understand the differences in the electrostatic adsorption of proteins/peptides onto cellular membranes versus complex formation between cationic membranes and DNA.     

Effects of phosphatidylserine and phosphatidylethanolamine content on partitioning of triflupromazine and chlorpromazine between phosphatidylcholine-aminophospholipid bilayer vesicles and water studied by second-derivative spectrophotometry

To assess the affinity of psychotropic phenothiazine drugs, triflupromazine (TFZ) and chlorpromazine (CPZ), for the membranes of central nervous system and the other organs in the body, the partition coefficients (Kps) of these drugs to phosphatidylcholine (PC)-phosphatidylserine (PS) and PC-phosphatidylethanolamine (PE) small and large unilamellar vesicles (SUV, LUV) were examined by a second-derivative spectrophotometric method, since PS is abundantly contained in the membranes of the central nervous system and PE is distributed widely in the membranes of the organs in the body. Size and preparation methods of the vesicles did not affect the Kp values at each aminophospholipid content suggesting that the partition of the phenothiazine drugs was not affected by the structural differences in the vesicles such as their curvature or asymmetric distribution of the phospholipids between the outer and inner layers of the bilayer membranes. However, the Kp values of both drugs increased remarkably according to the PS content in the bilayer membranes, i.e., the Kp values for the vesicles of 30 mol% PS content were about 3 times of that for the vesicles of PC alone, while both Kp values slightly reduced with the increase in the content of PE in the bilayer membranes of PC-PE vesicles. The results indicate that both drugs have higher affinity for the PC-PS bilayer membranes than for the PC and PC-PE membranes, which can offer an evidence for the fact that TFZ and CPZ are predominantly distributed and accumulated in the brain and nerve cell membranes that contain PS abundantly.     

Design,synthesis and characterization of artificial phospholipids as model membrane receptors for specific binding of rabbit C-reactive protein

C-reactive protein (CRP) is an acute phase reactive protein, which has been shown to specifically bind to phosphorylcholine (PC) and phosphorylethanoamine (PE) moieties in the presence of calcium. In order to investigate the effect of steric hindrance on the specific binding of CRP to membranes, we designed and synthesized six phospholipids, each containing a long-arm spacer of 3, 6 or 8 atoms between the head group and hydrophobic tail. By mixing synthesized lipids and natural lipids the ligand-containing monolayers were prepared, which have PC or PE groups protruding out of the membrane surface. To characterize of the synthesized phospholipids, the thickness of the lipid monolayers was measured by surface plasmon resonance (SPR) technique, the phase behavior of the lipid monolayer at air/water interface was studied by pressure–area analysis, and the specific binding of rabbit C-reactive protein to the synthesized lipid containing membranes was studied by imaging ellipsometry.     

Asymmetric ABC-triblock copolymer membranes induce a directed insertion of membrane proteins

Asymmetric molecules and materials provide an important basis for the organization and function of biological systems. It is well known that, for example, the inner and outer leaflets of biological membranes are strictly asymmetric with respect to lipid composition and distribution. This plays a crucial role for many membrane-related processes like carrier-mediated transport or insertion and orientation of integral membrane proteins. Most artificial membrane systems are, however, symmetric with respect to their midplane and membrane proteins are incorporated with random orientation. Here we describe a new approach to induce a directed insertion of membrane proteins into asymmetric membranes formed by amphiphilic ABC triblock copolymers with two chemically different water-soluble blocks A and C. In a comparative study we have reconstituted His-tag labeled Aquaporin 0 in lipid, ABA block copolymer, and ABC block copolymer vesicles. Immunolabeling, colorimetric, and fluorescence studies clearly show that a preferential orientation of the protein is only observed in the asymmetric ABC triblock copolymer membranes.     

Nanopore formation and phosphatidylserine externalization in a phospholipid bilayer at high transmembrane potential

Atomic-resolution molecular dynamics simulations of lipid bilayers containing 7% phosphatidylserine (PS) on one leaflet are consistent with experimental observations of membrane poration and PS externalization in living cells exposed to nanosecond, megavolt-per-meter electric pulses. Nanometer-diameter aqueous pores develop within nanoseconds after application of an electric field of 450 mV/nm, and electrophoretic transport of the anionic PS headgroup along the newly constructed hydrophilic pore surface commences even while pore formation is still in progress.