Conductivity and structural transitions in bilayer lipid membranes

5 structural transitions were found in bilayer lipid membranes (BLM) from egg lecithin (EL) within the temperature range 14-44 degrees C. In the transition zone BLM conductivity abruptly increases, in some cases current fluctuations of the order 150 pC of the channel type are initiated. The transition temperatures observed in BLM from EL coincide with those in biological membranes. The cause of this phenomenon is discussed, as well as possible use of these BLM in the region of structural transition as a model of cellular receptor to electromagnetic fields.     

Diacylglycerol causes major structural transitions in phospholipid bilayer membranes

We demonstrate for the first time that major structural changes are imposed on various phospholipid bilayers by diacylglycerol, a product of phosphatidylinositol metabolism. By 5 mole percent in phosphatidylethanolamine a lamellar to hexagonal transition starts that is complete at 10 mole percent. At 30 mole percent it causes the same transition in phosphatidylcholine and forms a cubic phase at 80 mole percent. Diacylglycerol disorders the phosphatidylserine lamellar phase. We view the formation of the non-lamellar phases as diagnostic of the destabilizations that diacylglycerol can cause in membranes. We suggest how DAG may act both in its specific activation of membrane enzymes and in inducing membrane fusion.     

Cyanine dye structural and voltage-induced variations in photo-voltages of bilayer membranes

Summary Flash illumination alters the voltage across bilayer lipid membranes in the presence of certain cyanine dyes. The waveforms of the photo-voltage vary systematically with dye structure and imposed transmembrane voltage. Experimental results are reported for 27 positively charged cyanine dyes, primarily oxazole derivatives, using lecithin/oxidized cholesterol bilayer membranes and 10-mm sodium chloride solutions. Several dyes do not induce any photo-voltages. Examples are 3,3 diethyl 9 ethyl 2,2 oxacarbocyanine iodide, 3,3 diethyl 2 oxa 2 thiacyanine iodide, and 3,3 dimethyl 2,2 indocarbocyanine iodide. Several dyes, when added to one side of the membranes, induce monophasic waveforms. Examples are 3,3 dimethyl 2,2 oxacarbocyanine chloride, and 3,4,3,4 tetramethyl 2,2 oxazalinocarbocyanine iodide. Other dyes induce a photo-voltage only if transmembrane voltages are imposed. These waveforms are biphasic with some dyes (3,3 diethyl 2,2 oxacarbocyanine iodide, for example) and monophasic with other dyes (3,3 dibutyl 2,2 oxacarbocyanine iodide, for example).The photo-voltage waveforms are explained by models that consider the movement of charged dye molecules within the membrane, following optical excitation. The dye movements are probably induced through charge rearrangements in the dye associated with long-lived triplet states, isomerization, or through excimer formation. These results provide information on the location and orientation of the dye molecules within bilayer membranes. The variations which occur in the waveforms with applied voltage indicate that these membranes are fluid in the direction perpendicular to the membrane plane.     

Influence of structural modifications on the phase behavior of semi-synthetic cerebroside sulfate

Cerebroside sulfate (galactosylceramide I3-sulfate) containing alpha-hydroxy lignoceric acid (C24:0h-CBS), nervonic acid (C24:1-CBS), or hexacosanoic acid (C26:0-CBS) was prepared by a semi-synthetic procedure and studied by differential scanning calorimetry. The phase behavior of these species in 2 M KCl was compared to that of shorter chain length hydroxy and non-hydroxy fatty acid species reported earlier. All three of the new lipids undergo metastable phase behavior similar but not identical to the other species. In addition, the metastable phase behavior of all of the non-hydroxy fatty acid species was found to be more complex than previously thought, with several phases of high transition temperatures and enthalpies possible. Fatty acid hydroxylation inhibits the transition from the metastable to some of the more stable phases. It also significantly increases the phase transition temperatures of both the metastable and stable phases indicating that it contributes to the hydrogen bonding network formed between the lipid molecules and helps overcome the lateral repulsive effect of the negatively charged sulfate. The C-15 cis double bond significantly lowers the temperature and enthalpy of the phase transition indicating that it increases the lateral separation of the lipid molecules and decreases the intermolecular hydrogen bonding interactions. However, it does not prevent formation of a more stable phase. By comparing the effect of various structural modifications reported here and earlier it could be concluded that fatty acid chain length has little effect on the phase transition temperature and enthalpy. This suggests that the forces between the lipid molecules may be dominated by head group interactions rather than interactions between the lipid chains. However, fatty acid chain length has a significant effect on the tendency of the hydroxy fatty acid species to form the more stable phase. The ease of formation of the stable phase increases with increase in chain length. Thus an increase in chain length helps overcome the kinetic barrier to stable phase formation presented by hydroxylation of the fatty acid.     

