Tristearin bilayers: structure of the aqueous interface and stability in the presence of surfactants

We report results of atomistic molecular dynamics simulations of an industrially-relevant, exemplar triacylglycerol (TAG), namely tristearin (TS), under aqueous conditions, at different temperatures and in the presence of an anionic surfactant, sodium dodecylbenzene sulphonate (SDBS). We predict the TS bilayers to be stable and in a gel phase at temperatures of 350 K and below. At 370 K the lipid bilayer was able to melt, but does not feature a stable liquid-crystalline phase bilayer at this elevated temperature. We also predict the structural characteristics of TS bilayers in the presence of SDBS molecules under aqueous conditions, where surfactant molecules are found to spontaneously insert into the TS bilayers. We model TS bilayers containing different amounts of SDBS, with the presence of SDBS imparting only a moderate effect on the structure of the system. Our study represents the first step in applying atomistic molecular dynamics simulations to the investigation of TAG-aqueous interfaces. Our results suggest that the CHARMM36 force-field appears suitable for the simulation of such systems, although the phase behaviour of the system may be shifted to lower temperatures than is the case for the actual system. Our findings provide a foundation for further simulation studies of the TS-aqueous interface.

Papel da localização da carga em determinação dos parâmetros do canal iônico

To aureus α-HL channel, we used the cysteine-scanning mutagenesis technique. Twenty-four mutants were produced from the substitution of a single aminoacid of the primary structure of the α-HL pro this yzed after the incorporation of a mutant channel in planar lipid bilayer membranes. The modified proteins were studied in the absence and presence of watersoluble specific sulphydryl-specific reagents, in order to introduce a strong positive or negative harge at positions of substitution. The introduction of a negative charge in the stem region onverted the selectivity of the channel from weak anionic to more cationic. However, the troduction of a positive charge increased its selectivity to the anion. The degree of these alterations was inversely dependent on the channel radius at the position of the introduced harge (selectivity). As to the asymmetry of the conductance-voltage, the influence of the harge was more complex. The introduction of the negative charge in the stem region (the trans art of the pore) provoked a decrease. The intensity of these alterations depended on the radius, and on the type of free charge at the pore entrance. These results suggest that the free charge at surrounds the pore wall is responsible for the cation-anion selectivity of the channel. The istribution of the charges between the entrances is crucial for determining the asymmetry of e conductance-voltage curves. We hope that these results serve as a model for studies with other nanometric channels, in biological or planar lipid bilayer membranes or in iotechnological applications

Preferential location of prilocaine and etidocaine in phospholipid bilayers: A molecular dynamics study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and H-2 NMR

We have examined the effect of the uncharged species of lidocaine (LDC) and etidocaine (EDC) on the acyl chain moiety of egg phosphatidylcholine liposomes. Changes in membrane organization caused by both anesthetics were detected through the use of EPR spin labels (5, 7 and 12 doxyl stearic acid methyl ester) or fluorescence probes (4, 6, 10, 16 pyrene-fatty acids). The disturbance caused by the LA was greater when the probes were inserted in more external positions of the acyl chain and decreased towards the hydrophobic core of the membrane. The results indicate a preferential insertion of LDC at the polar interface of the bilayer and in the first half of the acyl chain, for EDC. Additionally, 2 H NMR spectra of multilamellar liposomes composed by acyl chain-perdeutero DMPC and EPC (1:4 mol%) allowed the determination of the segmental order (S-mol) and dynamics (T-1) of the acyl chain region. In accordance to the fluorescence and EPR results, changes in molecular orientation and dynamics are more prominent if the LA preferential location is more superficial, as for LDC while EDC seems to organize the acyl chain region between carbons 2-8, which is indicative of its positioning. We propose that the preferential location of LDC and EDC inside the bilayers creates a "transient site", which is related to the anesthetic potency since it could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel. (C) 2007 Elsevier B.V. All rights reserved.

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper. we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4 h of incubation at 25 degreesC. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395 nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degreesC.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.

