Effect of hydrostatic pressure on the bilayer phase behavior of symmetric and asymmetric phospholipids with the same total chain length

The bilayer phase transitions of palmitoylstearoyl-phosphatidylcholine (PSPC), diheptadecanoyl-PC (C17PC) and stearoylpalmitoyl-PC (SPPC) which have the same total carbon numbers in the two acyl chains were observed by differential scanning calorimetry and high-pressure optical method. As the temperature increased, these bilayers exhibited four phases of the subgel (Lc), lamellar gel (L beta'), ripple gel (P beta') and liquid crystal (L alpha), in turn. The Lc phase was observed only in the first heating scan after cold storage. The temperatures of the phase transitions were almost linearly elevated by applying pressure. The temperature-pressure phase diagrams and the thermodynamic quantities associated with the phase transitions were compared among the lipid bilayers. For all the bilayers studied, the pressure-induced interdigitated gel (L beta I) phase appeared above the critical interdigitation pressure (CIP) between the L beta' and P beta' phases. The CIPs for the PSPC, C17PC and SPPC bilayers were found to be 50.6, 79.1 and 93.0 MPa, respectively. Contribution of two acyl chains to thermodynamic properties for the phase transitions of asymmetric PSPC and SPPC bilayers was not even. The sn-2 acyl chain lengths of asymmetric PCs governed primarily the bilayer properties. The fluorescence spectra of Prodan in lipid bilayers showed the emission maxima characteristic of bilayer phases, which were dependent on the location of Prodan in the bilayers. Second derivative of fluorescent spectrum exhibited the original emission spectrum of Prodan to be composed of the distribution of Prodan into multiple locations in the lipid bilayer. The F'497/F'430 value, a ratio of second derivative of fluorescence intensity at 497 nm to that at 430 nm, is decisive evidence whether bilayer interdigitation will occur. With respect to the L beta'/L beta I phase transition in the SPPC bilayer, the emission maximum of Prodan exhibited the narrow-range red-shift from 441 to 449 nm, indicating that the L beta I phase in the SPPC bilayer has a less polar "pocket" formed by a space between uneven terminal methyl ends of the sn-1 and sn-2 chains, in which the Prodan molecule remains stably.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe₂PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L_β) or ripple gel (P_β′) phase to the liquid crystalline (L_α) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa⁻¹ depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L_c) phase to the lamellar gel (L_β or L_β′) phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L_c/L_β transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L_c/L_α transition was observed at a temperature higher than the main-transition temperature. The main (L_β/L_α) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L_β′, P_β′ and pressure-induced interdigitated gel (L_βI) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe₂PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

Chain asymmetry alters thermotropic and barotropic properties of phospholipid bilayer membranes

The alignment of the sn-1 and sn-2 acyl chains at the terminal methyl ends generally produces significant influence on the thermodynamic properties of the bilayer phase transitions. We investigated the bilayer phase behavior of asymmetric phospholipids, myristoylpalmitoylphosphatidylcholine and palmitoylmyristoylphosphatidylcholine, by high-pressure light-transmittance and Prodan-fluorescence techniques and differential scanning calorimetry. Constructed temperature-pressure phase diagrams revealed that no stable phase can exist in the whole pressure range because of the formation of the most stable L_c phase. Nevertheless, the pretransition, the detection of which is severely hampered by the exceptionally prompt formation of the L_c phase, was successfully observed. Moreover, the effect of the total and difference of the sn-1 and sn-2 acyl chain lengths on minimal interdigitation pressure (MIP) was summarized in a MIP vs. chain-length inequivalence parameter plot, where the effect was proved to be classified in three zones depending on the alignment of both terminal methyl ends.     

Investigation of phase transitions of saturated phosphocholine lipid bilayers via molecular dynamics simulations

Lipid bilayers play an important role in biological systems as they protect cells against unwanted chemicals and provide a barrier for material inside a cell from leaking out. In this paper, nearly 30 μs of molecular dynamics (MD) simulations were performed to investigate phase transitions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC) lipid bilayers from the liquid crystalline (L_α) to the ripple (P_β) and to the gel phase (L_β). Our MD simulations accurately predict the main transition temperature for the single-component bilayers. A key focus of this work is to quantify the structure of the P_β phase for DMPC and compare with measures from x-ray experiments. The P_β major arm has similar structure to that of the L_β, while the thinner minor arm has interdigitated chains and the transition region between these two regions has large chain splay and disorder. At lower temperatures, our MD simulations predict the formation of the L_β phase with tilted fatty acid chains. The P_β and L_β phases are studied for mixtures of DMPC and DPPC and compare favorably with experiment. Overall, our MD simulations provide evidence for the relevancy of the CHARMM36 lipid force field for structures and add to our understanding of the less-defined P_β phase.     

