The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Δν, and the time course of the correlation function, C(t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.     

Influence of the curvature on the water structure in the headgroup region of phospholipid bilayer studied by the solvent relaxation technique

Solvent relaxation (SR) in 1,2-dioleoyl-palmitoyl-sn-glycero-3-phosphocholine (DOPC) unilamellar vesicles of different size was probed by 6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalene chloride (Patman), 6-propionyl-2-dimethylaminonaphthalene (Prodan) and 4-[(n-dodecylthio)methyl]-7-(N,N-dimethylamino)-coumarin (DTMAC). Patman probes the amount and mobility of the bound water molecules located at the carbonyl region of the bilayer. Membrane curvature significantly accelerates the solvent relaxation process, but does not influence the total Stokes shift, showing that membrane curvature increases the mobility, without affecting the amount of water molecules present in the headgroup region. This pattern was also verified for other phosphatidylcholines. Prodan is located in the phosphate region of the bilayer and probes a more polar, mobile and heterogeneous environment than Patman. The influence of membrane curvature on SR probed by Prodan is similar, however, less pronounced compared to Patman. DTMAC (first time used in SR) shows a broad distribution of locations along the z-axis. A substantial amount of the coumarin chromophores face bulk water. No effect of curvature on SR probed by DTMAC is detectable.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

Molecular interpretation of fluorescence solvent relaxation of Patman and 2H NMR experiments in phosphatidylcholine bilayers

The analysis of time-dependent fluorescence shifts of the bilayer probe 6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalene chloride (Patman) offers valuable information on the hydration and dynamics of phospholipid headgroups. Quenching studies on vesicles composed of four phosphatidylcholines with different hydrocarbon chains (18:1c9/18:1c9, DOPC; 16:0/18:1c9, POPC; 18:1c9/16:0, OPPC; 18:1c6/18:1c6, PCDelta6) show that the chromophore of Patman is defined located at the level of the sn-1 ester-group in the phospholipid, which is invariant to the hydrocarbon chain. The so-called solvent relaxation (SR) approach as well as solid-state 2H NMR reveals that DOPC and PCDelta6 are more hydrated than POPC and OPPC. A strong dependence of SR kinetics on the position of double bond in the investigated fatty acid chains was observed. Apparently, the closer the double bond is located to the hydrated sn-1 ester-group, the more mobile this group becomes. This work demonstrates that the SR approach can report mobility changes within phospholipid bilayers with a remarkable molecular resolution.     

Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations

Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shifts in the fluorescence emission of a chromophore, yielding polarity and viscosity information about the chromophore’s immediate environment. A chromophore applied to a phospholipid bilayer at a well-defined location (with respect to the z-axis of the bilayer) allows monitoring of the hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) probes the hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan provides information on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers. Those changes were predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. This experimental finding can be explained by MD simulations, which indicate that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful in understanding salt effects on the structure and dynamics of lipid bilayers. For example, such experiments demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, an observation which could not be explained by standard MD simulations. If polarizability is introduced into the applied force field, however, MD simulations show that big soft polarizable anions are able to interact with the hydrophilic/hydrophobic interface of the lipid bilayer, penetrating to the level probed by Laurdan, and that they expand and destabilize the bilayer making it more hydrated and mobile.     

Examining the effects of cholesterol on model membranes at high temperatures: Laurdan and Patman see it differently

