Binary Lipid Mixtures Research Articles

Effects of Low Concentrations of Docosahexaenoic Acid on the Structure and Phase Behavior of Model Lipid Membranes.

In this paper we report an X-ray diffraction study on the phase behavior of binary lipid mixtures of 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (DHA-PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at low concentrations below 5.0 mol% DHA-PE. Our results show that DHA-PE induces phase separation into a DHA rich liquid crystalline (Lα) phase and a DHA poor gel (Lβ′) phase at overall DHA-PE concentrations as low as 0.1 mol%. In addition, we find that the structure of the Lβ′ phase, from which the DHA-PE molecules are largely excluded, is modified in the phase-separated state at low DHA-PE concentrations, with a decrease in bilayer thickness of 1.34 nm for 0.1 mol% at room temperature, compared to pure DPPC bilayers. This result is contrary to that seen in similar studies on mono-unsaturated lipids where an increase in bilayer thickness is observed. The surprising effect of such low DHA-PE concentrations on membrane structure may be important in understanding the role of highly polyunsaturated lipids in biological membrane-based structures and similar artificial surfactant systems.

Investigation of Binary Lipid Mixtures of a Three-Chain Cationic Lipid with Phospholipids Suitable for Gene Delivery.

In the present work, we characterize binary lipid mixtures consisting of a three-chain amino-functionalized cationic lipid (DiTT4) with different phospholipids, namely, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The mixing behavior was investigated by differential scanning calorimetry (DSC). Additionally, aqueous dispersions of the binary mixtures were characterized by means of dynamic light scattering (DLS), laser Doppler electrophoresis, and transmission electron microscopy (TEM) to get further information about particle size, charge, and shape. The complex formation between different binary lipid mixtures and plasmid DNA (pDNA) was investigated by zeta-(ζ)-potential (laser Doppler electrophoresis) and DLS measurements, and the lipid/DNA complexes (lipoplexes) were screened for efficient DNA transfer (transfection) in cell culture. Finally, efficient lipid compositions were investigated with respect to serum stability. This work provides a detailed characterization of the cationic lipid mixtures as foundation for further research. Efficient gene transfer in the presence of serum was demonstrated for selected lipoplexes showing their capability to be used as high-potency gene delivery vehicles.

Citrem modulates internal nanostructure of glyceryl monooleate dispersions and bypasses complement activation: Towards development of safe tunable intravenous lipid nanocarriers

Lyotropic non-lamellar liquid crystalline (LLC) aqueous nanodispersions hold a great promise in drug solubilization and delivery, but these nanosystems often induce severe hemolysis and complement activation, which limit their applications for safe intravenous administration. Here, we engineer and characterize LLC aqueous nanodispersions from a binary lipid mixture consisting of 2,3-dihydroxypropyl oleate (glyceryl monooleate) and medium-chain triglycerides with tunable internal nanostructures and improved hemocompatibility controlled by citrem as stabilizer. Citrem, in a concentration-dependent manner, modulates the internal nanostructure of LLC dispersions from a biphasic H2/L2 feature to a neat L2 phase, where the latter resembles “thread-like” swollen micelles. Citrem stabilization totally overcomes hemolysis and complement activation, thus realizing the potential of the engineered LLC aqueous nanodispersions for exploitation in intravenous delivery of drugs and contrast agents. From the Clinical EditorThe complement system often gets activated after intravenous injection of nano drug-carriers. This may result in detrimental systemic effects. The authors described in this article the use of citrem as a stabilizing agent and showed the ability of this agent to abolish complement activation. Hence, citrem may prove to be an important component of tunable LLC nanocarriers that may be useful in future clinical setting.

Enzymatic action of phospholipase A2 on liposomal drug delivery systems

The overexpression of secretory phospholipase A2 (sPLA2) in tumors has opened new avenues for enzyme-triggered active unloading of liposomal antitumor drug carriers selectively at the target tumor. However, the effects of the liposome composition, drug encapsulation, and tumor microenvironment on the activity of sPLA2 are still not well understood. We carried out a physico-chemical study to characterize the sPLA2-assisted breakdown of liposomes using dye-release assays in the context of drug delivery and under physiologically relevant conditions. The influence of temperature, lipid concentration, enzyme concentration, and drug loading on the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm=42°C) liposomes with snake venom sPLA2 was investigated. The sensitivity of human sPLA2 to the liposome composition was checked using binary lipid mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) phospholipids with C14 and C16 acyl chains. Increasing temperature (36–41°C) was found to mainly shorten the enzyme lag-time, whereas the effect on lipid hydrolysis rate was modest. The enzyme lag-time was also found to be inversely dependent on the lipid-to-enzyme ratio. Drug encapsulation can alter the hydrolysis profile of the carrier liposomes. The activity of human sPLA2 was highly sensitive to the phospholipid acyl-chain length and negative surface charge density of the liposomes. We believe our work will prove useful for the optimization of sPLA2-susceptible liposomal formulations as well as will provide a solid ground for predicting the hydrolysis profile of the liposomes in vivo at the target site.

