Lipid fluorination enables phase separation from fluid phospholipid bilayers

To probe the effect of lipid fluorination on the formation of lipid domains in phospholipid bilayers, several new fluorinated and non-fluorinated synthetic lipids were synthesised, and the extent of phase separation of these lipids from phospholipid bilayers of different compositions was determined. At membrane concentrations as low as 1% mol/mol, both fluorinated and non-fluorinated lipids were observed to phase separate from a gel-phase (solid ordered) phospholipid matrix, but bilayers in a liquid disordered state caused no phase separation; if the gel-phase samples were heated above the transition temperature, then phase separation was lost. We found incorporation of perfluoroalkyl groups into the lipid enhanced phase separation, to such an extent that phase separation was observed from cholesterol containing bilayers in the liquid ordered phase.     

Fluidity modulation of phospholipid bilayers by electrolyte ions: insights from fluorescence microscopy and microslit electrokinetic experiments

Fluidity and charging of supported bilayer lipid membranes (sBLMs) prepared from 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) were studied by fluorescence recovery after photobleaching (FRAP) and microslit electrokinetic measurements at varying pH and ionic composition of the electrolyte. Measurements in neutral electrolytes (KCl, NaCl) revealed a strong correlation between the membrane fluidity and the membrane charging due to unsymmetrical water ion adsorption (OH(-) ? H(3)O(+)). The membrane fluidity significantly decreased below the isoelectric point of 3.9, suggesting a phase transition in the bilayer. The interactions of both chaotropic anions and strongly kosmotropic cations with the zwitterionic lipids were found to be related with nearly unhindered lipid mobility in the acidic pH range. While for the chaotropic anions the observed effect correlates with the increased negative net charge at low pH, no correlation was found between the changes in the membrane fluidity and charge in the presence of kosmotropic cations. We discuss the observed phenomena with respect to the interaction of the electrolyte ions with the lipid headgroup and the influence of this process on the headgroup orientation and hydration as well as on the lipid packaging.     

Cholesterol supports headgroup superlattice domain formation in fluid phospholipid/cholesterol bilayers

Fluorescence and Fourier transform infrared (FTIR) spectroscopic techniques were used to explore the effect of added cholesterol on the composition-dependent formation of putative phospholipid headgroup superlattices in fluid 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers. Steady-state fluorescence anisotropy measurements of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) revealed significant dips at several POPE-to-phospholipid mole fractions (X(PE)'s) when the cholesterol-to-lipid mole fraction (X(CHOL)) was fixed at 0.00, 0.35, 0.40, and 0.50. Most of the observed dips occur at or close to critical X(PE)'s predicted by the Headgroup Superlattice (SL) model, suggesting that phospholipid headgroups of different structures tend to adopt regular distributions even in the presence of cholesterol. Time-resolved fluorescence anisotropy measurements revealed that DPH-PC senses a disordered and highly mobile microenvironment in the POPE/POPC/CHOL bilayers at those critical X(PE)'s, indicating that this probe may partition to defect regions in the bilayers. The presence of coexisting packing defect regions and regularly distributed SL domains is a key feature predicted by the Headgroup SL model. Importantly, probe-free FTIR measurements of acyl chain C-H, interfacial carbonyl, and headgroup phosphate stretching peak frequencies revealed the presence of abrupt changes at X(PE)'s close to those observed in the fluorescence data. When X(PE) was varied from 0.60 to 0.72 and X(CHOL) from 0.34 to 0.46, a clear dip at the lipid composition coordinates (X(PE), X(CHOL)) approximately (0.68, 0.40) was observed in the three-dimensional surface plots of DPH-PC anisotropy as well as the carbonyl and phosphate stretching frequencies. The critical X(CHOL) at 0.40 agrees with the Cholesterol SL model, which assumes that cholesterol and phospholipid form SL domains at the lipid acyl chain level. In conclusion, this study provides evidence that cholesterol supports formation of phospholipid headgroup SLs in fluid state ternary lipid bilayers. The feasibility of the parallel existence of SLs at the lipid headgroup and acyl chain levels supports the relevance of the lipid SL model for the membranes of eukaryotic cells that typically contain significant amounts of cholesterol. We speculate that lipid SL formation may play a central role in the regulation of membrane lipid compositions, maintenance of organelle boundaries, and other crucial phenomena in those cells.     