The intrinsic structural asymmetry of highly curved phospholipid bilayer membranes

Phosphorus-31 NMR studies of solutions of small L-alpha-dipalmitoyl phosphatidylcholine bilayer vesicles containing sodium dimethyl phosphate uniformly distributed between the continuous external and the intravesicular aqueous spaces, with the paramagnetic shift reagent Pr3+ present only in the external space, are reported. These studies give the distribution both of dipalmitoyl phosphatidylcholine in the vesicle inner and outer monolayers and of dimethyl phosphate in the aqueous spaces. With the third necessary parameter obtained from the vesicle sedimentation coefficient, the very different packing parameters of dipalmitoyl phosphatidylcholine in inner and outer monolayers can be determined. The vesicle outer radius is 109 A. Although the total bilayer thickness is virtually identical to that of planar bilayers, the outer monolayer is thicker (20 A) and the inner monolayer thinner (15 A). The area per head group at the inner surface, 68 A2, is like the planar value, but the tails are much more folded, so as to decrease the radial lengths and increase the tangential spreat (to 94A2). The reverse is true in the outer layer: the surface per head group is 76 A2, tapering to 51 A2 in the tail region, so that outer layer tails are relatively extended. The difference is equivalent to a shift of about two 2g1 kinks from outer to inner layers; the uneven packing certainly affects fluidity, and may have important biological consequences.     

Thermodynamic and structural properties of phosphatidylserine bilayer membranes in the presence of lithium ions and protons

The binding of lithium ions to phosphatidylserine has been studied by differential scanning calorimetry for dialkyl and diacyl lipid forms and by X-ray diffraction for dihexadecylphosphatidylserine (DHPS). On first mixing DHPS with LiCl solutions an ordered L_β (L_c) phase is formed with a bilayer repeat distance of 5.55 nm and one strong wide-angle, chain-chain reflection at 0.405 nm (26°C), corresponding to bilayers of little, (mono)hydrated lipid with chains approximately perpendicular to the membrane surface. On heating, this phase transforms to an inverted hexagonal phase (H₁₁, H_α) with a repeat distance of 3.75 nm, at a chain-melting transition temperature of approximately 90°C (DHPS). Cooling, after equilibration of the DHPS⁻·Li⁺ sample in the fluid phase, creates a new low-temperature phase (L_c') which has a repeat distance of 4.0 nm, corresponding to strongly tilted chains (ϕ=42°). The L_c phase also transforms on heating to the H_α phase, but at a considerably lower chain-melting temperature of approx. 70°C (DHPS). The calorimetric behavior as a function of Li⁺ concentration is qualitatively very similar for the different dialkyl- and diacylphosphatidylserines studied, and is analogous to the results obtained on pH titration. After an initial small increase in transition temperature, that is caused by coulombic ion binding and concomitant surface charge neutralization, a much larger increase in the chain-melting transition temperature occurs, caused by dehydration of the lipid, as a consequence of a further stereospecific ion binding. This suggests that Li⁺ and H⁺ have similar binding sites on the PS headgroup.     

Electrochemical study of ion channel behavior in incorporated poly L-glutamate bilayer lipid membranes

The lipid layer membranes were fabricated on the glassy carbon electrode (GC) and demonstrated to be bilayer lipid membranes by impedance spectroscopy. The formation of incorporated poly L-glutamate bilayer lipid membrane was achieved. The ion channel behavior of the incorporated poly L-glutamate membrane was determined. When the stimulus calcium cations were added into the electrolyte, the ion channel was opened immediately and exhibited distinct channel current. Otherwise, the ion channel was closed. The cyclic voltammogram at the GC electrode coated with incorporated poly L-glutamate DMPC film response to calcium ion is very fast compared with that at the GC electrode coated only with DMPC film. Ion channel current is not dependent on the time but on the concentration of calcium. The mechanism of the ion channel formation was investigated.     

