Molecular dynamics simulations and 2H NMR study of the GalCer/DPPG lipid bilayer

Molecular dynamics simulations were performed on a two-component lipid bilayer system in the liquid crystalline phase at constant pressure and constant temperature. The lipid bilayers were composed of a mixture of neutral galactosylceramide (GalCer) and charged dipalmitoylphosphatidylglycerol (DPPG) lipid molecules. Two lipid bilayer systems were prepared with GalCer:DPPG ratio 9:1 (10%-DPPG system) and 3:1 (25%-DPPG system). The 10%-DPPG system represents a collapsed state lipid bilayer, with a narrow water space between the bilayers, and the 25%-DPPG system represents an expanded state with a fluid space of approximately 10 nm. The number of lipid molecules used in each simulation was 1024, and the length of the production run simulation was 10 ns. The simulations were validated by comparing the results with experimental data for several important aspects of the bilayer structure and dynamics. Deuterium order parameters obtained from (2)H NMR experiments for DPPG chains are in a very good agreement with those obtained from molecular dynamics simulations. The surface area per GalCer lipid molecule was estimated to be 0.608 +/- 0.011 nm(2). From the simulated electron density profiles, the bilayer thickness defined as the distance between the phosphorus peaks across the bilayer was calculated to be 4.21 nm. Both simulation systems revealed a tendency for cooperative bilayer undulations, as expected in the liquid crystalline phase. The interaction of water with the GalCer and DPPG oxygen atoms results in a strong water ordering in a spherical hydration shell and the formation of hydrogen bonds (H-bonds). Each GalCer lipid molecule makes 8.6 +/- 0.1 H-bonds with the surrounding water, whereas each DPPG lipid molecule makes 8.3 +/- 0.1 H-bonds. The number of water molecules per GalCer or DPPG in the hydration shell was estimated to be 10-11 from an analysis of the radial distribution functions. The formation of the intermolecular hydrogen bonds was observed between hydroxyl groups from the opposing GalCer sugar headgroups, giving an energy of adhesion in the range between -1.0 and -3.4 erg/cm(2). We suggest that this value is the contribution of the hydrogen-bond component to the net adhesion energy between GalCer bilayers in the liquid crystalline phase.     

Molecular Interactions of the Major Myelin Glycosphingolipids and Myelin Basic Protein in Model Membranes

The molecular organization, interactions, phase state and membrane-membrane interactions of model membranes containing cerebroside (GalCer), sulfatide (Sulf) and myelin basic protein (MBP) were investigated. Sulf shows a larger cross-sectional area than GalCer, in keeping with the lateral electrostatic repulsions in the negatively charged polar head group. The interactions of GalCer with different phospholipids are similar while those with Sulf depend on the phosphoryl choline moiety in the phospholipid. MBP induces a decrease of the phase transition temperature in both lipids but with Sulf this occurs at lower proportions of MBP. In mixtures of Sulf with phosphatidylcholine MBP induces phase separation among Sulf-rich and PC-rich domains. Extensive apposition of bilayers containing Sulf is induced by MBP while GalCer interferes with this process. Few membrane interactions proceed to bilayer merging or whole bilayer fusion and the glycosphingolipids help preserve the membrane integrity.     

Range of the solvation pressure between lipid membranes: dependence on the packing density of solvent molecules

