FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.     

Phase behavior of hydrated lipid bilayer composed of binary mixture of phospholipids with different head groups

Phase behavior of hydrated lipid bilayer was investigated for the mixtures of two phospholipid species chosen from phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) with the same acyl chains. The pseudo-binary phase diagrams constructed by a differential scanning calorimetry (DSC) were analyzed based on a thermodynamic model applying the Bragg–Williams approximation for non-ideality of mixing. The interchange energy parameters, ρ₀, derived from this approach were positive for all mixture systems in both gel and liquid–crystalline phase bilayers, and increased in the order PG/PE<PC/PA<PC/PE<PG/PA with a few exception. This suggests that the energetical disadvantage for the mixed-pair formation relative to the like-pair formation in the hydrated bilayer increases in this order. In addition, the ρ₀ values increased with the increase in the acyl chain length of the phospholipids. These experimental results were discussed in terms of an intermolecular interaction of the phospholipid species in hydrated bilayer.     

A Langmuir film approach to elucidating interactions in lipid membranes: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/cholesterol/metal cation systems

The interactions between two membrane lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and cholesterol (CHOL), were studied in Langmuir films using surface pressure isotherms and Brewster angle microscopy. The DPPE/CHOL interactions were probed for chosen monolayer and subphase (Na(+), Ca(2+)) composition at 20, 25, and 30 degrees C. The results obtained show that DPPE and CHOL are miscible for the cholesterol mol fractions x(CHOL)=0.3-0.5. Cholesterol induces condensation of the DPPE monolayers. The most significant condensation of the DPPE/CHOL monolayers was observed in the presence of Ca(2+) ions in the subphase at x(CHOL)=0.4. The negative deviation of the molecular surface area (MMA) additivity from the ideal behavior together with negative values of excess free enthalpy of mixing in the monolayers were interpreted in terms of attractive interactions between lipid molecules.     

Phase equilibria of model milk membrane lipid systems

The phase behaviour of mixtures of recombined milk membrane lipids dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), dioleoylphosphatidylethanolamine (DOPE), phosphatidylinositol (PI) and dioleoylphosphatidylserine (DOPS) in 60% water was examined as a function of temperature between 5 and 90 degrees C. The aim was to examine under which lipid composition the average properties turn from balanced over to hydrophobic. The phase boundaries were determined by small angle X-ray diffraction (SAXD) and differential scanning calorimetry (DSC). The lamellar phase was dominating in the DOPC/SM/DOPE system. The phase boundary for the reversed hexagonal phase was only observed at high DOPE content within the examined temperature interval. The anionic phospholipids PI and DOPS induced a swollen lamellar phase, but no significant change of the transition between the lamellar phase and the reversed hexagonal phase was observed.     

Phase behavior and dynamic heterogeneities in lipids: A coarse-grained simulation study of DPPC-DPPE mixtures

The coarse-grained Marrink-model for biomembrane simulation is used to study mixtures of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) at various concentrations and temperatures. At high temperatures close to ideal mixing is observed. In the low temperature ordered phase dynamic heterogeneities are identified under some conditions. These are correlated with heterogeneities in the local order and define local neighborhoods.     

The cryoprotectant trehalose destabilises the bilayer organisation of Escherichia coli-derived membrane systems at elevated temperatures as determined by H and P-NMR