Miscibility of Sphingomyelins and Phosphatidylcholines in Unsaturated Phosphatidylcholine Bilayers

Polyunsaturated phospholipids are common in biological membranes and affect the lateral structure of bilayers. We have examined how saturated sphingomyelin (SM; palmitoyl and stearoyl SM (PSM and SSM, respectively)) and phosphatidylcholine (PC; dipalmitoyl PC and 1-palmitoyl-2-stearoyl PC (DPPC and PSPC, respectively)) segregate laterally to form ordered gel phases in increasingly unsaturated PC bilayers (sn-1: 16:0 and sn-2: 18:1...22:6; or sn-1 and sn-2: 18:1…22:6). The formation of gel phases was determined from the lifetime analysis of trans-parinaric acid. Using calorimetry, we also determined gel phase formation by PSM and DPPC in unsaturated PC mixed bilayers. Comparing PSM with DPPC, we observed that PSM formed a gel phase with less order than DPPC at comparable bilayer concentrations. The same was true when SSM was compared with PSPC. Furthermore, we observed that at equal saturated phospholipid concentration, the gel phases formed were less ordered in unsaturated PCs having 16:0 in sn-1, as compared to PCs having unsaturated acyl chains in both sn-1 and sn-2. The gel phases formed by the saturated phospholipids in unsaturated PC bilayers did not appear to achieve properties similar to pure saturated phospholipid bilayers, suggesting that complete lateral phase separation did not occur. Based on scanning calorimetry analysis, the melting of the gel phases formed by PSM and DPPC in unsaturated PC mixed bilayers (at 45 mol % saturated phospholipid) had low cooperativity and hence most likely were of mixed composition, in good agreement with trans-parinaric acid lifetime data. We conclude that both interfacial properties of the saturated phospholipids and their chain length, as well as the presence of 16:0 in sn-1 of the unsaturated PCs and the total number of cis unsaturations and acyl chain length (18 to 22) of the unsaturated PCs, all affected the formation of gel phases enriched in saturated phospholipids, under the conditions used.     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro

Cytosolic phospholipase A₂ alpha (cPLA₂α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA₂α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA₂α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA₂α. Such promiscuous binding would explain the previous finding that cPLA₂α has both PLA₁ and PLA₂ activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA₂α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA₂α.     

How does acyl chain length affect thermotropic phase behavior of saturated diacylphosphatidylcholine–cholesterol binary bilayers?

Thermotropic phase behavior of diacylphosphatidylcholine (CnPC)–cholesterol binary bilayers (n = 14–16) was examined by fluorescence spectroscopy using 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and differential scanning calorimetry. The former technique can detect structural changes of the bilayer in response to the changes in polarity around Prodan molecules partitioned in a relatively hydrophilic region of the bilayer, while the latter is sensitive to the conformational changes of the acyl chains. On the basis of the data from both techniques, we propose possible temperature T–cholesterol composition X_ch phase diagrams for these binary bilayers. A notable feature of our phase diagrams, including our previous results for diheptadecanoylphosphatidylcholine (C17PC) and distearoylphosphatidylcholine (C18PC), is that there is a peritectic-like point around X_ch = 0.15, which can be interpreted as indicating the formation of a 1:6-complex of cholesterol and CnPCs within the binary bilayer irrespective of the acyl chain length. We could give a reasonable explanation for such complex formation using the modified superlattice view. Our results also showed that the X_ch value of the abolition of the main transition is almost constant for n = 14–17 (ca. 0.33), while it increases to ca. 0.50 for n = 18. By contrast, a biphasic n-dependence of X_ch was observed for the abolition of the pretransition, suggesting that there are at least two antagonistic n-dependent factors. We speculate that this could be explained by the enhancement of the van der Waals interaction with increases in n and the weakening of the repulsion between the neighboring headgroups with decreases in n.     