At high temperature, the presence of cholesterol in phospholipid membranes alters the influence of membrane dipoles, including water molecules, on naphthalene-based fluorescent probes such as Laurdan and Patman (solvatochromism). Although both of these probes report identical changes to their emission spectra as a function of temperature in pure phosphatidylcholine bilayers, they differ in their response to cholesterol. Computer simulations of the spectra based on a simple model of solvatochromism indicated that the presence of cholesterol reduces the probability of bilayer dipole relaxation and also blunts the tendency of heat to enhance that probability. While the overall effect of cholesterol on membrane dipoles was detected identically by the two probes, Laurdan was influenced much more by the additional effect on temperature sensitivity than was Patman. A comparison of the fluorescence data with simulations using a coarse-grained bilayer model (de Meyer et al., 2010) suggested that these probes may be differentially sensitive to two closely related properties distinguishable in the presence of cholesterol. Specifically, Patman fluorescence correlated best with the average phospholipid acyl chain order. On the other hand, Laurdan fluorescence tracked more closely with the area per lipid molecule which, although affected generally by chain order, is also impacted by additional membrane-condensing effects of cholesterol. We postulate that this difference between Laurdan and Patman may be attributed to the bulkier charged headgroup of Patman which may cause the probe to preferentially locate in juxtaposition to the diminutive headgroup of cholesterol as the membrane condenses.     

Structure, dynamics, and hydration of POPC/POPS bilayers suspended in NaCl, KCl, and CsCl solutions

Effects of alkali metal chlorides on the properties of mixed negatively charged lipid bilayers are experimentally measured and numerically simulated. Addition of 20mol% of negatively charged phosphatidylserine to zwitterionic phosphatidylcholine strengthens adsorption of monovalent cations revealing their specificity, in the following order: Cs(+)相似文献   

Mixed micelles and other structures in the solubilization of bilayer lipid membranes by surfactants

The solubilization of lipid bilayers by surfactants is accompanied by morphological changes of the bilayer and the emergence of mixed micelles. From a phase equilibrium perspective, the lipid/surfactant/water system is in a two-phase area during the solubilization: a phase containing mixed micelles is in equilibrium with bilayer structures of the lamellar phase. In some cases three phases are present, the single micelle phase replaced by a concentrated and a dilute solution phase. In the case of non-ionic surfactants, the lipid bilayers reach saturation when mixed micelles, often flexible rod-like or thread-like, start to form in the aqueous solution, at a constant chemical potential of the surfactant. The composition of the bilayers also remains fixed during the dissolution. The phase behavior encountered with many charged surfactants is different. The lamellar phase becomes destabilized at a certain content of surfactant in the membrane, and then disintegrates, forming mixed micelles, or a hexagonal phase, or an intermediate phase. Defective bilayer intermediates, such as perforated vesicles, have been found in several systems, mainly with charged surfactants. The perforated membranes, in some systems, go over into thread-like micelles via lace-like structures, often without a clear two-phase region. Intermediates in the form of disks, either micelles or bilayer fragments, have been observed in several cases. Most noteworthy are the planar and circular disks found in systems containing a large fraction of cholesterol in the bilayer. Bile salts are a special class of surfactants that seem to break down the bilayer at low additions. Originally, disk-like mixed micelles were conjectured, with polar membrane lipids building the disk, and the bile salts covering the hydrophobic rim. Later work has shown that flexible cylinders are the dominant intermediates also in these systems, even if the disk-like structures have been re-established as transients in the transformation from mixed micelles to vesicles.     

Supported phospholipid/alkanethiol biomimetic membranes: insulating properties.

A novel model lipid bilayer membrane is prepared by the addition of phospholipid vesicles to alkanethiol monolayers on gold. This supported hybrid bilayer membrane is rugged, easily and reproducibly prepared in the absence of organic solvent, and is stable for very long periods of time. We have characterized the insulating characteristics of this membrane by examining the rate of electron transfer and by impedance spectroscopy. Supported hybrid bilayers formed from phospholipids and alkanethiols are pinhole-free and demonstrate measured values of conductivity and resistivity which are within an order of magnitude of that reported for black lipid membranes. Capacitance values suggest a dielectric constant of 2.7 for phospholipid membranes in the absence of organic solvent. The protein toxin, melittin, destroys the insulating capability of the phospholipid layer without significantly altering the bilayer structure. This model membrane will allow the assessment of the effect of lipid membrane perturbants on the insulating properties of natural lipid membranes.     

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance.

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.     