In this study, ²H and ³¹P-NMR techniques were used to study the effects of trehalose and glycerol on phase transitions and lipid acyl chain order of membrane systems derived from cells of E. coli unsaturated fatty acid auxotroph strain K1059, which was grown in the presence of [11,11-²H₂]-oleic acid or [11,11-²H₂]-elaidic acid. From an analysis of the temperature dependence of the quadrupolar splitting it could be concluded that neither 1 M trehalose or glycerol generally had any significant effect on the temperature of the lamellar gel to liquid-crystalline phase transition. In the case of the oleate-containing hydrated total lipid extract, glycerol but not trehalose caused a 5°C increase of this transition temperature. In general, both cryoprotectants induced an ordering of the acyl chains in the liquid-crystalline state. Trehalose and glycerol both decrease the bilayer to non-bilayer transition temperature of the hydrated lipid extract of oleate-grown cells by about 5°C, but only trehalose in addition induces an isotropic to hexagonal (H_II) phase transition. In the biological membranes, trehalose and not glycerol destabilised the lipid bilayer, and in the case of the E. coli spheroplasts, part of the induced non-bilayer structures is ascribed to a hexagonal (H_II) phase in analogy with the total lipids. Interestingly, 1 mM Mg²⁺ was a prerequisite for the destabilisation of the lipid bilayer. In the hydrated total lipid extract of E. coli grown on the more ordered elaidic acid, both transition temperatures were shifted about 20°C upwards compared with the oleate-containing lipid, but the effect of trehalose on the lipid phase behaviour was similar. The bilayer destabilising ability of trehalose might have implications for the possible protection of biological systems by (cryo-)protectants during dehydration, in that protection is unlikely to be caused by preventing the occurrence of polymorphic phase transitions.     

Enzyme purification with aqueous two-phase systems: comparison between systems composed of pure polymers and systems composed of crude polymers

The main drawback when using aqueous two-phase systems for macromolecule purification is the high cost of most polymers used. The purification of an enzyme, alcohol dehydrogenase, from a crude extract of Saccharomyces cerevisiae was tested in systems composed of poly(ethylene glycol) and a crude hydroxypropyl starch or Reppal PES 100, a purified fraction of hydroxypropyl starch. Purification factors measured for the enzyme were very similar in both systems (between 0.8 and 1.4 for both systems in the upper phase). However, systems composed of Reppal PES present a greater recovery of enzyme, between 77% and 100% versus 60% and 100%, while systems composed of crude hydroxypropyl starch exhibit a larger Δlog K for the tested ligand, 1.26 versus 0.81.     

Miscibility of DPPC and DPPA in monolayers at the air/water interface

Monolayers of mixtures of 1,2-dipalmitoylphosphatidylcholine (DPPC) as the substrate and 1,2-dipalmitoylphosphatidic acid (DPPA) as the product of the hydrolysis reaction catalyzed by phospholipase D (PLD) were investigated in the presence of Ca2+. The miscibility behavior and the microstructure of mixed domains have been studied by grazing incidence X-ray diffraction (GIXD), Brewster angle microscopy and film balance measurements. The phase diagram reveals partial miscibility on both sides and a wide miscibility gap, which becomes narrower at high pressure. At low pressure, the segregation of condensed DPPA-rich domains in a fluid-like DPPC matrix was detected already at small DPPA concentrations and their structure was determined. A small amount of DPPC mixed into the segregated DPPA domains induces the transformation from rectangular to an oblique unit cell and increases the tilt angle in the condensed domains. At high pressure, two types of condensed phase domains were found: DPPC-rich and DPPA-rich. A drastic reduction of the tilt angle in the DPPC-rich domains with increasing amount of DPPA was observed. The decrease of the tilt angle must be connected with a change of the head group conformation of DPPC in such mixed domains.     

AFM study of the thermotropic behaviour of supported DPPC bilayers with and without the model peptide WALP23