Neutron diffraction studies on phosphatidylcholine model membranes. II. Chain conformation and segmental disorder

In this paper neutron diffraction experiments on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine selectively deuterated in the hydrocarbon chains are reported. The experiments were carried out in the gel phase L_β′ and in the liquid crystalline phase L_α. The labelled segments were assumed to have a Gaussian distribution in the projection on the bilayer normal and their mean positions were derived with an accuracy of ±1 ångström unit from a fit to the observed structure factors. The values obtained in the L_β′ phase confirm the model with chains in all trans configuration tilted with respect to the bilayer normal by an angle that increases with water content. From samples that were deuterated in both chains separately and studied at low water content it was seen that the chains of the molecule are out of step by as much as 1.5 carbon-carbon bond lengths. A constant width of the label distribution in the projection on the bilayer normal was observed for segments at the beginning and end of the chains. This is an additional indication for the chains being in the all trans state in L_β′ phase. In the L_α phase, the present experiments show that consecutive segments are well-separated in the profile. The whole chain region is shortened by a factor of ~0.75 compared to the L_β′ phase. In contrast to the gel phase, the width of the label distribution is not constant over the entire region, but is found to be increased by more than a factor of two at the end of the chains. This complements the picture derived by deuterium magnetic resonance experiments, where order parameters and correlation times of segmental motions along the chains, which essentially determine the orientational disorder and angular fluctuations of the segments, were obtained.     

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.     

A time-resolved synchrotron X-ray study of a crystalline phase bilayer transition and packing in a saturated monogalactosyldiacylglycerol-water system

The structural changes associated with a phase transition between the gel-phase bilayer (L_β) in which the acyl chains pack in a hexagonal subcell, and a crystalline bilayer phase (L_C1) where the acyl chains are packed in an orthorhombic subcell in a saturated monogalactosyldiacylglycero-water system are reported. The phase change is cooperative and takes place isothermally after the lamellar-gel phase has been held at 20°C for about 8 min. The transformation of the acyl chain subcell from hexagonal to orthorhombic induces a change in diffraction maxima observed in the region 0.6–0.7 nm which is interpreted as a change in packing of the galactose residues from an orthorhombic to hexagonal subcell. We conclude that the rearrangement of the acyl chains into a more closely packed subcell requires the head groups to reorient to reduce the steric hindrance between the bulky galactose residues.     

Acyl-chain methyl distributions of liquid-ordered and -disordered membranes

A central feature of the lipid raft concept is the formation of cholesterol-rich lipid domains. The introduction of relatively rigid cholesterol molecules into fluid liquid-disordered (L_d) phospholipid bilayers can produce liquid-ordered (L_o) mixtures in which the rigidity of cholesterol causes partial ordering of the flexible hydrocarbon acyl chains of the phospholipids. Several lines of evidence support this concept, but direct structural information about L_o membranes is lacking. Here we present the structure of L_o membranes formed from cholesterol and dioleoylphosphatidylcholine (DOPC). Specific deuteration of the DOPC acyl-chain methyl groups and neutron diffraction measurements reveal an extraordinary disorder of the acyl chains of neat L_d DOPC bilayers. The disorder is so great that >20% of the methyl groups are in intimate contact with water in the bilayer interface. The ordering of the DOPC acyl chains by cholesterol leads to retraction of the methyl groups away from the interface. Molecular dynamics simulations based on experimental systems reveal asymmetric transbilayer distributions of the methyl groups associated with each bilayer leaflet.     

Pressure-induced phase transitions of lipid bilayers observed by fluorescent probes Prodan and Laurdan

The fluorescence spectra of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) and 6-dodecanoyl-2-(dimethylamino)naphthalene (Laurdan) in bilayer membranes of 1,2-distearoylphosphatidylcholine (DSPC) were observed as a function of pressure at constant temperature. The emission spectra of Prodan and Laurdan varied with the pressure-induced states of bilayer membranes. The maximum emission wavelength (lambda(max)) of Prodan characteristic of the liquid crystalline (L(alpha)), lamellar gel (L(beta)') and pressure-induced interdigitated gel (L(beta)I) phases of the DSPC bilayer was 480, 440 and 500 nm, respectively. On the other hand, the lambda(max) of Laurdan characteristic of the L(alpha) and L(beta)' phases was 480 and 440 nm in a similar manner as Prodan probe. However, no change in the lambda(max) was observed in spite of the occurrence of the interdigitation of bilayer. Since the lambda(max) reflects the solvent property around the probe molecules, we could speculate about the location of fluorescent probe in the bilayer membranes. In the L(alpha) phase the same chromophore group of Prodan and Laurdan probes distributes around phosphate group of lipid (i.e., polar region). The transformation of bilayer into the L(beta)' phase causes the Prodan and Laurdan molecules to move into the glycerol backbone (i.e., less polar) region. In the ripple gel (P(beta)') phase, the emission spectrum of Prodan shows a broad peak at about 480 nm and a shoulder around 440 nm, which means that the Prodan molecules are widespread over the wide range from the glycerol backbone to the hydrophilic part of bilayer. The P(beta)'/L(beta)I phase transition causes the Prodan molecule to squeeze out from the glycerol backbone region and to move the hydrophilic region near the bilayer surface. Contrarily, the Laurdan molecule was not squeezed out from the glycerol backbone region because the long acyl chain of Laurdan serves as an anchor in the hydrophobic core of bilayer. The ratio of fluorescence intensity of Prodan at 480 nm to that at 440 nm, F(480)/F(440), is available to observation of bilayer phase transitions. The plot of F(480)/F(440) versus pressure seems to be useful for the recognition of bilayer phase transition, especially the bilayer interdigitation.     

