Monounsaturated PE does not phase-separate from the lipid raft molecules sphingomyelin and cholesterol: role for polyunsaturation?

We investigated interactions of the lipid raft molecules sphingomyelin (SM) and cholesterol (CHOL) in monolayers and bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (POPE) or 1-palmitoyl-2-docosahexaenoyl-sn-glycerophosphatidylethanolamine (PDPE) at 35 degrees C. Techniques employed were pressure-area (pi-A) isotherms generated from Langmuir-Blodgett films, solid-state (2)H and (31)P NMR spectroscopies, and differential scanning calorimetry (DSC). Condensation calculated from pi-A isotherms and reduction in the enthalpy of the gel-liquid-crystalline transition in DSC scans showed CHOL has a strong affinity for POPE, comparable to that observed between SM-CHOL. Order parameters derived from (2)H NMR spectra of the perdeuterated sn-1 chain of POPE-d(31) increased by >50% upon addition of equimolar CHOL to POPE-d(31)/SM (1:1 mol) bilayers. Close proximity of CHOL to POPE even in the presence of SM is indicated. Chemical shift anisotropy (Deltasigma(csa)) measured from (1)H-decoupled (31)P NMR spectra also implied intimate lipid mixing in POPE/SM/CHOL (1:1:1 mol). In contrast, pi-A isotherms and corroborating DSC studies of PDPE/SM (1:1 mol) indicate phase separation between SM and PDPE, which was maintained in the presence of CHOL. The cholesterol-associated increase in order of the perdeuterated sn-1 chain of PDPE determined by (2)H NMR was 2-fold less for PDPE-d(31)/SM/CHOL (1:1:1 mol) than POPE-d(31)/SM/CHOL (1:1:1 mol). Our findings support the notion that acyl chain dependent lateral phase separation occurs in the presence of a docosahexaenoic acid (DHA)-containing phospholipid (PDPE), but not an oleic acid-containing phospholipid (POPE). We propose that monounsaturated lipids do not promote formation of stable lipid rafts and that polyunsaturation may be important for raft stability.     

Docosahexaenoic acid: membrane properties of a unique fatty acid

Docosahexaenoic acid (DHA) with 22-carbons and 6 double bonds is the extreme example of an omega-3 polyunsaturated fatty acid (PUFA). DHA has strong medical implications since its dietary presence has been positively linked to the prevention of numerous human afflictions including cancer and heart disease. The PUFA, moreover, is essential to neurological function. It is remarkable that one simple molecule has been reported to affect so many seemingly unrelated biological processes. Although details of a molecular mode of action remain elusive, DHA must be acting at a fundamental level common to many tissues that is related to the high degree of conformational flexibility that the multiple double bonds have been identified to confer. One likely target for DHA action is at the cell membrane where the fatty acid is known to readily incorporate into membrane phospholipids. Once esterified into phospholipids DHA has been demonstrated to significantly alter many basic properties of membranes including acyl chain order and "fluidity", phase behavior, elastic compressibility, permeability, fusion, flip-flop and protein activity. It is concluded that DHA's interaction with other membrane lipids, particularly cholesterol, may play a prominent role in modulating the local structure and function of cell membranes.     

Stable cubic phases in codispersions of glucocerebroside and palmitoyloleoylphosphatidylethanolamine

The effect of glucocerebroside (GlcCer) on the structure and thermotropic phase behavior of aqueous dispersions of palmitoyloleoylphosphatidylethanolamine (POPE) has been examined using simultaneous small-angle and wide-angle X-ray diffraction methods. Binary mixtures of GlcCer:POPE in molar ratios of 2:100, 5:100, 10:100, 20:100, 30:100, and 40:100 were examined in the temperature range 20-90 degrees C. Cubic phase has been observed in binary mixtures comprised of molar ratios greater than 5:100 in the temperature range of 60-90 degrees C upon heating at a rate of 2 degrees C/min. The cubic phase is relatively stable and coexists with inverted hexagonal or lamellar phases. It persists in the codispersions throughout subsequent cooling scans to 30 degrees C. The space group of the cubic phase is determined to be Pn3m or Pn3. The lattice constant of the Pn3m cubic phase was found to be almost constant when it coexists with lamellar liquid-crystal phase. Marked temperature-dependent changes were observed when cubic phase coexists with hexagonal phase or lamellar-gel phases. This is the first report of cubic phases formed by codispersions of glycosphingolipids and phospholipids. The mechanism of cubic phase formation and the interaction between GlcCer and POPE is discussed in terms of the putative biological functions of glycolipids.     