Temperature-controlled Atomic Force Microscopy (TC-AFM) in Contact Mode is used here to directly image the mechanisms by which melting and crystallization of supported, hydrated DPPC bilayers proceed in the presence and absence of the model peptide WALP23. Melting from the gel L_β′ to the liquid-crystalline L_α phase starts at pre-existing line-type packing defects (grain boundaries) in absence of the peptide. The exact transition temperature is shown to be influenced by the magnitude of the force exerted by the AFM probe on the bilayer, but is higher than the main transition temperature of non-supported DPPC vesicles in all cases due to bilayer–substrate interactions. Cooling of the fluid L_α bilayer shows the formation of the line-type defects at the borders between different gel-phase regions that originate from different nuclei. The number of these defects depends directly on the rate of cooling through the transition, as predicted by classical nucleation theory.The presence of the transmembrane, synthetic model peptide WALP23 is known to give rise to heterogeneity in the bilayer as microdomains with a striped appearance are formed in the DPPC bilayer. This striated phase consists of alternating lines of lipids and peptide. It is shown here that melting starts with the peptide-associated lipids in the domains, whose melting temperature is lowered by 0.8–2.0 °C compared to the remaining, peptide-free parts of the bilayer. The stabilization of the fluid phase is ascribed to adaptations of the lipids to the shorter peptide. The lipids not associated with the peptide melt at the same temperature as those in the pure DPPC supported bilayer.     

Composition dependence of vesicle morphology and mixing properties in a bacterial model membrane system

We have determined the mixing properties and lamellar organization of bacterial membrane mimetics composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol (POPG) at various molar ratios applying differential scanning calorimetry, small and wide-angle X-ray scattering, as well as optical phase contrast microscopy. Combining the experimental thermodynamic data with a simulation of the liquidus and solidus lines, we were able to construct a phase diagram. Using this approach, we find that the lipids mix in all phases non-ideally in the thermodynamic sense. As expected, pure POPE assembles into multilamellar and pure POPG into unilamellar vesicles, respectively, which are stable within the studied temperature range. In contrast, mixtures of the two components form oligolamellar vesicles consisting of about three to five bilayers. The layers within these oligolamellar liposomes are positionally correlated within the gel phase, but become uncorrelated within the fluid phase exhibiting freely fluctuating bilayers, while the vesicles as a whole remain intact and do not break up into unilamellar forms. X-ray, as well as DSC data, respectively, reveal a miscibility gap due to a lateral phase segregation at POPG concentrations above about 70 mol%, similar to previously reported data on mixtures composed of disaturated PEs and PGs. Hence, the existence of a region of immiscibility is a general feature of PE/PG mixtures and the mixing properties are dominated by PE/PG headgroup interactions, but are largely independent of the composition of the hydrocarbon chains. This is in accordance with a recent theoretical prediction.     

On the ordering of N-alkane and N-alcohol solutes in phospholipid bilayer model membrane systems

²H-NMR measurements of the ordering of several n-alkane and one n-alcohol solutes in lipid bilayers formed from dimyristoyl lecithin (DML) are reported. The results are consistent with orientation of the solutes between the lipid chains, the n-alkanes having a preference for the bilayer interior, while the n-octanol is anchored at the bilayer surface. Solubility of the short chain n-alkanes, n-hexane and n-octane as an ordered component in the L_α phase is more limited than that of n-dodecane. The NMR data are supported by low angle X-ray diffraction results which confirm that n-octanol has a more dramatic influence on bilayer area than the n-alkanes.     

Phospholipid matrix as a target for sulfur mustard (HD): NMR study in model membrane systems

Although the interactions of sulfur mustard (HD) with nucleic acids and proteins have been well studied, the toxic interactions with the membrane matrix and specially the phospholipid bilayer have so far been poorly investigated. We have used several NMR techniques to study these interactions: ¹H NMR to observe the localization of HD in membranes of small unilamellar vesicles (SUV) of lecithin; ³¹P NMR to verify the hypothesis of pore formation in membranes of large unilamellar vesicles (LUV); and pseudo solid state ³¹P and ²H NMR to analyze the dynamic consequences of the presence of HD in multilayer dispersions of dimyristoylphosphatidylcholine (DMPC). Immediate and late modifications of the DMPC–HD complexes have been observed at the macroscopic and microscopic levels. After intoxication, HD is spontaneously incorporated into the membrane and locates at the level of the chain methylene groups. This incorporation occurs without formation of pores in the membrane. The presence of HD in the phospholipid dispersion differentially increases the membrane fluidity depending upon the level involved. Weak at the superficial level (phosphate group), this increase is dose-dependent on progression into the membrane. This increase is related to a lowering of transition temperature when measured at the chain level. Macroscopically, HD induces dose- and time-dependent modifications of the DMPC–HD complexes, leading to the formation of an optically transparent gel. This gel formation is confirmed at a microscopic level, where all structures disappear after intoxication. This revised version was published online in July 2006 with corrections to the Cover Date.     