Buffer-induced swelling and vesicle budding in binary lipid mixtures of dioleoylphosphatidylcholine:dioleoylphosphatidylethanolamine and dioleoylphosphatidylcholine:lysophosphatidylcholine using small-angle X-ray scattering and ³¹P static NMR.

A large variety of data exists on lipid phase behavior; however, it is mostly in nonbuffered systems over nonbiological temperature ranges. We present biophysical data on lipid mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and lysophosphatidylcholine (LysoPC) examining their behaviors in excess water and buffer systems over the temperature range 4-34 °C. These mixtures are commonly used to investigate the effects of spontaneous curvature on integral membrane proteins. Using small-angle X-ray scattering (SAXS) and (31)P NMR, we observed lamellar and vesicle phases, with the buffer causing an increase in the layer spacing. Increasing amounts of DOPE in a DOPC bilayer decreased the layer spacing of the mesophase, while the opposite trend was observed for increasing amounts of LysoPC. (31)P static NMR was used to analyze the DOPC:LysoPC samples to investigate the vesicle sizes present, with evidence of vesicle budding observed at LysoPC concentrations above 30 mol %. NMR line shapes were fitted using an adapted program accounting for the distortion of the lipids within the magnetic field. The distortion of the vesicle, because of magnetic susceptibility, varied with LysoPC content, and a discontinuity was found in both the water and buffer samples. Generally, the distortion increased with LysoPC content; however, at a ratio of DOPC:LysoPC 60:40, the sample showed a level of distortion of the vesicle similar to that of pure DOPC. This implies an increased flexibility in the membrane at this point. Commonly, the assumption is that for increasing LysoPC concentration there is a reduction in membrane tension, implying that estimations of membrane tension based on spontaneous curvature assumptions may not be accurate.

Membrane Resistance to Detergent-Induced Solubilization as a Matter of Physical Phase in Binary Lipid Mixtures

Molecular biology protocols routinely use detergents for extraction of membrane or intracellular components. However, detergent-induced solubilization usually results in the presence of membrane debris, with similar composition to membrane rafts and liquid-ordered (Lo) phase. In the present work we have studied the solubilization by the detergent Triton X-100 (TX-100) of membranes composed of different lipid mixtures and phases. The membrane compositions evaluated were palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:cholesterol (7:3) (liquid disordered phase, Ld), Sphingomyelin (SM, Gel phase) and SM:cholesterol (7:3) (Lo phase). The solubilization was followed by optical microscopy of giant unilamellar vesicles (GUVs), showing that POPC and SM membranes are totally solubilized by TX-100, while POPC:cholesterol membranes are partially solubilized and SM:cholesterol membranes are completely insoluble. The incorporation of TX-100 in GUVs was measured trough area increase for vesicles in fluid phases, showing that for POPC membranes, the addition of cholesterol diminishes the incorporation of TX-100 and for vesicles of SM:cholesterol the area increase very low. Light scattering of large unilamellar vesicles (LUVs) titrated with TX-100 confirmed the results of optical microscopy. The isothermal titration calorimetry (ITC) of LUVs with detergent showed that the addition of cholesterol diminishes the partition coefficient of TX-100 in both POPC and SM membranes. In addition, enthalpic changes are observed in the same detergent-lipid molar ratios associated with the onset and completion of the solubilization process. Differential scanning calorimetry of SM LUVs with detergent show detergent-induced fluidification of the gel phase, which could account for part of the large endothermic contribution measured with ITC. Our result suggests that cholesterol increases membrane resistance to detergent-induced solubilization and confirmed that the Lo phase is virtually insoluble. Supported by: FAPESP and CNPq