Bilayer thickness modulates the conductance of the BK channel in model membranes

The conductance of the BK channel was evaluated in reconstituted bilayers made of POPE/POPS (3.3:1), or POPE/POPS with an added 20% of either SPM (3.3:1:1), CER (3.3:1:1), or CHL (3.3:1:1). The presence of SPM, which is known to increase bilayer thickness, significantly reduced the conductance of the BK channel. To directly test the role of membrane thickness, the conductance of the BK channel was measured in bilayers formed from PCs with acyl chains of increasing length (C14:1-C24:1), all in the absence of SPM. Slope conductance was maximal at a chain length of (C18:1) and much reduced for both thinner (C14:1) and thicker (C24:1) bilayers, indicating that membrane thickness alone can modify slope conductance. Further, in a simplified binary mixture of DOPE/SPM that forms a confined, phase-separated bilayer, the measured conductance of BK channels shows a clear bimodal distribution. In contrast, the addition of CER, which has an acyl chain structure similar to SPM but without its bulky polar head group to POPE/POPS, was without effect, as was the addition of CHL. The surface structure of membranes made from these same lipid mixtures was examined with AFM. Incorporation of both SPM and CER resulted in the formation of microdomains in POPE/POPS monolayers, but only SPM promoted a substantial increase in the amount of the high phase observed for the corresponding bilayers. The addition of CHL to POPE/POPS eliminated the phase separation observed in the POPE/POPS bilayer. The decrease in channel conductance observed with the incorporation of SPM into POPE/POPS membranes was, therefore, attributed to larger SPM-rich domains that appear thicker than the neighboring bilayer.     

Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane

It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE). Two techniques are employed, pressure-area isotherms on monolayers to examine condensation and interfacial elasticity behavior, and differential scanning calorimetry (DSC) on bilayers to evaluate phase separations. Significant levels of condensation are observed for mixtures of SM and cholesterol. Surface elasticity measurements indicate that cholesterol decreases and SDPE increases the in-plane elasticity of SM monolayers. At X(SDPE)> or =0.15 in SM, a more horizontal region emerges in the pressure-area isotherms indicating 'squeeze out' of SDPE from the monolayers. Addition of cholesterol to equimolar amounts of SM and SDPE further increases the amount of 'squeeze out', supporting the concept of phase separation into a cholesterol- and SM-rich liquid ordered phase and a SDPE-rich liquid disordered phase. This conclusion is corroborated by DSC studies where as little as X(Chol)=0.0025 induces a phase separation between the two lipids.     

A 2H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

A H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

Cubic phase is induced by cholesterol in the dispersion of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine

The effect of cholesterol, a major constituent of eukaryotic cell membranes, on the structure and thermotropic phase behaviour of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) dispersed in excess water was examined by synchrotron X-ray diffraction methods. Temperature scans over the range 10-75 degrees C showed that the gel to liquid-crystalline phase transition decreased from 25 to 10 degrees C in the presence of 20 mol% cholesterol, and no gel phase could be detected in the wide-angle X-ray scattering (WAXS) intensity profile of mixtures containing 35 mol% cholesterol. The small-angle X-ray scattering (SAXS) intensity profiles showed that the lamellar to nonlamellar phase transition temperature was also decreased in mixtures containing up to 30 mol% cholesterol but the trend was reversed in mixtures containing a higher proportion of cholesterol. There was evidence that the transition of the lamellar liquid-crystal phase is to cubic phases in mixtures containing less than 30 mol% cholesterol. The space group of one of these cubic phases was assigned as Pn3m. This effect of cholesterol on non-bilayer-forming phospholipids is considered in the context of the role of cholesterol in membrane organization and function.     

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (α-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%～5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.     

Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes

Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures. Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L. monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L. monocytogenes, presumably by maintaining membrane fluidity. The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L. monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region. tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C. However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1. No evidence was seen for a clear lipid phase transition in any sample. We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L. monocytogenes membrane that permits growth at refrigeration temperatures.     