A class of supported membranes: formation of fluid phospholipid bilayers on photonic band gap colloidal crystals

We report the formation of a new class of supported membranes consisting of a fluid phospholipid bilayer coupled directly to a broadly tunable colloidal crystal with a well-defined photonic band gap. For nanoscale colloidal crystals exhibiting a band gap at the optical frequencies, substrate-induced vesicle fusion gives rise to a surface bilayer riding onto the crystal surface. The bilayer is two-dimensionally continuous, spanning multiple beads with lateral mobilities which reflect the coupling between the bilayer topography and the curvature of the supporting colloidal surface. In contrast, the spreading of vesicles on micrometer scale colloidal crystals results in the formation of bilayers wrapping individual colloidal beads. We show that simple UV photolithography of colloidal crystals produces binary patterns of crystal wettabilities, photonic stopbands, and corresponding patterns of lipid mono- and bilayer morphologies. We envisage that these approaches will be exploitable for the development of optical transduction assays and microarrays for many membrane-mediated processes, including transport and receptor-ligand interactions.     

Cation and anion responses of zwitterion phospholipid bilayers

The electrochemical behavior of zwitterion phospholipid bilayers as determined with the help of membrane potential data, is examined. The synthetic DL-β-γ-dipalmitoyl-α-phosphatidylcoline bilayer shows at 30°C nernstian and sub-nernstian anion responses which depend on the cation present in the electrolytes (LiCl, NaCl, KCl, CsCl, CaCl₂, CeCl₃), and in some cases on the concentration of the solutions considered. On the other hand, nernstian cation responses have been observed in non-isothermal membrane-potential measurements performed on the same lipidic films. These experimental results are explained on the assumption that, from a potentiometric viewpoint the phospholipid membrane behaves as a neutral carrier membrane electrode. Accordingly, the form of the potential equations, and then the anion response of the bilayer, follows from the chemistry of the phospholipid molecules.     

Penetration and saturation of lysozyme in phospholipid bilayers

We show that the combination of X-ray reflectivity, tryptophan fluorescence spectrum, and fluorescence quenching by bromine provides a useful tool to probe the location of lysozyme in lipid bilayers. To this end, we prepare lamellar complexes composed of phospholipids and lysozyme on solid surfaces using a solution-casting method. The proteins lie spontaneously between adjacent bilayers in the complexes. The results indicate that lysozyme may penetrate into the lipid bilayers. But the penetration depth is very shallow, and the tryptophan residues do not penetrate beyond the interface between the hydrocardon core and the headgroup region of the lipid bilayer. The penetration becomes saturated when more proteins are incorporated into the lamellar complex. The excess proteins stay in the interlamellar aqueous layers.     

New GHK hydrophobic derivatives: interaction with phospholipid bilayers

Three hydrophobic derivatives of GHK peptide containing either N-terminal hexanoyl, decanoyl or myristoyl acyl moieties were synthesized. The binding of these peptidolipids to phospholipid bilayers as well as their hemolytic activity were determined. Moreover, the influence of these peptidolipids on several physicochemical properties of liposomes was studied. Binding experiments indicate a high affinity of these peptidolipids for lipids ordered in liposomes. Nevertheless, this interaction does not promote the release of entrapped carboxyfluorescein. Experiments carried out by the asymmetric membrane method (NBD-PE/dithionite) and quenching studies (PC-pyrene/KI) indicate that this association has a protective effect suggesting that the hydrophobic moiety inserts in the external part of the bilayer and the peptide chain remains protruding from the surface hindering the entrance or the approach of reactants to it. The microviscosity of DPPC bilayers determined using TMA-DPH as fluorescent marker was not affected by the presence of peptidolipids. Besides, results indicate that myristoyl-GHK produces total hemolysis at 2.5 × 10⁻⁴ M but decanoyl and hexanoyl derivatives at 5 × 10⁻⁴ M induce only 10% of hemolysis.     