Single Molecule Diffusion Studies of PTEN: Insights into Membrane Binding

PTEN, a tumor suppressor gene that encodes a dual specificity phosphatase that dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3), is one of the most frequent genes deleted or mutated in a wide variety of tumors. PTEN acts as an antagonist to phosphoinositide-3-kinase (PI3K) signaling, thereby affecting various cellular processes such as cell proliferation and survival. The activity of PTEN is regulated by dynamic shuttling between the cytoplasm and the plasma membrane. The membrane association of PTEN strongly depends on the composition and lateral distribution of the lipids in the membrane. Several biophysical techniques have been used to characterize PTEN-membrane interaction. Here, we use single-molecule total internal reflection fluorescence microscopy (TIRFM), which is a powerful tool to study the molecular mechanism of membrane targeted proteins. The single-molecule TIRFM allows us to observe single PTEN molecules as they dynamically associate/dissociate and laterally diffuse along the lipid bilayer membrane. PTEN lipid binding is investigated on supported lipid bilayers of binary and ternary lipid mixtures of PC with physiological relevant levels of PS, PI(4,5)P2 and PI. We tracked individual PTEN molecules and statistically determine the lateral diffusion and dwell time of PTEN on heterogeneous lipid bilayers. We find significant differences in PTEN dynamic behavior when bound to the different membrane environments. Furthermore, to gain insight into the molecular mechanisms of PTEN membrane association, we compared the lipid binding of wt PTEN, an N-terminally truncated PTEN-(Δ1–15 AA) that lacks the PI(4,5)P2 binding site, and the recently discovered, PTEN-L with a 173 AA N-terminal extension. We find profound differences in the dynamic behavior of these PTEN derivatives at the membrane. It has recently been suggested that PTEN associates as a dimer with the membrane. We find that the tendency to form membrane bound dimers varies among these PTEN variants.

Phase Transitions of Binary Lipid Mixtures: A Combined Study by Adiabatic Scanning Calorimetry and Quartz Crystal Microbalance with Dissipation Monitoring

The phase transitions of binary lipid mixtures are studied by a combination of Peltier-element-based adiabatic scanning calorimetry (pASC) and quartz crystal microbalance with dissipation monitoring (QCM-D). pASC, a novel type of calorimeter, provides valuable and unambiguous information on the heat capacity and the enthalpy, whereas QCM-D is proposed as a genuine way of determining phase diagrams by analysing the temperature dependence of the viscosity. Two binary mixtures of phospholipids with the same polar head and differing in the alkyl chain length, DMPC + DPPC and DMPC + DSPC, are discussed. Both techniques give consistent phase diagrams, which compare well with literature results, showing their capability to map the phase behaviour of pure lipids as well as lipid mixtures. This work can be considered as a departure point for further investigations on more complex lipid mixtures displaying relevant phases such as the liquid-ordered phase and solid-lipid interfaces with biologically functional importance.

Solubilization of binary lipid mixtures by the detergent Triton X-100: the role of cholesterol.

The solubilization of lipid bilayers of different composition and phase by the detergent Triton X-100 (Triton X-100) was investigated using optical and fluorescence microscopy of giant unilamellar vesicles (GUVs) and light scattering of large unilamellar vesicles (LUVs). The compositions explored were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in the liquid-disordered (Ld) phase, sphingomyelin (SM), in the gel phase, and binary mixtures of these phospholipids with 30 mol % cholesterol (chol), resulting in bilayers in the Ld and liquid-ordered (Lo) phases, respectively. We show that the phospholipid bilayers are completely soluble in TX-100, but optical microscopy reveals that whereas fluid POPC is gradually solubilized by TX-100, gel SM is first shattered in bilayer fragments. Incorporation of TX-100 in the membrane leads to increase in GUV area, which was quantified and expressed as bound detergent-to-lipid molar ratio. The partition of TX-100 in POPC is very high, decreases in POPC/chol, and is negligible in SM/chol. Fluorescence microscopy shows that TX-100 induces Lo/Ld phase separation in previously homogeneous POPC/chol GUVs, and insoluble bilayer fragments/vesicles are detected with optical microscopy and light scattering. Vesicles of SM/chol, in the Lo phase, are virtually insoluble in TX-100. Taken together, our results show that the presence of cholesterol is the origin of membrane resistance to solubilization, which depending on the specific lipid mixture can result in either partially (POPC/chol) or completely (SM/chol) insoluble mixtures.