Well-ordered multilamellar arrays of liquid-crystalline phosphatidylcholine and equimolar phosphatidylcholine-cholesterol bilayers have been formed in the nonaqueous solvents formamide and 1,3-propanediol. The organization of these bilayers and the interactions between apposing bilayer surfaces have been investigated by X-ray diffraction analysis of liposomes compressed by applied osmotic pressures up to 6 X 10(7) dyn/cm2 (60 atm). The structure of egg phosphatidylcholine (EPC) bilayers in these solvents is quite different than in water, with the bilayer thickness being largest in water, 3 A narrower in formamide, and 6 A narrower in 1,3-propanediol. The incorporation of equimolar cholesterol increases the thickness of EPC bilayers immersed in each solvent, by over 10 A in the case of 1,3-propanediol. The osmotic pressures of various concentrations of the neutral polymer poly(vinylpyrrolidone) dissolved in formamide or 1,3-propanediol have been measured with a custom-built membrane osmometer. These measurements are used to obtain the distance dependence of the repulsive solvation pressure between apposing bilayer surfaces. For each solvent, the solvation pressure decreases exponentially with distance between bilayer surfaces. However, for both EPC and EPC-cholesterol bilayers, the decay length and magnitude of this repulsive pressure strongly depend on the solvent. The decay length for EPC bilayers in water, formamide, and 1,3-propanediol is found to be 1.7, 2.4, and 2.6 A, respectively, whereas the decay length for equimolar EPC-cholesterol bilayers in water, formamide, and 1,3-propanediol is found to be 2.1, 2.9, and 3.1 A, respectively. These data indicate that the decay length is inversely proportional to the cube root of the number of solvent molecules per unit volume.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes

Pressure versus distance relationships have been obtained for egg phosphatidylcholine bilayers containing a range of cholesterol concentrations. Water was removed from between adjacent bilayers by the application of osmotic pressures in the range of 0.4-2600 atm (4 x 10(5)-2.6 x 10(9) dyn/cm2), and the distance between adjacent bilayers was obtained by Fourier analysis of X-ray diffraction data. For applied pressures up to about 50 atm and bilayer surface separations of 15-5 A, the incorporation of up to equimolar cholesterol has little influence on plots of pressure versus bilayer separation. However, for the higher applied pressures, cholesterol reduces the interbilayer separation distance by an amount that depends on the cholesterol concentration in the bilayer. For example, the incorporation of equimolar cholesterol reduces the distance between bilayers by as much as 6 A at an applied pressure of 2600 atm. At this applied pressure, electron density profiles show that the high-density head-group peaks from apposing bilayers have merged. This indicates that equimolar concentrations of cholesterol spread the lipid molecules apart in the plane of the bilayer enough to allow the phosphatidylcholine head groups from apposing bilayers to interpenetrate as the bilayers are squeezed together. All of these X-ray and pressure-distance data indicate that, by reducing the volume fraction of phospholipid head groups, cholesterol markedly reduces the steric repulsion between apposing bilayers but has a much smaller effect on the sum of the longer ranged repulsive hydration and fluctuation pressures. Increasing concentrations of cholesterol monotonically increase the dipole potential of egg phosphatidylcholine monolayers, from 415 mV with no cholesterol to 493 mV with equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of polyphenols with bilayers: conditions for increasing bilayer adhesion.

Because proteins and other molecules with a high polyphenol content are commonly involved in adhesion processes, we are investigating the interactions between polyphenols and biological materials. A naturally occurring polyphenol that binds a variety of proteins and lipids is tannic acid (TA), which contains five digallic acid residues covalently linked to a central D-glucose. A previous study has shown that TA increases the adhesion between apposing phosphatidylcholine (PC) bilayers and over a very narrow concentration range collapses the interbilayer fluid space from about 15 A to 5 A. To determine the chemical requirements a polyphenolic molecule must possess to increase bilayer adhesion, we have synthesized several simpler TA analogs that vary in their size, shape, and number of gallic acid and hydroxyl groups. X-ray diffraction, absorbance, binding, and differential scanning calorimetry measurements were used to investigate the interaction of these polyphenolic molecules with egg PC (EPC) and dipalmitoyl PC (DPPC) bilayers. Of these synthetic polyphenols, only penta-O-galloyl-alpha-D-glucose (PGG) was able to completely mimic the effects of TA by collapsing the interbilayer fluid space from 15 A to 5 A, decreasing the dipole potential by about 300 mV, increasing the transition enthalpy of DPPC liposomes, and inducing an interdigitated phase in DPPC. Binding studies indicated that the fluid space was reduced to 5 A at an EPC:PGG mole ratio of 5:1. We conclude that these polyphenols collapse the fluid space of PC bilayers because they 1) are amphipathic and partition into the bilayers interfacial region, 2) are long enough to span the interbilayer space, 3) contain several gallic acids distributed so that they can partition simultaneously into apposing bilayers, and 4) have sufficient gallic acid residues to interact with all lipid headgroups and cover the bilayer surface. Under these conditions we conclude that the polyphenols from interbilayer bridges. We argue that these bridges are stabilized by increased adhesion arising from an increased van der Waals interaction between apposing bilayers, electrostatic interactions between the pi electrons in the phenol ring and the -(N+CH3)3 groups on the PC headgroups, decreased hydration repulsion between bilayers, and hydrogen bonds between the H-bond-donating moieties on the polyphenols and H-bond-accepting groups in the bilayer.     