Direct photochemical patterning and refunctionalization of supported phospholipid bilayers

A wet photolithographic route for micropatterning fluid phospholipid bilayers is demonstrated in which spatially directed illumination by short-wavelength ultraviolet radiation results in highly localized photochemical degradation of the exposed lipids. Using this method, we can directly engineer patterns of hydrophilic voids within a fluid membrane as well as isolated membrane corrals over large substrate areas. We show that the lipid-free regions can be refilled by the same or other lipids and lipid mixtures which establish contiguity with the existing membrane, thereby providing a synthetic means for manipulating membrane compositions, engineering metastable membrane microdomains, probing 2D lipid-lipid mixing, and designing membrane-embedded arrays of soluble proteins. Following this route, new constructs can be envisaged for high-throughput membrane proteomic, biosensor array, and spatially directed, aqueous-phase material synthesis.     

Stability and permeability of amphiphile bilayers

In this review the rupture and permeability of bilayers are considered on the basis of a mechanism of the formation of microscopic holes as fluctuations in the bilayers. The hole formation is treated as a nucleation process of a new phase in a two-dimensional system with short-range intermolecular forces. Free rupture and deliberate rupture (by α-particles) of foam bilayers (Newtonian black films) are discussed. A comparison is made between the rupture of foam and emulsion bilayers. Experimental methods for obtaining foam and emulsion bilayers from thin liquid films are considered. Methods for investigating the stability and permeability of foam bilayers, which are based on a microscopic model allowing the use of amphiphile solutions with very low concentrations, are described. Experimental dependences of the lifetime of bilayers, the probability of observing the foam bilayer in a foam film, the gas permeability of bilayers, etc. on the concentration of amphiphile molecules in the solution are reported. The influence of temperature and external impact (e.g. α-particle irradiation) have also been experimentally studied. A good agreement between theory and experiment is established, allowing determination of several characteristics of foam and emulsion bilayers obtained from ionics or non-ionics: the specific edge energy of bilayer holes, equilibrium surfactant concentration below which the bilayer is thermodynamically metastable, work for the formation of a nucleus hole, number of vacancies in the nucleus hole, coefficient of gas diffusion through the bilayer, etc. On the basis of the effect of temperature on the rupture of foam bilayers the binding energy of a surfactant molecule in the bilayer is determined. The adsorption isotherm of surfactant vacancies in the foam bilayer is obtained which shows a first-order phase transition. Some applications to scientific, technological and medical problems are considered. The foam bilayer is used as a model for investigating short-range forces in biological structures, the interaction between membranes and cell fusion. It is also shown that the foam bilayer is a suitable model for studying the alveolar surface and stability. On that basis a clinical diagnostic method is developed for assessment of the human foetal lung maturity.     

Curvature effect on the structure of phospholipid bilayers

High-resolution small-angle X-ray scattering (SAXS), complemented by small-angle neutron scattering (SANS) and dynamic light scattering (DLS) experiments, was used to study the effect of curvature on the bilayer structure of dioleoyl-phosphatidylcholine (DOPC) and dioleoyl-phosphatidylserine (DOPS) unilamellar vesicles (ULVs). Bilayer curvature, as a result of finite vesicle size, was varied as a function of vesicle radius and determined by DLS and SANS measurements. Unilamellarity of large DOPC ULVs was achieved by the addition of small amounts (up to 4 mol %) of the charged lipid, DOPS. A comparison of SANS data over the range of 0.02 < q <0.2 A-1 indicated no change in the overall bilayer thickness as a function of ULV diameter (620 to 1840 A). SANS data were corroborated by high-resolution (0.06 < q <0.6 A-1) SAXS data for the same diameter ULVs and data obtained from planar samples of aligned bilayers. Both the inner and outer leaflets of the bilayer were found to be indistinguishable. This observation agrees well with simple geometric models describing the effect of vesicle curvature. However, 1220-A-diameter pure DOPS ULVs form asymmetric bilayers whose structure can most likely be rationalized in terms of geometrical constraints coupled with electrostatic interactions, rather than curvature alone.     