Study of the interaction between cardiolipin bilayers and methylene blue in polymer-based Layer-by-Layer and Langmuir films applied as membrane mimetic systems

An increase of the reports involving mimetic systems has been observed. Briefly, these systems use biological phospholipids to exploit specific interactions between membrane-models and drugs. Here, the Layer-by-Layer (LbL) and Langmuir techniques were used to investigate the interaction between cardiolipin (CLP-negative phospholipid) and a cationic-like drug methylene blue (MB). Supported by a cationic polyelectrolyte (PAH), LbL films containing PAH/(CLP + MB) and PAH/(CLP + MB + AgNP) were grown up to 14 bilayers. The optical microscopy analysis revealed a decrease of the CLP vesicle sizes in the presence of MB as a possible consequence of the MB action onto the mechanical properties of the CLP membrane. From FTIR spectra, changes mainly related to peak position and band intensity and shape were observed in the spectra from PAH/CLP when in the presence of MB. The latter supports that the interactions between the phosphate and amine charged groups from CLP and PAH, respectively, established during the LbL film fabrication, besides the CLP hydrocarbon environment, are influenced by the presence of MB. Using the micro-Raman technique, a chemical mapping was build based on MB spectrum by resonance Raman scattering (RRS) and surface-enhanced resonance Raman scattering (SERRS). The later phenomenon was activated by Ag nanoparticles (AgNPs) trapped within the LbL film allowing collecting spectra for a single bilayer of PAH/(CLP + MB + AgNP). A rough estimation showed a SERRS amplification of 10(3) in comparison to RRS spectra. As a complementary approach, Langmuir films of CLP in the presence of co-spread MB were investigated through surface pressure vs mean molecular area (pi-A) isotherms. The results showed that for concentrations of MB below 100 mol%, the drug is expelled to water subphase for high values of surface pressure (condensed phase). For concentration at 100% and higher, the MB keeps bound to CLP floating monolayer. (C) 2010 Elsevier B.V. All rights reserved.

Studies on the encapsulation of diclofenac in small unilamellar liposomes of soya phosphatidylcholine

The encapsulation of acid (AD) and sodium diclofenac (SD) in small unilamellar liposomes (SUV) as well as the interactions of the drug with the bilayer was studied. SUV was prepared by sonication from multilamellar liposomes containing soya phosphatidylcholine and diclofenac at various proportions. The size distribution obtained from dynamic light scattering showed that the incorporation of SD decreases significantly the size of the liposomes suggesting that the drug interacts with the bilayer of the liposomes. This size decrease is related with the phase transition of liposomes to mixed micelar solution. The encapsulation of the hydrophilic dye indocyanine green in the aqueous compartment of liposomes showed that the rate of captured dye decreases with SD concentration suggesting the transition of liposomes to mixed micelles. The P-31 NMR analysis indicates that SD interacts with the phosphate of phosphatidylcholine head groups. A schematic model for interaction of SD with phosphatidylcholine of the liposomes in which the diclofenac anion interacts with the ammonium group of the phospholipid and the dichloropheryl ring occupies a more internal site of bilayer near phosphate group was proposed. (C) 2004 Elsevier B.V. All rights reserved.

Effect of vesicles of dimethyldioctadecylammonium chloride and phospholipids on the rate of decarboxylation of 6-nitrobenzisoxazole-3-carboxylate

Sonicated mixtures of dimethyldioctadecylammonium chloride (DODAC), egg phosphatidylcholine (PC), dimyristoyl phosphatidylcholine (DMPC), and dipalmitoyl phosphatidylcholine (DPPC) were used to analyze vesicle effects on the rate of decarboxylation of 6-nitrobenzisoxazol-3-carboxylic acid (Nboc). Electron microscopic images of the vesicles were obtained with trehalose, a know cryoprotector. Phase diagrams and phase transitions temperatures of the vesicle bilayers were determined. Nboc decarboxylation rates increased in the presence of vesicles prepared with both phospholipids and DODAC/phospholipid mixtures. Quantitative analysis of vesicular effects was done using pseudophase models. Phospholipids catalyzed up to 140-fold while the maximum catalysis by DODAC/lipid vesicles reached 800-fold. Acceleration depends on alkyl chain length, fatty acid insaturation of the lipids, and the DODAC/phospholipid molar ratio. Catalysis is not related to the liquid crystalline-gel state of the bilayer and may be related to the relative position of Nboc with respect to the interface.