Area per molecule and distribution of water in fully hydrated dilauroylphosphatidylethanolamine bilayers

The area per lipid molecule for fully hydrated dilauroylphosphatidylethanolamine (DLPE) has been obtained in both the gel and liquid-crystalline states by combining wide-angle X-ray diffraction, electron density profiles, and previously published dilatometry results [Wilkinson, D. A., & Nagle, J. F. (1981) Biochemistry 20, 187-192]. The molecular area increases from 41.0 +/- 0.2 to 49.1 +/- 1.2 A2 upon melting from the gel to liquid-crystalline phase. The thickness of the bilayer, as measured from the electron density profiles, decreases about 4 A upon melting, from 45.2 +/- 0.3 to 41.0 +/- 0.6 A. A somewhat unexpected result is that the fluid layer between fully hydrated bilayers is the same in both gel and liquid-crystalline phases and is only about 5 A thick. From these data, plus the volume of the anhydrous DLPE molecule, it is possible to determine the number of water molecules per lipid and their approximate distribution relative to the lipid molecule. Our analysis shows that there are about 7 and 9 waters per DLPE molecule in the gel and liquid-crystalline phases, respectively. About half of the water is located in the fluid space between adjacent bilayers, and the remaining waters are intercalated into the bilayer, presumably in the head group region. There are significantly fewer water molecules in the fluid spaces between DLPE bilayers than in the fluid spaces in gel- or liquid-crystalline-phase phosphatidylcholine bilayers. This small fluid space in PE bilayers could arise from interbilayer hydrogen bond formation through the water molecules or electrostatic interactions between the amine and phosphate groups on apposing bilayers.     

Galactocerebroside-phospholipid interactions in bilayer membranes.

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

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer-sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1, and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in supported lipid bilayers. Using "slow-cooled vesicle fusion" to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area/perimeter ratio (A(D)/P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), "quenched vesicle fusion", and find that the microstructures are remarkably similar above a cholesterol mol fraction of approximately 0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose-specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection.     

Atomistic-level study of the interactions between hIAPP protofibrils and membranes: Influence of pH and lipid composition

The pathology of type 2 diabetes mellitus is associated with the aggregation of human islet amyloid polypeptide (hIAPP) and aggregation-mediated membrane disruption. The interactions of hIAPP aggregates with lipid membrane, as well as the effects of pH and lipid composition at the atomic level, remain elusive. Herein, using molecular dynamics simulations, we investigate the interactions of hIAPP protofibrillar oligomers with lipids, and the membrane perturbation that they induce, when they are partially inserted in an anionic dipalmitoyl-phosphatidylglycerol (DPPG) membrane or a mixed dipalmitoyl-phosphatidylcholine (DPPC)/DPPG (7:3) lipid bilayer under acidic/neutral pH conditions. We observed that the tilt angles and insertion depths of the hIAPP protofibril are strongly correlated with the pH and lipid composition. At neutral pH, the tilt angle and insertion depth of hIAPP protofibrils at a DPPG bilayer reach ~52° and ~1.62 nm with respect to the membrane surface, while they become ~77° and ~1.75 nm at a mixed DPPC/DPPG membrane. The calculated tilt angle of hIAPP at DPPG membrane is consistent with a recent chiral sum frequency generation spectroscopic study. The acidic pH induces a smaller tilt angle of ~40° and a shallower insertion depth (~1.24 nm) of hIAPP at the DPPG membrane surface, mainly due to protonation of His18 near the turn region. These differences mainly result from a combination of distinct electrostatic, van der Waals, hydrogen bonding and salt-bridge interactions between hIAPP and lipid bilayers. The hIAPP-membrane interaction energy analysis reveals that besides charged residues K1, R11 and H18, aromatic residues Phe15 and Phe23 also exhibit strong interactions with lipid bilayers, revealing the crucial role of aromatic residues in stabilizing the membrane-bound hIAPP protofibrils. hIAPP-membrane interactions disturb the lipid ordering and the local bilayer thickness around the peptides. Our results provide atomic-level information of membrane interaction of hIAPP protofibrils, revealing pH-dependent and membrane-modulated hIAPP aggregation at the early stage.     

