Investigation of the structure of multilayer lipid membranes by real-time neutron diffraction

This paper reports on the results of neutron diffraction experiments on the study of structural changes in multilayer lipid membranes as a function of the degree of hydration. The experiments have been performed on a time-of-flight diffractometer at the IBR-2 pulsed reactor (Joint Institute for Nuclear Research, Dubna, Russia) with a dipalmitoylphosphatidylcholine (DPPC) multilayer membrane oriented on a quartz plate. The structural parameters have been determined from the simultaneously measured several (up to five) diffraction orders of reflection, which has made it possible to calculate the profile of the DPPC bilayer structure. A high rate of the measurement of diffraction patterns has allowed one to trace the evolution the lamellar structure of the lipid bilayer in the course of its hydration and dehydration. The minimum time of accumulation of the necessary statistics is 3 min, which has made it possible to determine the characteristic times of transition processes in the membrane with a high accuracy.     

Permeation of nanocrystals across lipid membranes

Biological membranes are one of the major structural elements of cells, and play a key role as a selective barrier and substrate for many proteins that facilitate transport and signaling processes. Understanding the structural and mechanical properties of lipid membranes during permeation of nanomaterials is of prime importance in determining the toxicity of nanomaterials to living cells. It has been shown that the interaction between lipid membranes and nanomaterials and the disruption of lipid membranes are often determined by physicochemical properties of nanomaterials, such as size, shape and surface composition. In this work, molecular dynamic simulations were carried out using various sizes of nanocrystals as a probe to explore the transport of nanomaterials across dipalmitoylphosphatidylcholine (DPPC) bilayers and the changes in the structural and mechanical properties of DPPC bilayers during the permeation. A coarse-grained model was used to provide insight at large time and length scales. In this work, an external driving force helps the nanocrystals across the lipid bilayer. The minimum forces needed to penetrate the model membrane and the interaction of nanocrystals and lipid bilayers were investigated in simulations. The elastic and dynamic properties of lipid bilayers, including the local and bulk properties during the permeation of the nanocrystals, which are of considerable fundamental interest, were also studied. The findings described will lead to better understanding of nanomaterial–lipid membrane interactions and the mechanical and dynamic properties of lipid membranes under permeation.     

Lateral diffusion in equimolar mixtures of natural sphingomyelins with dioleoylphosphatidylcholine

Cellular membranes of mammals are composed of a complex assembly of diverse phospholipids. Sphingomyelin (SM) and phosphatidylcholine (PC) are important lipids of eukaryotic cellular membranes and neuronal tissues, and presumably participate in the formation of membrane domains, known as "rafts," through intermolecular interaction and lateral microphase decomposition. In these two-dimensional membrane systems, lateral diffusion of lipids is an essential dynamic factor, which might even be indicative of lipid phase separation process. Here, we used pulsed field gradient nuclear magnetic resonance to study lateral diffusion of lipid components in macroscopically oriented bilayers composed of equimolar mixtures of natural SMs of egg yolk, bovine brain, bovine milk and dipalmitoylphosphatidylcholine (DPPC) with dioleoylphosphatidylcholine (DOPC). In addition, differential scanning calorimetry was used as a complementary technique to characterize the phase state of the lipid bilayers. In fully liquid bilayers, the lateral diffusion coefficients in both DOPC/DPPC and DOPC/SM systems exhibit mean values of the pure bilayers. For DOPC/SM bilayer system, this behavior can be explained by a model where most SM molecules form short-lived lateral domains with preferential SM-SM interactions occurring within them. However, for bilayers in the presence of their low-temperature gel phase, lateral diffusion becomes complicated and cannot simply be understood solely by a simple change in the liquid phase decomposition.     