Polymorphism and kinetic behavior of binary mixtures of triglycerides

The work is aimed at investigating the polymorphism and the phase transition kinetics of binary lipid mixtures with potential application in controlled drug delivery. The lipid systems, constituted of glyceryl tristearate (GTS) added with different amounts (1.0–7.5% w/w) of a medium-chain liquid triglyceride (C10–C12 acyl derivative – MCT), were studied by differential scanning calorimetry, by X-ray diffraction and hot-stage microscopy. The liquid lipid, although present in small amount, modified the thermal profile and the diffraction pattern of the systems, indicating that it promoted the formation of the GTS stable polymorph, β, during the re-solidification of the melted mixture. This promotion effect of MCT was concentration-dependent and evident for systems containing MCT>2.5%. Also the kinetics of transformation of GTS polymorphs was affected by the percentage of the liquid component. The α→β-transition was a biphasic process which for GTS–MCT mixture (99:1) superimposed that of pure GTS, while followed a different trend for systems containing percentages of MCT higher than 2.5.

A theoretical study of biological membrane response to temperature gradients at the single-cell level

Recent experimental studies provide evidence for the existence of a spatially non-uniform temperature field in living cells and in particular in their plasma membrane. These findings have led to the development of a new and exciting field: thermal biology at the single-cell level. Here, we examine theoretically a specific aspect of this field, i.e. how temperature gradients at the single-cell level affect the phase behaviour and geometry of heterogeneous membranes. We address this issue by using the Onsager reciprocal relations combined with a simple model for a binary lipid mixture. We demonstrate that even small temperature variations along the membrane may introduce intriguing phenomena, such as phase separation above the critical temperature and unusual shape response. These results also suggest that the shape of a membrane can be manipulated by dynamically controlling the temperature field in its vicinity. The effects of intramembranous temperature gradients have never been studied experimentally. Thus, the predictions of the current contribution are of a somewhat speculative nature. Experimental verification of these results could mark the beginning of a new line of research in the field of biological membranes. We report our findings with the hope of inspiring others to perform such experiments.

Biophysical investigations of the structure and function of the tear fluid lipid layers and the effect of ectoine. Part B: Artificial lipid films

The tear fluid lipid layer is present at the outermost part of the tear film which lines the ocular surface and functions to maintain the corneal surface moist by retarding evaporation. Instability in the structure of the tear fluid lipid layer can cause an increased rate of evaporation and thus dry eye syndrome. Ectoine has been previously shown to fluidize lipid monolayers and alter the phase behavior. In the current study we have investigated the effect of ectoine on the artificial tear fluid lipid layer composed of binary and ternary lipid mixtures of dipalmitoyl phosphatidylcholine (DPPC), cholesteryl esters and tri-acyl-glycerols. The focus of our study was mainly the structural and the biophysical aspects of the artificial tear fluid lipid layer using surface activity studies and topology analysis. The presence of ectoine consistently causes an expansion of the pressure–area isotherm indicating increased intermolecular spacing. The topology studies showed the formation of droplet-like structures due to the addition of ectoine only when tri-acyl-glycerol is present in the mixture of DPPC and chol-palmitate, similar to the natural meibomian lipids. Consequently, the hypothesis of an exclusion of tri/di-acyl-glycerol from the meibomian lipid film in the presence of ectoine in the subphase is confirmed. A model describing the effect of ectoine on meibomian lipid films is further presented which may have an application for the use of ectoines in eye drops as a treatment for the dry eye syndrome.

Demixing and crystallization of DODAB in DPPC-DODAB binary mixtures.

The crystallization mechanism of one lipid component within multicomponent lipid mixtures remains unclear. To shed light on this issue, we studied the demixing and crystallization behaviors of a binary lipid system using neutral dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DODAB) as model molecules. The results indicate that when DODAB is no more than equimolar (e.g., DPPC/DODAB = 2/1 and 1/1), DPPC is miscible with DODAB and hinders the crystallization of DODAB, and the samples undergo reversible gel-fluid phase transitions upon heating and cooling. However, when DODAB is dominant in the mixture (DPPC/DODAB = 1/2), cooling of the mixed fluid phase results in the formation of a DODAB-rich gel domain and a DPPC-DODAB mixed gel domain. Such phase-separated mixed gels can undergo further demixing and crystallization, producing a DODAB-rich crystalline domain and a DPPC-rich tilted gel domain upon prolonged (or plus low-temperature) incubation. Besides, evidence has been given that the crystallized DODAB-rich domain remains in the same lipid bilayer as the DPPC-rich domain. All the three binary lipid mixtures can hold large amounts of water in the lipid interlamellar regions, allowing the incorporation of a large number of water-soluble substances such as DNA or proteins, which can be used for the fabrication of functional biofilms and biomaterials. Influences of water content and salt concentration on the phase structures (e.g., repeat distances) of the binary mixtures have also been studied.