Bilayer interfacial properties modulate the binding of amphipathic peptides

The free energy of transfer (DeltaG degrees ) from water to lipid bilayers was measured for two amphipathic peptides, the presequence of the mitochondrial peptide rhodanese (MPR) and melittin. Experiments were designed to determine the effects on peptide partitioning of the addition of lipids that produce structural modifications to the bilayer/water interface. In particular, the addition of cholesterol or the cholesterol analog 6-ketocholestanol increases the bilayer area compressibility modulus, indicating that these molecules modify lipid-lipid interactions in the plane of the bilayer. The addition of 6-ketocholestanol or lipids with attached polyethylene glycol chains (PEG-lipids) modify the effective thickness of the interfacial region; 6-ketocholestanol increases the width of hydrophilic headgroup region in the direction of the acyl chains whereas the protruding PEG chains of PEG-lipids increase the structural width of the headgroup region into the surrounding aqueous phase. The incorporation of PEG-lipids with PEG molecular weights of 2000 or 5000 had no appreciable effect on peptide partitioning that could not be accounted for by the presence of surface charge. However, for both MPR and melittin DeltaG degrees decreased linearly with increasing bilayer compressibility modulus, demonstrating the importance of bilayer mechanical properties in the binding of amphipathic peptides.     

beta-Sheet structured beta-amyloid(1-40) perturbs phosphatidylcholine model membranes

The disruption of intracellular calcium homeostasis plays a central role in the pathology of Alzheimer's disease, which is also characterized by accumulation of the amyloid-beta peptides Abeta40 and Abeta42. These amphipathic peptides may become associated with neuronal membranes and affect their barrier function, resulting in the loss of calcium homeostasis. This suggestion has been extensively investigated by exposing protein-free model membranes, either vesicles or planar bilayers, to soluble Abeta. Primarily unstructured Abeta has been shown to undergo a membrane-induced conformational change to either primarily beta-structure or helical structure, depending, among other factors, on the model membrane composition. Association of Abeta renders lipid bilayers permeable to ions but there is dispute whether this is due to the formation of discrete transmembrane ion channels of Abeta peptides, or to a non-specific perturbation of bilayer integrity by lipid head group-associated Abeta. Here, we have attempted incorporation of Abeta in the hydrophobic core of zwitterionic bilayers, the most simple model membrane system, by preparing proteoliposomes by hydration of a mixed film of Abeta peptides and phosphatidylcholine (PC) lipids. Despite the use of a solvent mixture in which Abeta40 and Abeta42 are almost entirely helical, the Abeta analogs were beta-structured in the resulting vesicle dispersions. When Abeta40-containing vesicles were fused into a zwitterionic planar bilayer, the typical irregular "single channel-like" conductance of Abeta was observed. The maximum conductance increased with additional vesicle fusion, while still exhibiting single channel-like behavior. Supported bilayers formed from Abeta40/PC vesicles did not exhibit any channel-like topological features, but the bilayer destabilized in time. Abeta40 was present primarily as beta-sheets in supported multilayers formed from the same vesicles. The combined observations argue for a non-specific perturbation of zwitterionic bilayers by surface association of small amphipathic Abeta40 assemblies.     

Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state (31)P and (13)C NMR spectroscopy

The conformation and dynamics of melittin bound to the dimyristoylphosphatidylcholine (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state (31)P and (13)C NMR spectroscopy. Using (31)P NMR, it was found that melittin-lipid bilayers form magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature (T(m) = 24 degrees C). The conformation, orientation, and dynamics of melittin bound to the membrane were further determined by using this magnetically oriented lipid bilayer system. For this purpose, the (13)C NMR spectra of site-specifically (13)C-labeled melittin bound to the membrane in the static, fast magic angle spinning (MAS) and slow MAS conditions were measured. Subsequently, we analyzed the (13)C chemical shift tensors of carbonyl carbons in the peptide backbone under the conditions where they form an alpha-helix and reorient rapidly about the average helical axis. Finally, it was found that melittin adopts a transmembrane alpha-helix whose average axis is parallel to the bilayer normal. The kink angle between the N- and C-terminal helical rods of melittin in the lipid bilayer is approximately 140 degrees or approximately 160 degrees, which is larger than the value of 120 degrees determined by x-ray diffraction studies. Pore formation was clearly observed below the T(m) in the initial stage of lysis by microscope. This is considered to be caused by the association of melittin molecules in the lipid bilayer.     