Human pharmacokinetics of ethynyl estradiol 3-sulfate and 17-sulfate

Pharmacokinetic parameters of ethynyl estradiol 3-sulfate (EE-3) and 17-sulfate (EE-17) were estimated. Each sulfate was administered orally and intravenously to five ovariectomized volunteer women. Blood samples were taken over a period of 24 h. Radioimmunoassay for free and sulfoconjugated ethynyl estradiol (EE) was performed. The analysis of the plasma concentrations obtained after administration of EE-3 and EE-17 indicates significant differences in their pharmacokinetic profiles. EE-3 is cleared more rapidly from the central compartment (systemic circulation), which may indicate that differences in protein binding, tissue binding, metabolism, and distribution exist between EE-3 and EE-17. It has been suggested that these conjugates are a slow-release reservoir for maintenance of blood levels of free EE itself. However, previous studies in baboons have shown that the half-lives of the free and sulfoconjugated EE are similar (ranging from 8.8 to 11.2 h), which is not consistent with this hypothesis. The t1/2 beta (mean 9.28 h) of the 17-sulfate after IV administration was almost identical in women and baboons, and similar to the t1/2 beta of free EE, confirming the previous observation. Only 3.4% of IV and 11.4% of the orally administered 17-sulfate appeared in the blood as free EE; with the 3-sulfate, the conversions were 13.7 and 20.7%, respectively, suggesting that these sulfates are not important slow-release reservoirs. The similarity of pharmacokinetic parameters between women and baboons suggests that this species of nonhuman primate is, in important respects, a suitable animal model for clinical pharmacology.     

Effects of pentanol isomers on the phase behavior of phospholipid bilayer membranes

Differential scanning calorimetry (DSC) was used to analyze the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of pentanol isomers. The concentration of each pentanol isomer needed to induce the interdigitated phase was determined by the appearance of a biphasic effect in the main transition temperatures, the onset of a hysteresis associated with the main transition from the gel-to-liquid crystalline phase, and the disappearance of the pretransition. Lower threshold concentrations were found to correlate with isomers of greater alkyl chain length while branching of the alkyl chain was found to increase biphasic behavior. The addition of a methyl group to butanol systems drastically decreased threshold concentrations. However, as demonstrated in the DPPC/neopentanol system, branching of the alkyl chain away from the -OH group lowers the threshold concentration while maintaining a biphasic effect.     

Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The beta-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D2O for days and is assigned to the transmembrane beta-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D2O are assigned to the extracellular and periplasmic loops. The two-dimensional 1H/15N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5 degrees tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to alpha-helical membrane proteins.     

NMR structural studies of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes

The β-barrels found in the outer membranes of prokaryotic and eukaryotic organisms constitute an important functional class of proteins. Here we present solid-state NMR spectra of the bacterial outer membrane protein OmpX in oriented lipid bilayer membranes. We show that OmpX is folded in both glass-supported oriented lipid bilayers and in lipid bicelles that can be magnetically oriented with the membrane plane parallel or perpendicular to the direction of the magnetic field. The presence of resolved peaks in these spectra demonstrates that OmpX undergoes rotational diffusion around an axis perpendicular to the membrane surface. A tightly hydrogen-bonded domain of OmpX resists exchange with D₂O for days and is assigned to the transmembrane β-barrel, while peaks at isotropic resonance frequencies that disappear rapidly in D₂O are assigned to the extracellular and periplasmic loops. The two-dimensional ¹H/¹⁵N separated local field spectra of OmpX have several resolved peaks, and agree well with the spectra calculated from the crystal structure of OmpX rotated with the barrel axis nearly parallel (5° tilt) to the direction of the magnetic field. The data indicate that it will be possible to obtain site-specific resonance assignments and to determine the structure, tilt, and rotation of OmpX in membranes using the solid-state NMR methods that are currently being applied to α-helical membrane proteins.     

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.     

Dynamical behavior of lipid bilayer membranes for taste substances under random membrane-potential fluctuations

The dynamical behavior of the lipid bilayer membranes was experimentally studied under superposition of random or periodic membrane-potential fluctuations. The analysis of the mutual information has revealed that, in less than 10 Hz of random fluctuations, each of the time series of the mutual information of the transmembrane current for the five chemical substances (taste substances) has its inherent pattern, but not in a periodic fluctuation. On the other hand, the analysis of the power spectrum of the frequency could not distinguish those five basic taste substances in both random and periodic fluctuations. We provide the new detection idea of chemical substances by random fluctuations.     