Ethanol and methanol induced changes in phospholipid monolayer

The main components of cell membranes are phospholipids and proteins. The aim of our study was to examine structural changes of dipalmitoyl-phosphatidylcholine (DPPC) monolayer as a simple model system of a cell membrane in different environments. Pure water, ethanol and methanol solutions were used as subphases of Langmuir films as a membrane models. For detection of changes in charge states of the molecules as well as relation with structural and conformational changes, a contactless method Maxwell's displacement currents (MDC) was used. Behaviour of DPPC molecules on two different subphases is substantialy different. In DPPC monolayer on the subphase of methanol-water, a gradual absorption (incorporation, penetration) of methanol molecules into the layer can appear. In DPPC monolayer on the subphase of ethanol-water adsorption of ethanol molecules on the layer can be observed. The membrane permeability might change. At both subphases (ethanol-water and methanol-water) the elasticity modulus of the monolayer decreases leading to the loss of membrane elasticity.     

Miscibility phase diagrams of giant vesicles containing sphingomyelin

Saturated sphingomyelin (SM) lipids are implicated in lipid rafts in cell plasma membranes. Here we use fluorescence microscopy to observe coexisting liquid domains in vesicles containing SM, an unsaturated phosphatidylcholine lipid (either DOPC or POPC), and cholesterol. We note similar phase behavior in a model membrane mixture without SM (DOPC/DPPC/Chol), but find no micron-scale liquid domains in membranes of POPC/PSM/Chol. We delineate the onset of solid phases below the miscibility transition temperature, and detail indirect evidence for a three-phase coexistence of one solid and two liquid phases.     

Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

Myelin basic protein(MBP) is an essential structure involved in the generation of central nervous system(CNS)myelin.Myelin shape has been described as liquid crystal structure of biological membrane.The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin.In this paper,we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy(AFM).By analyzing the pressure–area(π–A) and pressure–time(π–T) isotherms,univariate linear regression equation was obtained.In addition,the elastic modulus,surface pressure increase,maximal insertion pressure,and synergy factor of monolayers were detected.These parameters can be used to modulate the monolayers binding of protein,and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3-phosphoserine(DPPS) monolayer,followed by DPPC/DPPS mixed and1,2-dipalmitoyl-sn-glycero-3-phospho-choline(DPPC) monolayers via electrostatic and hydrophobic interactions.AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP(5 n M) show a phase separation texture at the surface pressure of 20 m N/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure.MBP is not an integral membrane protein but,due to its positive charge,interacts with the lipid head groups and stabilizes the membranes.The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value.     

31P NMR and FTIR studies on the interaction of phosphorylacetohydrazides with dipalmitoyl phosphatidylcholine model membranes

The effect of original synthetic nootropic drugs (phosphorylacetohydrazides) on the physical properties of the dipalmitoyl phosphatidylcholine (DPPC) membranes was studied by³¹P nuclear magnetic resonance and Fourier transform infrared spectroscopies. It has been shown that the tested preparations reduce the phase transition temperature, widen the transition interval, suppress pretransition, render some condensing effect on the gel phase and essentially disorder the lipid bilayer in the liquid-crystalline state. The obtained results are in agreement with the suggested mechanism of the primary pharmacological effect of nootropic preparations. According to this mechanism, the drug molecules penetrate into the hydrophilic region of the bilayer, interacting closely with the polar groups of DPPC, disturbing the bilayer organization and leading to polymorphism. By taking into account that the main property of nootropic preparations is to improve processes of training and memory, we suppose that the new lipid-drug organization of phospholipidic membranes and lipid polymorphism are the necessary steps of the nootropic activity. The similarity of molecular mechanisms of various nootropic drug effects on the lipid bilayer allows us to suppose that the positive effect of nootropics on the synaptic transmission may be governed by their influence on the phase transition of the lipid component of the synaptic membranes at the stage of the neurotransmitter release.     

Probing lateral diffusion in lipid membranes on nanoscale by PFG NMR with high gradient strength

This work demonstrates the feasibility of noninvasive studies of diffusion on a submicrometer length scale in aligned model lipid membranes using pulsed field gradient nuclear magnetic resonance with ultrahigh (up to 35 T/m) gradient strength. Application of such gradients allows the use of sufficiently small diffusion times under conditions of narrow-pulse approximation. As a result, monitoring anomalous or restricted diffusion in lipid membranes on a length scale in the range of 100 nm becomes possible. The ability to study diffusion in lipid membranes on this length scale is very important because it is comparable with the size of biologically relevant domains (i.e., rafts), which are believed to exist in biomembranes.     