Integrated membrane systems for gas separation in biotechnology: potential and prospects

Integrated non-porous membrane systems were applied for microbial combustible gas separation processes. Methane/CO₂ mixtures of various concentrations from methane fermentation processes (biogas) were separated using a membrane-separation complex of permabsorber type into individual components of technical grade (more than 95% purity). In experiments with three-component mixtures, using a selective membrane valve with various liquid carriers, all the gases of interest (H₂, CH₄ and CO₂) were obtained at greater than 90% purity in one separation step. The perspectives for the further application of non-porous membrane separating devices for various gaseous mixtures from different microbial processes are discussed.V. Teplyakov and E. Sostina are with the A.V. Topchiev institute of Petrochemical Synthesis, Russian Academy of Sciences, Membrane Research Center, Moscow 117912, Russia. E. Sostina is also, and A. Netrusov is with the Microbiology Department, Moscow University, Moscow 119899, Russia. I. Beckman is with the Chemistry Department, Moscow University, Moscow 119899, Russia.     

Location and orientation of Triclosan in phospholipid model membranes

Triclosan is a hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. Although the molecular mechanism of action of this molecule has been attributed to inhibition of fatty acid biosynthesis, earlier work in our laboratories strongly suggested that the antibacterial action of Triclosan is mediated at least partly through its membranotropic effects. In order to assess its location in phospholipid membranes, high-resolution magic-angle spinning natural abundance ¹³C NMR of Triclosan embedded within egg yolk lecithin model membranes has been used to obtain ¹³C spin–lattice relaxation times for both Triclosan and lecithin carbon atoms in the presence of Gd³⁺ ions. The results indicate that Triclosan is localized in the upper region of the phospholipid membrane, its hydroxyl group residing in the vicinity of the C=O/C2 carbon atoms of the acyl chain of the phospholipid, and the rest of the Triclosan molecule is probably aligned in a nearly perpendicular orientation with respect to the phospholipid molecule. Intercalation of Triclosan into bacterial cell membranes likely compromises the functional integrity of those membranes, thereby accounting for at least some of this compounds antibacterial effects.Abbreviations COLOC correlation by long-range coupling - EYL egg yolk lecithin - HETCOR heteronuclear chemical-shift correlation - MAS magic-angle spinning - MLV multilamellar vesicles     

Photosystem II in different parts of the thylakoid membrane: a functional comparison between different domains

The electron transport properties of photosystem II (PSII) from five different domains of the thylakoid membrane were analyzed by flash-induced fluorescence kinetics. These domains are the entire grana, the grana core, the margins from the grana, the stroma lamellae, and the Y100 fraction (which represent more purified stroma lamellae). The two first fractions originate from appressed grana membranes and have PSII with a high proportion of O(2)-evolving centers (80-90%) and efficient electron transport on the acceptor side. About 30% of the granal PSII centers were found in the margin fraction. Two-thirds of those PSII centers evolve O(2), but the electron transfer on the acceptor side is slowed. PSII from the stroma lamellae was less active. The fraction containing the entire stroma has only 43% O(2)-evolving PSII centers and slow electron transfer on the acceptor side. In contrast, PSII centers of the Y100 fraction show no O(2) evolution and were unable to reduce Q(B). Flash-induced fluorescence decay measurements in the presence of DCMU give information about the integrity of the donor side of PSII. We were able to distinguish between PSII centers with a functional Mn cluster and without any Mn cluster, and PSII centers which undergo photoactivation and have a partially assembled Mn cluster. From this analysis, we propose the existence of a PSII activity gradient in the thylakoid membrane. The gradient is directed from the stroma lamellae, where the Mn cluster is absent or inactive, via the margins where photoactivation accelerates, to the grana core domain where PSII is fully photoactivated. The photoactivation process correlates to the PSII diffusion along the membrane and is initiated in the stroma lamellae while the final steps take place in the appressed regions of the grana core. The margin domain is seemingly very important in this process.     