Lipid-polypeptide interactions in bilayer lipid membranes

Summary The modifications of the electrical properties of bilayer lipid membranes (BLM) composed of cholesterol and an ionic surfactant upon interaction with charged polypeptides were studied. The addition of 10^–8 m polylysine (Ps⁺) to one side of anionic cholesterol dodecylphosphate BLM increases the specific membrane conductance over 1000-fold (from 10^–8 to 10^–5 mho/cm²) and develops a cationic transmembrane potential larger than 50 mV. This potential is reverted by addition of polyanions such as RNA, polyglutamic or polyadenilic acid to the same side on which Ps⁺ is present, by addition of Ps⁺ to the opposite side, or by addition of trypsin to either side. Both conductance and potential changes are hindered by increasing the ionic strength or by raising the pH of the bathing medium, disappearing above pH 11.5 where it is known that Ps⁺ folds into an -helix. The interaction of polyglutamic acid (PGA) with a cationic cholesterol-hexadecyltrimethylammonium bromide BLM results in increased membrane conductance and development of an anionic transmembrane potential which is reverted by addition of polycations to the same aqueous phase where PGA is present. Addition of either Ps⁺ or PGA to one or both sides of a neutral BLM composed of 7-dehydrocholesterol induces no significant change. The observations suggest the formation of a lipid polymer membrane resultant from the interaction, predominantly electrostatic, of the isolated components. The implications of these results are discussed in terms of the current models of membrane structure.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the interaction of hydrated N-palmitoylgalactosylsphingosine (NPGS) and dipalmitoylphosphatidylcholine (DPPC). For mixtures containing less than 23 mol% NPGS, complete miscibility of NPGS into hydrated DPPC bilayers is observed in both the bilayer gel and liquid-crystal phases. X-ray diffraction data demonstrate insignificant differences in the DPPC-bilayer gel-phase parameters on incorporation of up to 23 mol% NPGS. At greater than 23 mol% NPGS, additional high-temperature transitions occur, indicating phase separation of cerebroside. For these cerebroside concentrations, at 20 degrees C, x-ray diffraction shows two lamellar phases, hydrated DPPC-NPGS gel bilayers (d = 64 A) containing 23 mol% NPGS, and NPGS "crystal" bilayers (d = 55 A). On heating to temperatures greater than 45 degrees C, the mixed DPPC-NPGS bilayer phase undergoes chain melting, and on further increasing the temperature progressively more NPGS is incorporated into the liquid-crystal DPPC-NPGS bilayer phase. At temperatures greater than 82 degrees C (the transition temperature of hydrated NPGS), complete lipid miscibility is observed at all DPPC/NPGS molar ratios.     

Interactions between digitonin and bilayer membranes

The morphology of interactions between digitonin and cholesterol has been investigated. When precipitated from ethanolic solutions, digitonin-cholesterol complexes form in flat lamellar sheets. In contrast, when the complex is formed in a bilayer membrane, the membrane is deformed into corrugations of hemitubules. The polarity of the deformations formed in bilayer membranes is highly correlated with the direction of entry of digitonin into the membrane. We suggest that the morphology of digitonin/cholesterol hemitubules is dependent upon the complex being formed within a bilayer and, in addition, is not correlated with asymmetry of cholesterol concentration across the membrane.     

Conformational partitioning of the fusion peptide of HIV-1 gp41 and its structural analogs in bilayer membranes

Experiments have shown that the ability of the HIV-1 virus to infect cells can be greatly diminished by deactivation of the N-terminal (fusion) peptide of its glycoprotein gp41. Deactivation can be achieved by the deletion of several amino acid residues, or replacement of a hydrophobic residue with a polar residue, to form mutant variants of the wild-type peptide. We report Monte Carlo simulation studies of a simplified peptide/membrane model, representing the interaction of an HIV-1 fusion peptide (FP) and four closely related mutagens with a lipid bilayer. In agreement with experimental results, we show that FP inserts deeply into the bilayer at approximately 40 degrees to the bilayer normal. We also show a previously unreported behavior of membrane peptides, namely their equilibrium partitioning between several distinct conformations within the bilayer. We quantify this partitioning behavior and characterize each conformation in terms of its geometry, energy, and entropy. The diminished ability of FP mutagens to hemolyse and aggregate red blood cells due to their partitioning into unfavorable conformations, is also discussed. Our analysis supports a negative curvature mechanism for red blood cell hemolysis by FP. We also suggest that the small repulsive forces between surface-adsorbed peptides in opposing membrane surfaces may block aggregation.