Magnetoresistance measurements of graphene at the charge neutrality point

We report on transport measurements of the insulating state that forms at the charge neutrality point of graphene in a magnetic field. Using both conventional two-terminal measurements, sensitive to bulk and edge conductance, and Corbino measurements, sensitive only to the bulk conductance, we observed a vanishing conductance with increasing magnetic fields. By examining the resistance changes of this insulating state with varying perpendicular and in-plane fields, we probe the spin-active components of the excitations in total fields of up to 45?T. Our results indicate that the ν=0 quantum Hall state in single layer graphene is not spin-polarized.     

Interactions of lactoferricin B derivatives with model cell membrane studied by Raman spectroscopy

In this work, we employed Raman spectroscopy to study the effect of the antimicrobial peptide lactoferricin B (LfB) on model cell membranes. We used two derivatives of LfB (RRWQWRMKKLG and RRWQWR) with broad‐spectrum activity against gram‐positive and gram‐negative bacteria, fungus, viruses and tumors. Raman spectra of the peptides showed no conformational change in the temperature range 4–60 °C. The positions of the amide I and amide III bands suggest that in an aqueous solution these peptides preferentially adopt a random coil‐like conformation. We also investigated the effect the peptides had on the melting behavior of model cell membranes composed of zwitterionic lipid dipalmitoylglycero‐phosphocholine (DPPC) and anionic lipid dipalmitoylglycero‐phosphoglycerol (DPPG). Raman CH stretching bands were used to follow the melting of the lipid vesicles. We found that the melting of DPPC lipid vesicles is not affected by the presence of the peptides, while the presence of the peptides reduced cooperativity of the phase transition for anionic DPPG vesicle, suggesting that both peptides interact strongly and specifically with this model cell membrane composed of anionic lipid. Copyright © 2006 John Wiley & Sons, Ltd.     

A virtual instrumentation based protocol for the automated implementation of the inner field compensation method

One influential parameter which mediates interactions between many types of molecules and biological membranes stems from the lumped contributions of the transmembrane potential, dipole potential and the difference in the surface potentials on both sides of a membrane. With relevance to cell physiology, such electrical features of a biomembrane are prone to undergoing changes as a result of interactions with the aqueous surrounding. Among the most useful tools devoted to exploring changes of electrical parameters of a lipid membrane induced by certain extracellular ions, lipid composition, and embedded membrane peptides and proteins, are spectroscopic imaging and the inner field compensation (IFC) method. In this work we layout the principles of a fully computerized version of the IFC method, which makes it more readily available to users. As a direct application, we deployed this improved version of the IFC method to time-resolve changes induced by alamethicin monomers upon membrane dipole potential, following their aggregation within an artificial lipid membrane. Intriguingly, even prior crossing the membrane core, the membrane-bound alamethicin monomers are shown to significantly increase the dipole potential of the monolayer they reside in. Such data further emphasize the yet less-explored interplay between membrane-based protein and peptides, and the membrane dipole potential.     

Gallium nitride electrodes for membrane-based electrochemical biosensors

We report on the deposition of planar lipid bilayers (supported membranes) on gallium nitride (GaN) electrodes for potential applications as membrane-based biosensors. The kinetics of the lipid membrane formation upon vesicle fusion were monitored by simultaneous measurements of resistance and capacitance of the membrane using AC impedance spectroscopy in the frequency range between 50mHz and 50kHz. We could identify a two-step process of membrane spreading and self-healing. Despite its relatively low resistance, the membrane can be modeled by a parallel combination of an ideal resistor and capacitor, indicating that the membrane efficiently blocks the diffusion of ions.     