Spectroscopic probing of ortho-nitrophenol localization in phospholipid bilayers

In the present study we estimated the localization of ortho-nitrophenol (ONP) within model membranes through its efficiency to quench and to modify the anisotropy of DPH and TMA-DPH fluorescence. These fluorescent probes are known to sense the hydrocarbon core and the polar head group region of membranes, respectively. TMA-DPH fluorescence in MLVs was more efficiently quenched than DPH (K(q,TMA-DPH)=2.36 and K(q,DPH)=1.07 mM ns(-1)). Moreover, these results demonstrated the interfacial localization of ONP and may contribute to understand membrane-mediated mechanisms of ONP-induced toxicity and the behavior of ONP as a product of several enzymatic reactions occurring in the presence of lipid-water interfaces.     

Solubilization of unilamellar phospholipid bilayers by nonionic surfactants

The mechanisms governing the solubilization of neutral or electrically charged unilamellar liposomes by a series of octylphenol polyethoxylated surfactants (average of ethylene oxide units between 8.5 and 20.0) were investigated. Solubilization was detected as a decrease in light-scattering of liposome suspensions. To this end, in accordance with the nomenclature adopted by Lichtenberg, three parameters were considered as corresponding to the effective surfactant/lipid molar ratios (Re) at which the surfactant (a) saturated the liposomesRe _sat; (b) resulted in a 50% solubilization of vesiclesRe _50% and (c) led to a total solubilization of liposomesRe _sol. These parameters, corresponded to theRe at which light scattering starts to decrease, reaches 50% of the original value and shows no further decrease.It is noteworthy that theRe _sat parameter decreases as the EO contents or the surfactant critical micellar concentration (CMC) increases. However, theRe _50% and theRe _sol parameters show the lowest values for the surfactant with 12.5 EO units in its molecular structure regardless of the electrical charge of the lipid bilayers. As a consequence, these last parameters are not linearly correlated with the CMC of these surfactants. The CMC values of the surfactant/lipid systems at 0.5 mM lipid concentration corresponded in all cases to the surfactant concentration at which liposomes were saturated by surfactants (S _sat).     

515—Cation and anion responses of zwitterion phospholipid bilayers

The electrochemical behavior of zwitterion phospholipid bilayers as determined with the help of membrane potential data, is examined. The synthetic DL-β,γ-dipalmitoyl-α-phosphatidylcoline bilayer shows at 30°C nernstian and sub-nernstian anion responses which depend on the cation present in the electrolytes (LiCl, NaCl, KCl, CsCl, CaCl₂, CeCl₃), and in some cases on the concentration of the solutions considered. On the other hand, nernstian cation responses have been observed in non-isothermal membrane-potential measurements performed on the same lipidic films. These experimental results are explained on the assumption that, from a potentiometric viewpoint the phospholipid membrane behaves as a neutral carrier membrane electrode. Accordingly, the form of the potential equations, and then the anion response of the bilayer, follows from the chemistry of the phospholipid molecules.     

Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers

The lipid membranes found in archaea have high bilayer stability and low permeability. The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains. In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively. We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers. We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC. The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data.     

Effect of buffer gas on the fluorescence yield of trapped gas-phase ions

We investigated the dependence of three different gases, helium, argon, and nitrogen, on the fluorescence signal intensity of rhodamine 6G cations in the gas phase. The method is based on laser-induced fluorescence of ions trapped in a Fourier transform ion cyclotron mass spectrometer. We found that the use of helium results in the highest fluorescence signal, while no fluorescence was detected when using argon under the same conditions.     

The orientational ordering of benzyl alcohol in phospholipid bilayers

The α-methylene segment of benzyl alcohol has been deuterated at one site and the proton—deuterium dipole spin interaction measured for this compound dissolved in bilayers of dimyristoylphosphatidylcholine. These data, when combined with measured deuterium quadrupole interactions at other sites, allow for a more complete understanding of the orientational ordering and conformation of benzyl alcohol in the bilayers. Models for the rotary motion of the ring are discussed.     

Interaction of amphipathic model lipopeptides with phospholipid bilayers.