Development of aqueous two‐phase systems comprising poly ethylene glycol and dextran for purification of pullulan: Phase diagrams and fiscal analysis

Pullulan is a commercially important Exopolysaccharide (EPS) with wide‐spread applications which is produced by Aureobasidium pullulans. The alternative α (1 4) & α (1 6) configuration in pullulan provides it the specific structural and conformational properties. Pullulan is currently being exploited in food, health care, pharmacy, lithography, cosmetics. The fermented broth is processed by organic solvent precipitation for isolation and purification of pullulan. In this study, we have tried to analyze the potential of aqueous two phase system as an alternate technique to extract pullulan from fermented broth. Including this viability of ATPS was also compared with conventional organic solvent precipitation system in terms of cost and time. It was found that ATPS process produced a higher yield of pullulan (80.56%) than organic solvent precipitation method (71.6%). ATPS was also found more economical and less time consuming method.     

Role of cholesterol in lipid raft formation: lessons from lipid model systems

Biochemical and cell-biological experiments have identified cholesterol as an important component of lipid ‘rafts’ and related structures (e.g., caveolae) in mammalian cell membranes, and membrane cholesterol levels as a key factor in determining raft stability and organization. Studies using cholesterol-containing bilayers as model systems have provided important insights into the roles that cholesterol plays in determining lipid raft behavior. This review will discuss recent progress in understanding two aspects of lipid-cholesterol interactions that are particularly relevant to understanding the formation and properties of lipid rafts. First, we will consider evidence that cholesterol interacts differentially with different membrane lipids, associating particularly strongly with saturated, high-melting phospho- and sphingolipids and particularly weakly with highly unsaturated lipid species. Second, we will review recent progress in reconstituting and directly observing segregated raft-like (liquid-ordered) domains in model membranes that mimic the lipid compositions of natural membranes incorporating raft domains.     

Detergent solubilization of bovine erythrocytes. Comparison between the insoluble material and the intact membrane

Early works have shown that when biomembranes are extracted with the non-ionic detergent Triton X-100 at 4 degrees C, only a subset of the components is solubilized. The aim of this paper was to investigate the solubilization of a cell membrane at different Triton concentrations, and to compare the lipid composition and acyl chain order/mobility of the insoluble material with those of the original membrane. We choose bovine erythrocytes, because they have an uncommon composition, as they have a huge amount of sphingomyelin and phosphatidylcholine is almost absent. We determined the degree of order/mobility of the lipid acyl chains by EPR spectroscopy, using liposoluble spin labels. Incubation of bovine erythrocytes with increasing Triton X-100 concentrations yields decreasing amounts of insoluble material which is enriched in sphingomyelin and depleted in cholesterol. Complete lipid solubilization is achieved at a detergent/lipid ratio of about 60, which is much higher than the values reported for human erythrocytes, but is in line with results obtained in model systems. An insoluble pellet is still obtained at higher Triton concentrations, which seems to consist mainly of protein. A very high correlation is found between lipid chain mobility restrictions and sphingomyelin content in the lipid structures. The human erythrocyte membrane also fits well in this correlation, suggesting a significant role of sphingomyelin in determining acyl chain organization. The analogies and differences between our insoluble material and the detergent-resistant membranes (DRM) are discussed.