Enhancing low-field magnetoresistance of La0.67Ca0.33MnO3 films deposited on anodized aluminium-oxide membranes

In this paper we report a new method to fabricate nanostructured films, La0.67Ca0.33MnO3 （LCMO） nanostructured films have been fabricated by using pulsed electron beam deposition （PED） on anodized aluminium oxide （AAO） membranes, The magnetic and electronic transport properties are investigated by using the Quantum Design physics properties measurement system （PPMS） and magnetic properties measurement system （MPMS）. The resistance peak temperature （Tp） is about 85 K and the Curie temperature （To） is about 250 K for the LCMO film on an AAO membrane with a pore diameter of 20nm. Large magnetoresistance ratio （MR） is observed near Tp. The MR is as high as 85% under 1 T magnetic field. The great enhancement of MR at low magnetic fields could be attributed to the lattice distortion and the grain boundary that are induced by the nanopores on the AAO membrane.     

Diffusion of gases across lipid membranes with OmpA channel: a molecular dynamics study

Molecular transport across biological membranes occurs in a range of important chemical and biological processes. The biological membrane can usually be modelled as a phospholipid bilayer, but to correctly represent biological transport, the embedded transmembrane proteins must also be included. In previous molecular simulation studies on transport of small gas molecules in dipalmitoylphosphatidylcholine (DPPC) bilayer membrane, a coarse-grained model was used to provide direct insight into collective phenomena in biological membranes. Coarse graining allowed investigation of longer time and length scales by reducing the degrees of freedom and employing suitable potentials. In this work, membranes that include transmembrane proteins are modelled. This allows one to compare the molecular transport across a lipid membrane with and without the assistance of transmembrane channels. Outer membrane protein A (OmpA) – a porin from Escherichia coli with a small pore size – was chosen in this study because its detailed structure is known, it has high stability and is known to form a nonspecific diffusion channel that permits the penetration of various solutes. In this work the pore characteristics and interaction between lipid and protein were investigated and transport of water and other small gas molecules within the channel were studied. The MD simulation results obtained are compared with previous simulation results and available experimental data. The results obtained from this study will lead to better understanding of protein functionality and advance the development of biochips and drug delivery systems.     

Effect of some statin group of drugs on the phase profile of liposomal membrane – a fluorescence anisotropy study

Using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, we have investigated the effect of some statin group of drugs, widely used in hyperlipidemia, on the phase transition of model membranes such as dipalmitoyl phosphatidylcholine (DPPC) liposomes. The nature of the changes in the fluorescence anisotropy values suggested that the drugs simvastatin and mevastatin fluidized the membrane both before and after the phase transition temperature (T _m), whereas atorvastatin fluidized the membrane below T _m but rigidified the same above T _m, i.e., introduced an intermediate fluid condition within the lipid matrix. We have calculated the changes in van’t Hoff enthalpy values associated with the phase transitions due to chain melting in all the cases, and observed that the values of enthalpy decreased with increase in drug concentrations. In order to get a better insight, the fraction of motionally restricted lipid molecules was determined.     

High-field ESR on aligned membranes: a simple method to record spectra from different membrane orientations in the magnetic field

A combination of isopotential spin-dry ultracentrifugation (ISDU) and microtome techniques was used to facilitate the collection of high field/high frequency (170 GHz) ESR spectra corresponding to different orientations of the membrane normal relative to the magnetic field. This technique is particularly valuable for aligned biological samples in vitro. At 170 GHz, conventional sample preparation techniques based solely on ISDU constrained the sample to be oriented so that the membrane normal was parallel to the applied magnetic field due to the geometry and the millimeter wave field distribution of the Fabry-Pérot resonator used in these experiments. This orientational constraint limited the information that could be obtained from aligned membranes at high field. The combined ISDU/microtome technique overcame this limitation. Spectra from spin-labeled Gramicidin A and the spin label cholestane in aligned DPPC membranes provide a demonstration of the technique. We also discuss some virtues of high field/high frequency ESR on aligned membranes compared to X-band.     