Thermal and structural behavior of natural cerebroside 3-sulfate in bilayer membranes

Differential scanning calorimetry (DSC), polarizing microscopy and X-ray diffraction studies have been performed on dry and hydrated natural bovine brain sulfatides. Dry sulfatide fractions exhibit a high temperature transition (delta H = 6.6 kcal/mol sulfatide) at 87.3 degrees C. X-ray diffraction shows this transition to be associated with a hydrocarbon chain order-disorder transformation between two lamellar phases. Hydrated sulfatide dispersions undergo a complex chain order-disorder transition (delta H = 7.5 kcal/mol sulfatide) at 32 degrees C with two peak temperatures at 35 degrees C and 47 degrees C. Structural studies performed on hydrated liquid-crystal sulfatide dispersions at 75 degrees C verify the existence of a bilayer structure over the 16 wt.% to 50 wt.% phosphate buffer (pH = 7.4) range. The interbilayer separation between galactosyl-3-sulfate groups averages 48 A as the multilamellar bilayers swell with the addition of phosphate buffer. The formation of micellar phases is not observed at high water contents. The comparison of the structural characteristics of dry and hydrated sulfatides with structural data for dry and hydrated bovine brain non-sulfated glycolipid (cerebroside) is discussed in molecular terms.     

Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations

It has been established that the fusion of both biological membranes and phospholipid bilayers can be modulated by altering their lipid composition (Chernomordik et al., 1995 .J. Membr. Biol. 146:3). In particular, when added exogenously between apposing membranes, monomyristoylphosphatidylcholine (MMPC) inhibits membrane fusion, whereas glycerol monoleate (GMO), oleic acid (OA), and arachidonic acid (AA) promote fusion. This present study uses x-ray diffraction to investigate the effects of MMPC, GMO, OA, and AA on the bending and stability of lipid bilayers when bilayers are forced together with applied osmotic pressure. The addition of 10 and 30 mol% MMPC to egg phosphatidylcholine (EPC) bilayers maintains the bilayer structure, even when the interbilayer fluid spacing is reduced to approximately 3 A, and increases the repulsive pressure between bilayers so that the fluid spacing in excess water increases by 5 and 15 A, respectively. Thus MMPC increases the undulation pressure, implying that the addition of MMPC promotes out-of-plane bending and decreases the adhesion energy between bilayers. In contrast, the addition of GMO has minor effects on the undulation pressure; 10 and 50 mol% GMO increase the fluid spacing of EPC in excess water by 0 and 2 A, respectively. However, x-ray diffraction indicates that, at small interbilayer separations, GMO, OA, or AA converts the bilayer to a structure containing hexagonally packed scattering units approximately 50 A in diameter. Thus GMO, OA, or AA destabilizes bilayer structure as apposing bilayers are brought into contact, which could contribute to their role in promoting membrane fusion.     

Interactions between charged, uncharged, and zwitterionic bilayers containing phosphatidylglycerol.