Photoinduced hole-transfer in semiconducting polymer/low-bandgap cyanine dye blends: evidence for unit charge separation quantum yield

Power-conversion efficiencies of organic heterojunction solar cells can be increased by using semiconducting donor-acceptor materials with complementary absorption spectra extending to the near-infrared region. Here, we used continuous wave fluorescence and absorption, as well as nanosecond transient absorption spectroscopy to study the initial charge transfer step for blends of a donor poly(p-phenylenevinylene) derivative and low-band gap cyanine dyes serving as electron acceptors. Electron transfer is the dominant relaxation process after photoexcitation of the donor. Hole transfer after cyanine photoexcitation occurs with an efficiency close to unity up to dye concentrations of similar to 30 wt%. Cyanines present an efficient self-quenching mechanism of their fluorescence, and for higher dye loadings in the blend, or pure cyanine films, this process effectively reduces the hole transfer. Comparison between dye emission in an inert polystyrene matrix and the donor matrix allowed us to separate the influence of self-quenching and charge transfer mechanisms. Favorable photovoltaic bilayer performance, including high open-circuit voltages of similar to 1 V confirmed the results from optical experiments. The characteristics of solar cells using different dyes also highlighted the need for balanced adjustment of the energy levels and their offsets at the heterojunction when using low-bandgap materials, and accentuated important effects of interface interactions and solid-state packing on charge generation and transport.

Nucleação de vortices e paredes de domínio em nanoestruturas magnéticas

We report a theoretical investigation of the magnetic phases and hysteresis of exchange biased ferromagnetic (F) nanoelements for three di erent systems: exchange biased nanoparticles, exchange biased narrow ferromagnetic stripes and exchange biased thin ferromagnetic lms. In all cases the focus is on the new e ects produced by suitable patterns of the exchange energy coupling the ferromagnetic nanoelement with a large anisotropy antiferromagnetic (AF) substrate. We investigate the hysteresis of iron and permalloy nanoparticles with a square basis, with lateral dimensions between 45 nm and 120 nm and thickness between 12 nm and 21 nm. Interface bias is aimed at producing large domains in thin lms. Our results show that, contrary to intuition, the interface exchange coupling may generate vortex states along the hysteresis loop. Also, the threshold value of the interface eld strength for vortex nucleation is smaller for iron nanoelements. We investigate the nucleation and depinning of an array of domain walls pinned at interface defects of a vicinal stripe/AF bilayer. The interface exchange eld displays a periodic pattern corresponding to the topology of the AF vicinal substrate. The vicinal AF substrate consists of a sequence of terraces, each with spins from one AF subalattice, alternating one another. As a result the interface eld of neighboring terraces point in opposite direction, leading to the nucleation of a sequence of domain walls in the ferromagnetic stripe. We investigated iron an permalloy micrometric stripes, with width ranging from 100 nm and 300 nm and thickness of 5 nm. We focused in domain wall sequences with same chirality and alternate chirality. We have found that for 100nm terraces the same chiraility sequence is more stable, requiring a larger value of the external eld for depinning. The third system consists of an iron lm with a thickness of 5 nm, exchange coupled to an AF substrate with a periodic distribution of islands where the AF spins have the opposite direction of the spins in the background. This corresponds to a two-sublattice noncompensated AF plane (such as the surface of a (100) FeF2 lm), with monolayer-height islands containing spins of one sublattice on a surface containing spins of the opposite sublattice. The interface eld acting in the ferromagnetic spins over the islands points in the opposite direction of that in the spins over the background. This a model system for the investigation of interface roughness e ects. We have studied the coercicivity an exchange bias hysteresis shift as a function of the distance between the islands and the degree of interface roughness. We have found a relevant reduction of coercivity for nearly compensated interfaces. Also the e ective hysteresis shift is not proportional to the liquid moment of the AF plane. We also developed an analytical model which reproduces qualitatively the results of numerical simulations

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vesicle-micelle transition in aqueous mixtures of the cationic dioctadecyldimethylammonium and octadecyltrimethylammonium bromide surfactants