Suppression of Lα/Lβ Phase Coexistence in the Lipids of Pulmonary Surfactant

To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting L_α and L_β phases. In the L_β structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the L_β composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The L_β chains, however, lack the tilt of the L_β′ phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the L_β phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.     

Structural plasticity in self-assembling transmembrane β-sheets

Here we test the hypothesis that membrane-spanning β-sheets can exhibit structural plasticity in membranes due to their ability to shift hydrogen-bonding patterns. Transmembrane β-sheet forming peptides of the sequence AcWL_n, where n = 5, 6, or 7, which range from 21 to 27 Å in maximum length, were incorporated into bilayers made of phosphatidylcholine lipids with saturated acyl chains containing 14, 16, or 18 carbons, which are 36–50 Å in thickness. The effect of the peptide β-sheets on fluid- and gel-phase bilayers were studied with differential scanning calorimetry and circular dichroism spectroscopy. We show that AcWL₅ forms a stable, peptide-rich gel phase in all three lipids. The whole family of AcWL_n peptides appears to form similarly stable, nonmembrane-disrupting β-sheets in all bilayer phases and thicknesses. Bilayers containing up to 20 mol % peptide, which is the maximum concentration tested, formed gel phases with melting temperatures that were equal to, or slightly higher than, the pure lipid transitions. Given the range of peptide lengths and bilayer thicknesses tested, these experiments show that the AcWL_n family of membrane-inserted β-sheets exhibit remarkable structural plasticity in membranes.     

Reorientational and Conformational Ordering Processes at Elevated Pressures in 1,2-Dioleoyl Phosphatidylcholine: A Raman and Infrared Spectroscopic Study

Raman and infrared spectra of fully hydrated bilayers of 1,2-dioleoyl phosphatidylcholine (DOPC) were measured at increasing hydrostatic pressures up to -37 kbar. Under ambient conditions aqueous dispersions of DOPC are in the liquid crystalline state. The application of an external hydrostatic pressure induces conformational and dynamic ordering processes in DOPC, which trigger a first-order structural phase transition at 5 kbar from a disordered liquid crystalline state to a highly ordered gel state. In the gel phase the methylene chains of each molecule are fully extended and the two all-trans chain segments on both sides of the rigid cis double bond form a bent structure. The bent oleoyl chains in each molecule, as well as in neighboring molecules are packed parallel to each other. To achieve this parallel interchain packing, the double bonds of the sn-1 and sn-2 chains of each molecule must be aligned at the same position with respect to the bilayer interface which is achieved by a rotation of the C—C bonds in the glycerol moiety in the head group. The extremely strong interchain interactions in the gel phase of DOPC are unique for this lipid with cis dimono-unsaturated acyl chains. Our experimental results suggest that in the pressure-induced gel phase of DOPC the olefinic CH bonds are rotated out of the phase of the bent oleoyl chains and that the oleoyl chains of opposing bilayers bend towards opposite directions.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

Disc formation in cholesterol-free liposomes during phase transition

Cryogenic transmission electron microscopy (cryo-TEM) images of lysolipid-containing thermosensitive liposomes (LTSL) revealed that open liposomes and bilayer discs appeared when liposomes were cycled through the gel (L_β′) to liquid-crystalline (L_α) phase transition. The amount of bilayer discs generated was dependent on the combined presence of PEG-lipid and lysolipid in the membrane. We hypothesize that micelle-forming membrane components stabilize the rim of bilayer openings and membrane discs that form when liposomes are cycled through T_C.     

Membrane restructuring by phospholipase A2 is regulated by the presence of lipid domains

Secretory phospholipase A₂ (sPLA₂) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (L_α/L_β) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA₂. Specifically, we show that the presence of L_α/L_β and L_α/P_β′ phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich L_α regions, the L_β and P_β′ regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA₂ hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product.