Pressure vs. distance relationships have been obtained for phosphatidylglycerol bilayers, in both charged and uncharged states. Water was removed from the lipid multilayers by the application of osmotic pressures in the range of 0-2.7 x 10(9) dyn/cm2, and the distance between adjacent bilayers was obtained from Fourier analysis of lamellar x-ray diffraction data. For phosphatidylglycerol bilayers made electrically neutral either by lowering the pH or by adding equimolar concentrations of the positively charged lipid stearylamine, the pressure-distance data could be fit with a single exponential. The measured decay lengths were 1.1 A at low pH and 1.5 A with stearylamine, which are similar to decay lengths of the hydration pressure found for gel phases of other neutral bilayers. In addition, the magnitude of this repulsive pressure was proportional to the square of the Volta potential (equivalent to the dipole potential for electrically neutral bilayers) measured in monolayers in equilibrium with bilayers, in agreement with results previously found for the hydration pressure between phosphatidylcholine bilayers. For charged phosphatidylglycerol bilayers, the pressure-distance relation had two distinct regions. For bilayer separations greater than 10 A, the pressure-distance data had an exponential decay length (11 A) and a magnitude consistent with that expected for electrostatic repulsion from double-layer theory. For bilayer separations of 2-10 A, the pressure decayed much more rapidly with increasing bilayer separation (decay length less than 1 A). We interpret these data at low bilayer separations in terms of a combination of hydration repulsion and steric hindrance between the lipid head groups and the sodium ions trapped between apposing bilayers.     

Synthesis, structure, and thermal properties of 1,2-dipalmitoylgalloylglycerol (DPGG), a novel self-adhering lipid

A novel diacyl glycerol-based lipid with a polyphenolic head group has been synthesized and characterized. X-ray diffraction experiments show that this lipid, 1,2-dipalmitoylgalloylglycerol (DPGG), hydrates to form gel phase bilayers at 20 degrees C with extremely narrow interbilayer fluid separations, indicating that apposing DPGG bilayers strongly adhere to each other. Differential scanning calorimetry shows that fully hydrated DPGG exhibits a pretransition exotherm (3.7 kcal/mol) at 52 degrees C and a high enthalpy (11.3 kcal/mol) main endothermic transition at 69 degrees C. These thermal properties are similar to those of galactosylceramides with similar hydrocarbon chain compositions. The adhesive and thermal properties of DPGG are likely due to both intermolecular hydrogen-bonding and hydrophobic interactions between the aromatic rings on the gallic acids.     

Multibilayer structure of dimyristoylphosphatidylcholine dihydrate as determined by energy minimization

Complete energy minimization was carried out on the multibilayer crystal structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine dihydrate (DMPC.2H2O), starting from the X-ray structure determination reported by Pearson and Pascher (1979) Nature 281, 499-501. The asymmetric unit contains two nonidentical DMPC molecules and four water molecules. Minimization removed the acyl chain disorder present in the X-ray structure and caused the carbon planes of the acyl chains to become mutually parallel. Two energy-minimized structures (structures I and II) were found which mainly differed in the hydrogen-bonding arrangement of the waters of hydration. In structure I as in the X-ray structure, one of the water molecules forms a hydrogen-bonded bridge between successive bilayers; but in structure II, all hydrogen bonds are satisfied on the same bilayer. Structure II corresponds to the global energy minimum and is also a suitable structure for single bilayers. The lattice constants and cell volume of the minimized structures are close to the experimental values. The electrostatic force between DMPC bilayers is attractive. The mean hydration energy of the water is -14.2 kcal/mol, which is 2.5 kcal/mol lower than the binding energy of ice.     

Neutron and X-ray diffraction structural analysis of phosphatidylinositol bilayers

Phosphatidylinositol (PI) bilayers, squeezed together by applied osmotic pressures, were studied by both neutron diffraction and X-ray diffraction. The lamellar repeat period for PI bilayers decreased from 9.5 nm at an applied pressure of 1.1.10(6) dyn/cm2 (1.1 atm) to 5.4 nm at an applied pressure of 1.6.10(7) dyn/cm2 (16 atm). Further increases in applied pressure, up to 2.7.10(9) dyn/cm2 (2700 atm) reduced the repeat period by only about 0.3 nm, to 5.1 nm. Thus, a plot of applied pressure versus repeat period shows a sharp upward break for repeat periods less than about 5.4 nm. For repeat periods of less than 5.4 nm, analysis of neutron-scattering density profiles and electron-density profiles indicates that the structure of the PI bilayers changes as the bilayers are dehydrated, even though there are only small changes in the repeat period. These structural changes are most likely due to removal of water from the headgroup regions of the bilayer. D2O/H2O exchange experiments show that, at an applied pressure of 2.8.10(7) dyn/cm2, water is located between adjacent PI headgroups in the plane of the bilayer. We conclude that, although electrostatics provide the dominant long-range repulsive interaction, hydration repulsion and steric hindrance between PI headgroups from apposing bilayers provide the major barriers for the close approach of adjacent PI bilayers for repeat periods less than 5.4 nm. This structural analysis also indicates that the phosphoinositol group extends from the plane of the bilayer into the fluid space between adjacent bilayers. This extended orientation for the headgroup is consistent with electrophoretic measurements on PI vesicles.     