Molecular simulation study of phospholipid bilayers and insights of the interactions with disaccharides

Molecular simulations of hydrated dipalmitoylphosphatidylcholine lipid bilayers have been performed for temperatures in the range of 250-450 K. The area per headgroup increases with temperature from 58 to 77 A(2). Other properties such as hydration number, alkyl tail order parameter, diffusion coefficients, and radial distribution functions exhibit a clear dependence on temperature. Simulations of bilayers have also been performed in the presence of two disaccharides, namely trehalose and sucrose, at concentrations of up to 18 wt % (lipid-free basis). The simulated area per headgroup of the bilayer is not affected by the presence of the disaccharides, suggesting that the overall structure of the bilayer remains undisturbed. The results of simulations reveal that the interaction of disaccharide molecules with the bilayer occurs at the surface of the bilayer, and it is governed by the formation of multiple hydrogen bonds to specific groups of the lipid. Disaccharide molecules are observed to adopt specific conformations to fit onto the surface topology of the bilayer, often interacting with up to three different lipids simultaneously. At high disaccharide concentrations, the results of simulations indicate that disaccharides can serve as an effective replacement for water under anhydrous conditions, which helps explain their effectiveness as lyophilization agents for liposomes and cells.     

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.     

Lipid bilayer disruption by oligomeric α-synuclein depends on bilayer charge and accessibility of the hydrophobic core

Soluble oligomeric aggregates of α-synuclein have been implicated to play a central role in the pathogenesis of Parkinson's disease. Disruption and permeabilization of lipid bilayers by α-synuclein oligomers is postulated as a toxic mechanism, but the molecular details controlling the oligomer–membrane interaction are still unknown. Here we show that membrane disruption strongly depends on the accessibility of the hydrophobic membrane core and that charge interactions play an important but complex role. We systematically studied the influence of the physical membrane properties and solution conditions on lipid bilayer disruption by oligomers using a dye release assay. Varying the lipid headgroup composition revealed that membrane disruption only occurs for negatively charged bilayers. Furthermore, the electrostatic repulsion between the negatively charged α-synuclein and the negative surface charge of the bilayer inhibits vesicle disruption at low ionic strength. The disruption of negatively charged vesicles further depends on lipid packing parameters. Bilayer composition changes that result in an increased lipid headgroup spacing make vesicles more prone to disruption, suggesting that the accessibility of the bilayer hydrocarbon core modulates oligomer–membrane interaction. These data shed important new insights into the driving forces governing the highly debated process of oligomer–membrane interactions.     

Glycosphingolipids in membrane architecture

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin-labeled derivatives of 2 families of carbohydrate-bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip-flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca²⁺-induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.     

Binding and reorientation of melittin in a POPC bilayer: Computer simulations

We performed, using an all-atom force field, molecular dynamics computer simulations to study the binding of melittin to the POPC bilayer and its subsequent reorientation in this bilayer. The binding process involves a simultaneous folding and adsorption of the peptide to the bilayer, followed by the creation of a "U shaped" conformation. The reorientation of melittin from the parallel to the perpendicular conformation requires charged residues to cross the hydrophobic core of the bilayer. This is accomplished by a creation of defects in the bilayer that are filled out with water. The defects are caused by peptide charged residues dragging the lipid headgroup atoms along with them, as they reorient. With increased concentration of melittin water defects form stable pores; this makes it easier for the peptide N-terminus to reorient. Our results complement experimental and computational observations of the melittin/lipid bilayer interaction.