The vesicle-micelle transition in aqueous mixtures of dioctadecyidimethylammonium and octadecyltrimethylammonium bromide (DODAB and C(18)TAB) cationic surfactants, having respectively double and single chain, was investigated by differential scanning calorimetry (DSQ, steady-state fluorescence, dynamic light scattering (DLS) and surface tension. The experiments performed at constant total surfactant concentration, up to 1.0 mM, reveal that these homologous surfactants mix together to form mixed vesicles and/or micelles, depending on the relative amount of the surfactants. The melting temperature T-m of the mixed DODAB-C(18)TAB vesicles is larger than that for the neat DODAB in water owing to the incorporation of C(18)TAB in the vesicle bilayer. The surface tension decreases sigmoidally with C(18)TAB concentration and the inflection point lies around (XDODAB) approximate to 0.4, indicating the onset of micelle formation owing to saturation of DODAB vesicles by C(18)TAB molecules. When XDODAB > 0.5 C(18)TAB molecules are mainly solubilised by the vesicles, but when XDODAB < 0.25 micelles are dominant. Fluorescence data of the Nile Red probe incorporated in the system at different surfactant molar fractions indicate the formation of micelle and vesicle structures. These structures have apparent hydrodynamic radius RH of about 180 and 500-800 nm, respectively, as obtained by DLS measurements. (C) 2007 Elsevier B.V. All rights reserved.

Interaction of octaethylene glycol n-dodecyl monoether with dioctadecyldimethylammonium bromide and chloride vesicles

Several different methods were used to investigate the vesicle-to-micelle transition induced by the addition of the nonionic surfactant octaethylene glycol n-dodecyl monoether (C12E8) to spontaneously formed vesicle dispersions of dioctadecyldimethylammonium bromide and chloride (DODAX, X = Cl- and Br-). Dynamic light scattering reveals that fast mode micelles are formed upon addition of C12E6. The micellar mode becomes progressively dominant as the C12E8/DODAX molar ratio (R) is increased until the vesicle-to-micelle transition is complete. Turbidity, calorimetry, fluorescence quantum yield, and anisotropy measurements indicate two critical compositions: the first, R-sat, when the vesicle bilayer is saturated with C12E8 and the second, R-sol, which corresponds to the complete vesicle-to-micelle transition. Below R-sat the vesicles swell due to incorporation of the surfactant into the vesicle bilayer, and above R-sat mixed micelles and bilayer structures coexist, the determined R-sat and R-sol range from 0 to 1 and 4 to 6, respectively, depending on the surfactant counterion and the experimental method used. Cryo-transmission electron microscopy micrographs show that when R approximate to 4, micelles coexist with extended bilayer fragments. In pure DODAX (1.0 mM) dispersions, unilamellar vesicles are observed. According to the DSC results, C12E8 lowers the gel-to-liquid crystalline transition temperature, T-m, of DODAX and broadens the main transition peak which disappears around R approximate to 5 and 6 for DODAC and DODAB, respectively.

Initiating structural studies of Lys49-PLA2 homologues complexed with an anionic detergent, a fatty acid and a natural lipid

Lys49-Phospholipase A(2) (Lys49-PLA(2) - EC 3.1.1.4) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. Both MjTX-II from Bothrops moojeni and BthTX-I from Bothrops jararacussu are dimeric in solution and in the crystalline states, and a model for the Ca2+-independent membrane damaging mechanism has been suggested in which flexibility at the dimer interface region pert-nits quaternary structural transitions between open and closed membrane bound dimer conformations which results in the perturbation of membrane phospholipids and disruption of the bilayer structure [1]. With the aim of gaining insights into the structural determinants involved in protein/lipid association, we report here the crystallization and preliminary X-ray analysis of the (i) MjTX-II/SDS complex at a resolution of 2.78Angstrom, (ii) MjTX-II/STE complex at a resolution of 1.8 Angstrom and (W) BthTX-I/DMPC complex at 2.72Angstrom. These complexes were crystallized by the hanging drop vapour-diffusion technique in (i) HEPES buffer (pH 7.5) 1.8M ammonium sulfate with 2% (w/v) polyethyleneglycol 400, in (ii) 0.6-0.8 M sodium citrate as the precipitant (pH 6.0-6.5) and in (iii) sodium citrate buffer (pH 5.8) and PEG 4000 and 20% isopropanol, respectively. Single crystals of these complexes have been obtained and X-ray diffraction data have been collected at room temperature using a R-AXIS IV imaging plate system and graphite monochromated Cu Kalpha X-ray radiation generated by a Rigaku RU300 rotating anode generator for (i) and (W) and using using a Synchrotron Radiation Source (Laboratorio Nacional de Luz Sincrotron, LNLS, Campinas, Brazil) for (ii).