In order to investigate the conformation and localization of lipopeptides in lipid bilayers, a basic model peptide with a long alkyl chain, Ac-Ser-Val-Lys-Amy-Ser-Trp-Lys-Val-NHCH3 Amy-1; Amy = alpha-aminomyristic acid) was synthesized. Its interaction with neutral and acidic phospholipid bilayers was studied by circular dichroism (CD) spectroscopy, dye leakage and fluorescence measurements. Another peptide, Ac-Leu-Ala-Arg-Leu-Trp-Amy-Arg-Leu-Leu-Ala-Arg-Leu-NHCH3 (Amy-2), which was prepared previously, was used for comparison. The CD data indicated that Amy-1 took a beta-turn and/or a beta-structure in the absence and presence of liposomes. Amy-2 formed a beta-structure in aqueous solution and an alpha-helical structure in liposomes. The dye leakage ability of Amy-1 was much weaker than that of Amy-2. Fluorescence spectroscopic data suggest that the peptides are immersed in lipid bilayers. Based on these results, discussion is made in terms of localization of the peptides in lipid bilayers.     

Interface water dynamics and porating electric fields for phospholipid bilayers

Lipid bilayers, normally a barrier to charged species and large molecules, are permeabilized by electric fields, a phenomenon exploited by cell biologists and geneticists for porating and transfecting cells and tissues. Recent molecular simulation studies have advanced our understanding of electroporation, but the relative contributions of atomically local details (interface water and headgroup dipole and counterion configurations) and medium- and long-range electrostatic gradients and changes in membrane structural shifts to the initiating conditions and mechanisms of pore formation remain unclear. Molecular dynamics simulations of electroporation in several lipid systems presented here reveal the effects of lipid hydrocarbon tail length and composition on the magnitude of the field required for poration and on the location of the initial sites of field-driven water intrusion into the bilayer. Minimum porating external fields of 260 mV nm(-1), 280 mV nm(-1), 320 mV nm(-1), and 380 mV nm(-1) were found for 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), and 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC) bilayers, respectively, and correlated most strongly with the bilayer thickness. These phospholipid systems share several common features including a wide, dynamic distribution of the headgroup dipole angle with the bilayer normal ranging from 0 to 155 degrees that is only slightly shifted in a porating electric field, and similar electric field-induced shifts in water dipole orientation, although the mean water dipole moment profile at the aqueous-membrane interface is more sensitive to the electric field for DOPC than for the other phospholipids. The location of pore initiation, at the anode- or cathode-facing leaflet, varies with the composition of the bilayer and correlates with a change in the polarity of the localized electric field at the interface.     

Electron paramagnetic resonance studies of magnetically aligned phospholipid bilayers utilizing a phospholipid spin label

X-band electron paramagnetic resonance (EPR) spectroscopy was used to study the structural and dynamic properties of magnetically aligned phospholipid bilayers utilizing a variety of phosphocholine spin labels (PCSL) as a function oftemperature. 1-Palmitoyl-2-[n-(4,4-dimethyloxazolidine-N-oxyl)stearoyl]-sn-glycero-3-phosphocholine (n-PCSL) in which a nitroxide group was attached to the different acyl chain positions of the phospholipid (n = 5, 7, 12, and 14) were used as an EPR spin probe to investigate magnetically aligned phospholipid bilayers from the plateau (near to the headgroup) region to the end of the acyl chain (center of the bilayers). The addition of certain types of paramagnetic lanthanide ions changes the overall magnetic susceptibility anisotropy tensor of the bicelles, such that the bicelles flip with their bilayer normal either parallel or perpendicular to the magnetic field. The present study reveals for the first time that, in the case of the n-PCSL, the bilayer normal is aligned parallel and perpendicular to the magnetic field in the presence of lanthanide ions having positive delta(chi) (e.g., Tm3+) and negative delta(chi) (e.g., Dy3+), respectively. The magnetic alignment of the bilayers and the corresponding segmental molecular order parameter, S(mol), were investigated as a function of the temperature. The S(mol) values decrease in the following order, 5-PCSL > 7-PCSL > 12-PCSL > 14-PCSL, for the magnetically aligned phospholipid bilayers. Also, the variable temperature study indicates that, by increasing the temperature, the order parameters S(mol) decreased for all the n-PCSLs. The results indicate that magnetically aligned phospholipid bilayers represent an excellent model membrane system for X-band EPR studies.