2H and 13C nuclear magnetic resonance study of N-palmitoylgalactosylsphingosine (cerebroside)/cholesterol bilayers.

13C- and 2H-NMR experiments were used to examine the phase behavior and dynamic structures of N-palmitoylgalactosylsphingosine (NPGS) (cerebroside) and cholesterol (CHOL) in binary mixtures. 13C spectra of 13C=O-labeled and 2H spectra of [7,7-2H2] chain-labeled NPGS as well as 3 alpha-2H1 CHOL indicate that cerebroside and CHOL are immiscible in binary mixtures at temperatures less than 40 degrees C. In contrast, at 40 degrees C < t < or = T(C) (NPGS), up to 50 mol% CHOL can be incorporated into melted cerebroside bilayers. In addition, 13C and 2H spectra of melted NPGS/CHOL bilayers show a temperature and cholesterol concentration dependence. An analysis of spectra obtained from the melted 13C=O NPGS bilayer phase suggests that the planar NH-C=O group assumes an orientation tilted 40 degrees-55 degrees down from the bilayer interface. The similarity between the orientation of the amide group relative to the bilayer interface in melted bilayers and in the crystal structure of cerebroside suggests that the overall crystallographic conformation of cerebroside is preserved to a large degree in hydrated bilayers. Variation of temperature from 73 degrees to 86 degrees C and CHOL concentration from 0 to 51 mol% results in small changes in this general orientation of the amide group. 2H spectra of chain-labeled NPGS and labeled CHOL in NPGS/CHOL bilayer demonstrate that molecular exchange between the gel and liquid-gel (LG) phases is slow on the 2H time scale, and this facilitates the simulation of the two component 2H spectra of [7,7-2H2]NPGS/CHOL mixtures. Simulation parameters are used to quantitate the fractions of gel and LG cerebroside. The quadrupole splitting of [7,7-2H2]NPGS/CHOL mixtures and 2H simulations allows the LG phase bilayer fraction to be characterized as an equimolar mixture of cerebroside and CHOL.     

Effects of monovalent anions of the hofmeister series on DPPC lipid bilayers Part II: modeling the perpendicular and lateral equation-of-state

The effects of Hofmeister anions on the perpendicular and lateral equation-of-state (EOS) of the dipalmitoylphosphatidylcholine lamellar phase discussed in the companion article are here examined using appropriate free energy models for the intra- and interbilayer interactions. Minimizing the free energy with respect to the two basic geometrical parameters of the lamellar phase, which are the interbilayer water thickness, d(w), and the lipid headgroup area, a(L), provides the perpendicular (osmotic pressure balance) and lateral EOS. Standard models were used for the hydration, undulation, and Van der Waals attractive force between the bilayers in the presence of electrolytes whereas two alternative treatments of electrostatic interactions were used to obtain "binding" or "partitioning" constants of anions to the lipid bilayers both in the absence and in the presence of sodium binding. The computed binding constants depend on anion type and follow the Hofmeister series, but were found to increase with electrolyte concentration, implying that the local binding approximation cannot fit bilayer repulsion data. The partitioning model was also found inadequate at high electrolyte concentrations. The fitting attempts revealed two additional features worthy of future investigation. First, at maximum swelling in the presence of electrolytes the osmotic pressure of the bilayer system cannot be set equal to zero. Second, at high salt concentrations an additional repulsion appears to come into effect in the presence of strongly adsorbing anions such as I(-) or SCN(-). Both these phenomena may reflect an inconsistent treatment of the ion-surface interactions, which have an impact on the osmotic pressure. Alternatively, they may arise from bulk solution nonidealities that cannot be handled by the classical Poisson-Boltzmann formalism. The inability of current models to explain the "lateral" EOS by fitting the area per lipid headgroup as a function of salt type and concentration shows that current understanding of phospholipid-ion interactions is still very incomplete.     