Sensing and Stiffening of Tubular Membranes by the Syndapin 1 FBAR

Bin-Amphiphysin-Rvs (BAR) domains are essential components of the cellular machinery responsible for membrane deformation and were found to be sensitive sensors of membrane curvature. One common feature of BAR domains is a curved shape which correlates with high membrane curvatures often found in cells. We focused on characterizing the FBAR domain of Syndapin-1 in vitro, given its medical relevance to neurological diseases, using tubular membranes pulled out of Giant Unilamellar Vesicles (GUVs). Specifically, GUVs were synthesized using either porcine brain lipid extracts or the more conventional, binary lipid mixtures to systematically test how lipid composition affects curvature sensing activity. Using optical trapping coupled with force-spectroscopy and confocal microscopy, we discovered an inverse relationship between the curvature sensing activity of the FBAR domain and its equilibrium concentration in solution. At high bulk concentrations of protein, we explicitly measured an increase in the tube's persistence length, which can be understood as mechanical stiffening of the tube. Lastly, we used force spectroscopy to accurately test the effect of the protein on membrane relaxation dynamics in real time.

Effect of flavonoids on the phase separation in giant unilamellar vesicles formed from binary lipid mixtures

Confocal fluorescence microscopy have been employed to investigate phase separation in giant unilamellar vesicles prepared from binary mixtures of unsaturated dioleoylphosphocholine with saturated phosphocholines or brain sphingomyelin in the absence and presence of the flavonoids, biochanin A, phloretin, and myricetin. It has been demonstrated that biochanin A and phloretin make uncolored domains more circular or eliminate visible phase separation in liposomes while myricetin remains the irregular shape of fluorescence probe-excluding domains. Influence of the flavonoids on the endotherms of liposome suspension composed of dioleoylphosphocholine and dimyristoylphosphocholine was investigated by the differential scanning calorimetry. Calorimetry data do not contradict to confocal imaging results.

Lithographically Defined Macroscale Modulation of Lateral Fluidity and Phase Separation Realized via Patterned Nanoporous Silica-Supported Phospholipid Bilayers

Using lithographically defined surfaces consisting of hydrophilic patterns of nanoporous and nonporous (bulk) amorphous silica, we show that fusion of small, unilamellar lipid vesicles produces a single, contiguous, fluid bilayer phase experiencing a predetermined pattern of interfacial interactions. Although long-range lateral fluidity of the bilayer, characterized by fluorescence recovery after photobleaching, indicates a nominally single average diffusion constant, fluorescence microscopy-based measurements of temperature-dependent onset of fluidity reveals a locally enhanced fluidity for bilayer regions supported on nanoporous silica in the vicinity of the fluid-gel transition temperature. Furthermore, thermally quenching lipid bilayers composed of a binary lipid mixture below its apparent miscibility transition temperature induces qualitatively different lateral phase separation in each region of the supported bilayer: The nanoporous substrate produces large, microscopic domains (and domain-aggregates), whereas surface texture characterized by much smaller domains and devoid of any domain-aggregates appears on bulk glass-supported regions of the single-lipid bilayer. Interestingly, lateral distribution of the constituent molecules also reveals an enrichment of gel-phase lipids over nanoporous regions, presumably as a consequence of differential mobilities of constituent lipids across the topographic bulk/nanoporous boundary. Together, these results reveal that subtle local variations in constraints imposed at the bilayer interface, such as by spatial variations in roughness and substrate adhesion, can give rise to significant differences in macroscale biophysical properties of phospholipid bilayers even within a single, contiguous phase.

Non-aqueous microchip electrophoresis for characterization of lipid biomarkers

In vivo measurements of lipid biomarkers are hampered by their low solubility in aqueous solution, which limits the choices for molecular separations. Here, we introduce non-aqueous microchip electrophoretic separations of lipid mixtures performed in three-dimensional hybrid nanofluidic/microfluidic polymeric devices. Electrokinetic injection is used to reproducibly introduce discrete femtolitre to picolitre volumes of charged lipids into a separation microchannel containing low (100 μM-10 mM) concentration tetraalkylammonium tetraphenylborate background electrolyte (BGE) in N-methylformamide, supporting rapid electro-osmotic fluid flow in polydimethylsiloxane microchannels. The quality of the resulting electrophoretic separations depends on the voltage and timing of the injection pulse, the BGE concentration and the electric field strength. Injected volumes increase with longer injection pulse widths and higher injection pulse amplitudes. Separation efficiency, as measured by total plate number, N, increases with increasing electric field and with decreasing BGE concentration. Electrophoretic separations of binary and ternary lipid mixtures were achieved with high resolution (R s ∼ 5) and quality (N > 7.7 × 10(6) plates m(-1)). Rapid in vivo monitoring of lipid biomarkers requires high-quality separation and detection of lipids downstream of microdialysis sample collection, and the multilayered non-aqueous microfluidic devices studied here offer one possible avenue to swiftly process complex lipid samples. The resulting capability may make it possible to correlate oxidative stress with in vivo lipid biomarker levels.