Dietary supplementation of N-3 fatty acids and hydroperoxide levels in rat retinas.

Docosahexaenoic acid (DHA) plays an important role in visual and neural development in mammals. In the present study, effect of dietary supplementation with n-3 fatty acids, primarily docosahexaenoic acid (DHA) with high purity, on the fatty acid composition of photoreceptor cells of young rats (fed from 4 weeks) was investigated. DHA in rod outer segment (ROS) membranes was significantly increased in the group of high DHA feeding (9.69% total energy). Other n-3 fatty acids (alpha-linolenic acid (ALA) and eicosapentaenoic acid (EPA)) included in the diets with DHA (0.95%-5.63% total energy) also significantly increased the proportion of DHA compared with the linoleic acid diet groups. However, the proportions of arachidonic acid (ARA) and other long chain n-6 fatty acids (22:4n6 and 22:5n6) were suppressed in these n-3 fatty acids-fed groups. Phospholipid hydroperoxides in ROS membranes were determined using a highly sensitive analytical technique, chemiluminescence-high performance liquid chromatography (CL-HPLC). There was no increasing tendency in the hydroperoxide levels of ROS membranes containing high content of DHA, and phosphatidylethanolamine hydroperoxide (PEOOH) was much lower than phosphatidylcholine hydroperoxide (PCOOH) under normal light conditions, which implies that DHA supplementation does not much affect the peroxidizability of ROS membranes in vivo. But UV irradiation on separated ROS membranes accelerated the formation of phospholipid hydroperoxides in high DHA feeding rats, and PEOOH was produced more efficiently than PCOOH in vitro.     

Cholesterol's inhibition effect on entering of chlorzoxazone into phosphatidylethanolamine bilayer: Relevance to cytochrome P450 drug metabolism at endoplasmic reticulum membranes

Many drugs are metabolized by cytochrome P450 (CYP) in the endoplasmic reticulum (ER) membrane. Recent studies have shown that CYP-substrate drugs reach the CYP active site after entering the lipid hydrophobic part of the ER membrane. To clarify the role of cholesterol (Chol) in the CYP-related drug metabolic process, we investigated the lipid bilayer entry of CYP-substrate drugs using a model membrane system as follows. The model membrane system comprised palmitoyl-oleoyl-phosphatidylethanolamine (POPE) and Chol. Phosphatidylethanolamine is the second major phospholipid component of ER membranes. Chlorzoxazone (CZX) was used as the CYP-substrate drug. Calorimetric measurements showed that the addition of CZX to POPE bilayers decreased the gel–liquid crystal phase transition temperature; X-ray diffraction indicated that CZX distributes into the liquid crystal phase bilayers but not practically the gel phase POPE bilayers. In the presence of Chol, dialysis and X-ray structural analyses showed that Chol inhibited CZX entry into the bilayer with an increase in Chol concentration. The Chol concentration in the ER membrane (5–10 mol%) is much lower than that in the plasma membrane (approximately 30 mol%). This fact may allow CYP-substrate drugs to enter the hydrophobic portion of the ER membrane more easily than other organelle membranes, yielding efficient drug metabolism.     

Endothelial cell fatty acid unsaturation mediates cold-induced oxidative stress

Ultraprofound hypothermia (< 5 degrees C) induces changes to cell membranes such as liquid-to-gel lipid transitions and oxidative stress that have a negative effect on membrane function and cell survival. We hypothesized that fatty acid substitution of endothelial cell lipids and alterations in their unsaturation would modify cell survival at 0 degrees C, a temperature commonly used during storage and transportation of isolated cells or tissues and organs used in transplantation. Confluent bovine aortic endothelial cells were treated with 18-carbon fatty acids (C18:0, C18:1n-9, C18:2n-6, or C18:3n-3), C20:5n-3 or C22:6n-3 (DHA), and then stored at 0 degrees C without fatty acid supplements. Storage of control cells caused the release of lactate dehydrogenase (LDH) and a threefold increase in lipid peroxidation (LPO) when compared to control cells not exposed to cold. Pre-treating cells with C18:0 decreased the unsaturation of cell lipids and reduced LDH release at 0 degrees C by 50%, but all mono- or poly-unsaturated fatty acids increased injury in a concentration-dependent manner and as the extent of fatty acid unsaturation increased. DHA-treatment increased cell fatty acid unsaturation and caused maximal injury at 0 degrees C, which was prevented by lipophilic antioxidants BHT or vitamin E, the iron chelator deferoxamine, and to a lesser extent by vitamin C. Furthermore, the cold-induced increase in LPO was reduced by C18:0, vitamin E, or DFO but enhanced by DHA. In conclusion, the findings implicate iron catalyzed free radicals and LPO as a predominant mechanism of endothelial cell injury at 0 degrees C, which may be reduced by increasing lipid saturation or treating cells with antioxidants.     