Fluorescence Studies on New Potential Antitumoral Benzothienopyran-1-ones in Solution and in Liposomes

Fluorescence properties of four new potential antitumoral compounds, 3-arylbenzothieno[2,3-c]pyran-1-ones, were studied in solution and in lipid membranes of dipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC) and dioctadecyldimethylammonium bromide (DODAB). The 3-(4-methoxyphenyl)benzothieno[2,3-c]pyran-1-one (1c) exhibits the higher fluorescence quantum yields in all solvents studied. All compounds present a solvent sensitive emission, with significant red shifts in polar solvents for the methoxylated compounds. The results point to an ICT character of the excited state, more pronounced for compound 1c. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in liposomes of DPPC, DODAB and Egg-PC indicate that all compounds have two different locations, one due to a deep penetration in the lipid membrane and another corresponding to a more hydrated environment. In general, the methoxylated compounds prefer hydrated environments inside the liposomes. The 3-(4-fluorophenyl)benzothieno[2,3-c]pyran-1-one (1a) clearly prefers a hydrated environment, with some molecules located at the outer part of the liposome interface. On the contrary, the preferential location of 3-(2-fluorophenyl)benzothieno[2,3-c]pyran-1-one (1b) is in the region of lipid hydrophobic tails. Compounds with a planar geometry (1a and 1c) have higher mobility in the lipid membranes when phase transition occurs.     

Manifestations of Surface States in the Longitudinal Magnetoresistance of an Array of Bi Nanowires

The longitudinal magnetoresistance of the array of parallel-oriented bismuth nanowires each 100 nm in diameter grown by electrochemical deposition in nanopores of an Al₂O₃ membrane has been studied in magnetic fields up to 14 T and at temperatures down to 0.3 K. The resistance increases with the field and reaches a broad maximum in fields about 10 T. An anomalous increase in the resistance in weak fields is qualitatively consistent with the suppression of the antilocalization correction to the resistance, and the maximum is qualitatively associated with the classical size effect. Near the maximum at temperatures below 0.8 K, manifestations of reproducible magneto-oscillations of the resistance, which are periodic in field, have been detected. The period of these oscillations is close to a value corresponding to the passage of the flux quantum hc/e through the section of a nanowire. The Fourier analysis also confirms that the oscillations are periodic. This result is similar to the manifestation the Aharonov–Bohm effect caused by conducting surface states of Dirac fermions occupying L-valleys of bismuth.     

Developing DNP/Solid-State NMR Spectroscopy of Oriented Membranes

Dynamic nuclear polarization (DNP)/solid-state nuclear magnetic resonance (NMR) spectroscopy bears great potential for the investigation of membrane-associated polypeptides which can often be produced only in small amounts and which need to be ‘diluted’ in lipid bilayer environments to adopt or maintain their functional structure. Here we present investigations using biradicals, such as TOTAPOL and bTbK, for solid-state NMR signal enhancement using DNP in the context of lipid membranes. By transferring polarization from electron to nuclear spins using microwave irradiation signal enhancement factors of up to 13 are obtained with TOTAPOL and up to 17 with bTbK. The possible reasons why these factors are below those obtained in glassy samples of bulk solvents (40–60 under similar conditions) are evaluated and discussed. In order to further ameliorate the enhancement factors the physico-chemical characteristics of TEMPOL, TOTAPOL, bTbK, and bCTbK, such as their partitioning between hydrophilic and hydrophobic solvents or their stability under different environmental conditions are presented. Finally, having provided proof-of-concept that DNP/solid-state NMR measurements can be performed with oriented membrane samples work in progress is presented on the development of a flat-coil probe for DNP/solid-state NMR experiments on oriented membranes.     

拉曼光谱研究人参皂苷Rb1与DPPC双层膜的作用

为深入了解人参皂苷的分子药理学特性,阐明人参皂苷与细胞膜的作用机制,利用拉曼光谱从分子水平研究了不同浓度人参皂苷Rb1与DPPC(二棕榈酰磷脂酸胆碱)双层膜的作用.结果表明,人参皂苷Rb1没有改变DPPC的极性头部O-C-C-N+的稳定构象,极性头仍然平行于膜表面.并且,拉曼峰值比I1096/I1126/1096/I1062和I2848/I288/0随着药物浓度的增加而相应的变大,说明Rbl增加了烃链的无序度,增强了双层膜的流动性.由此推测该药物与DPPC的作用可能由于皂苷分子内及分子间的氢键与磷脂双层膜的极性头部相作用而停留在膜的表面.