Swelling of phospholipids by monovalent salt

Critical to biological processes such as membrane fusion and secretion, ion-lipid interactions at the membrane-water interface still raise many unanswered questions. Using reconstituted phosphatidylcholine membranes, we confirm here that multilamellar vesicles swell in salt solutions, a direct indication that salt modifies the interactions between neighboring membranes. By varying sample histories, and by comparing with data from ion carrier-containing bilayers, we eliminate the possibility that swelling is an equilibration artifact. Although both attractive and repulsive forces could be modified by salt, we show experimentally that swelling is driven primarily by weakening of the van der Waals attraction. To isolate the effect of salt on van der Waals interactions, we focus on high salt concentrations at which any possible electrostatic interactions are screened. By analysis of X-ray diffraction data, we show that salt does not alter membrane structure or bending rigidity, eliminating the possibility that repulsive fluctuation forces change with salt. By measuring changes in interbilayer separation with applied osmotic stress, we have determined, using the standard paradigm for bilayer interactions, that 1 M concentrations of KBr or KCl decrease the van der Waals strength by 50%. By weakening van der Waals attractions, salt increases energy barriers to membrane contact, possibly affecting cellular communication and biological signaling.     

SFG studies on interactions between antimicrobial peptides and supported lipid bilayers

The mode of action of antimicrobial peptides (AMPs) in disrupting cell membrane bilayers is of fundamental importance in understanding the efficiency of different AMPs, which is crucial to design antibiotics with improved properties. Recent developments in the field of sum frequency generation (SFG) vibrational spectroscopy have made it a powerful and unique biophysical technique in investigating the interactions between AMPs and a single substrate supported planar lipid bilayer. We will review some of the recent progress in applying SFG to study membrane lipid bilayers and discuss how SFG can provide novel information such as real-time bilayer structure change and AMP orientation during AMP-lipid bilayer interactions in a very biologically relevant manner. Several examples of applying SFG to monitor such interactions between AMPs and a dipalmitoyl phosphatidylglycerol (DPPG) bilayer are presented. Different modes of actions are observed for melittin, tachyplesin I, d-magainin 2, MSI-843, and a synthetic antibacterial oligomer, demonstrating that SFG is very effective in the study of AMPs and AMP-lipid bilayer interactions.     

Investigation of phospholipid area compression induced by calcium-mediated dextran sulfate interaction.

The association of anionic polyelectrolytes such as dextran sulfate (DS) to zwitterionic phospholipid surfaces via Ca(2+) bridges results in a perturbation of lipid packing at physiologically relevant Ca(2+) concentrations. Lipid area compression was investigated in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar bilayer dispersions by (2)H-NMR and in monolayer studies. Binding of DS to DMPC surfaces via Ca(2+) results in denser lipid packing, as indicated by higher lipid chain order. DMPC order parameters are homogeneously increased throughout the lipid bilayer. Higher order translates into more extended hydrocarbon chains and decreased average lipid area per molecule. Area compression is reported as a function of DS concentration and molecular weight. Altering the NaCl and Ca(2+) concentrations modified electrostatic interactions between DS and phospholipid. A maximal area reduction of DeltaA = 2.7 A(2) per DMPC molecule is observed. The lipid main-phase transition temperature increases upon formation of DMPC/Ca(2+)/DS-complexes. Lipid area compression after addition of DS and Ca(2+) to the subphase was also observed in monolayer experiments. A decrease in surface tension of up to 3.5 mN/m at constant molecular area was observed. DS binds to the lipid headgroups by formation of Ca(2+) bridges without penetrating the hydrophobic region. We suggest that area compression is the result of an attractive electrostatic interaction between neighboring lipid molecules induced by high local Ca(2+) concentration due to the presence of DS. X-ray diffraction experiments demonstrate that DS binding to apposing bilayers reduces bilayer separation. We speculate that DS binding alters the phase state of low-density lipoproteins that associate with polyelectrolytes of the arterial connective tissue in the early stages of arteriosclerosis.