Rab1a and Rab5a preferentially bind to binary lipid compositions with higher stored curvature elastic energy

Rab proteins are a large family of GTP-binding proteins that regulate cellular membrane traffic and organelle identity. Rab proteins cycle between association with membranes and binding to RabGDI. Bound on membranes, each Rab has a very specific cellular location and it is this remarkable degree of specificity with which Rab GTPases recognize distinct subsets of intracellular membranes that forms the basis of their ability to act as key cellular regulators, determining the recruitment of downstream effectors to the correct membrane at the correct time. The molecular mechanisms controlling Rab localization remain poorly understood. Here, we present a fluorescence-based assay to investigate Rab GTPase membrane extraction and delivery by RabGDI. Using EGFP-Rab fusion proteins the amount of Rab:GDI complex obtained by GDI extraction of Rab proteins from HEK293 membranes could be determined, enabling control of complex concentration. Subsequent partitioning of the Rab GTPases into vesicles made up of artificial binary lipid mixtures showed for the first time, that the composition of the target membrane plays a key role in the localization of Rab proteins by sensing the stored curvature elastic energy in the membrane.

Phospholipid–Lactose Permease Interaction As Reported by a Head-Labeled Pyrene Phosphatidylethanolamine: A FRET Study

Förster resonance energy transfer (FRET) measurements were performed in preceding works to study the selectivity between a single-tryptophan mutant of lactose permease (LacY) of Escherichia coli (used as the donor) and phospholipid probes labeled with pyrene at the acyl chain moiety (used as the acceptor). In the present work, we report the results obtained by using the same LacY mutant (W151/C154G) and binary lipid mixtures of phosphatidylethanolamine (PE) differing in the acyl chain composition and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (3:1 mol/mol) doped with a phospholipid probe labeled with pyrene at the headgroup. The use of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(1-pyrenesulfonyl) ammonium salt (HPyr-PE), which bears two unsaturated acyl chains, enabled the investigation of the specific interaction between LacY and HPyr-PE. The main conclusions raised from our results suggest that (i) for phase-separated systems, LacY would be located in fluid domains nominally enriched in POPG, and if a given proportion of PE is present in this phase, it will be mainly located around LacY; and (ii) in the absence of phase separation, LacY is preferentially surrounded by PE and, in particular, seems to be sensitive to the lipid spontaneous curvature.

Detection of Nanosized Ordered Domains in DOPC/DPPC and DOPC/Ch Binary Lipid Mixture Systems of Large Unilamellar Vesicles Using a TEMPO Quenching Method

Nanosized ordered domains formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC/DPPC) and DOPC/cholesterol (Ch) liposomes were characterized using a newly developed (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) quenching method. The membrane fluidity of the DOPC/DPPC liposomes, evaluated by the use of 1,6-diphenyl-1,3,5-hexatriene (DPH), increased significantly above their phase-transition temperature. The fluorescence spectra of 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated the formation of an immiscible ordered phase in the DOPC/DPPC (50/50) liposomal membrane at 30 °C. The analysis of the membrane polarity indicated that the surface of the liquid-disordered phase was hydrated whereas that of the ordered phase was dehydrated. DOPC/DPPC and DOPC/Ch (70/30) liposomes exhibited heterogeneous membranes, indicating that nanosized ordered domains formed on the surface of the DOPC/DPPC liposomes. The size of these nanosized ordered domains was estimated using the TEMPO quenching method. Because TEMPO can quench DPH distributed in the disordered phases, the remaining fluorescence from DPH is proportional to the size of the ordered domain. The domain sizes calculated for DOPC/DPPC (50/50), DOPC/DPPC (25/75), DOPC/Ch (70/30), and DOPC/DPPC/Ch (40/40/20) were 13.9, 36.2, 13.2, and 35.5 Å, respectively.