Influence of antimicrobial peptides on the formation of nonlamellar lipid mesophases

We have studied the influence of four antimicrobial peptides of different secondary and ternary structure - melittin (Mel), protegrin-1 (PG-1), peptidyl-glycylleucine-carboxyamide (PGLa), and gramicidin S (GS) - on the lamellar-to-nonlamellar transition of palmitoyloleoyl phosphatidylethanolamine (POPE) applying differential scanning calorimetry and small-angle X-ray diffraction. None of the peptides studied led to the formation of an inverted hexagonal phase observed for pure POPE at high temperatures. Instead either cubic or lamellar phases were stabilized to different degrees. GS was most effective in inducing a cubic phase, whereas Mel fully stabilized the lamellar phase. The behavior of POPE in the presence of PG-1 and PGLa was intermediate to GS and Mel. In addition to the known role of membrane elasticity we propose two mechanisms, which cause stabilization of the lamellar phase: electrostatic repulsion and lipid/peptide pore formation. Both mechanisms prevent transmembrane contact required to form either an inverted hexagonal phase or fusion pores, as precursors of the cubic phase.     

Effects of fatty acid unsaturation numbers on membrane fluidity and α-secretase-dependent amyloid precursor protein processing

Fatty acids may integrate into cell membranes to change physical properties of cell membranes, and subsequently alter cell functions in an unsaturation number-dependent manner. To address the roles of fatty acid unsaturation numbers in cellular pathways of Alzheimer's disease (AD), we systematically investigated the effects of fatty acids on cell membrane fluidity and α-secretase-cleaved soluble amyloid precursor protein (sAPP(α)) secretion in relation to unsaturation numbers using stearic acid (SA, 18:0), oleic acid (OA, 18:1), linoleic acid (LA, 18:2), α-linolenic acid (ALA, 18:3), arachidonic acid (AA, 20:4), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6). Treatments of differentiated human neuroblastoma (SH-SY5Y cells) with AA, EPA and DHA for 24h increased sAPP(α) secretion and membrane fluidity, whereas those treatments with SA, OA, LA and ALA did not. Treatments with AA and DHA did not alter the total expressions of amyloid precursor protein (APP) and α-secretases in SH-SY5Y cells. These results suggested that not all unsaturated fatty acids but only those with 4 or more double bonds, such as AA, EPA and DHA, are able to increase membrane fluidity and lead to increase in sAPP(α) secretion. This study provides insights into dietary strategies for the prevention of AD.     

Role of cholesterol in the formation and nature of lipid rafts in planar and spherical model membranes

Sterols play a crucial regulatory and structural role in the lateral organization of eukaryotic cell membranes. Cholesterol has been connected to the possible formation of ordered lipid domains (rafts) in mammalian cell membranes. Lipid rafts are composed of lipids in the liquid-ordered (l(o)) phase and are surrounded with lipids in the liquid-disordered (l(d)) phase. Cholesterol and sphingomyelin are thought to be the principal components of lipid rafts in cell and model membranes. We have used fluorescence microscopy and fluorescence recovery after photobleaching in planar supported lipid bilayers composed of porcine brain phosphatidylcholine (bPC), porcine brain sphingomyelin (bSM), and cholesterol to map the composition-dependence of l(d)/l(o) phase coexistence. Cholesterol decreases the fluidity of bPC bilayers, but disrupts the highly ordered gel phase of bSM, leading to a more fluid membrane. When mixed with bPC/bSM (1:1) or bPC/bSM (2:1), cholesterol induces the formation of l(o) phase domains. The fraction of the membrane in the l(o) phase was found to be directly proportional to the cholesterol concentration in both phospholipid mixtures, which implies that a significant fraction of bPC cosegregates into l(o) phase domains. Images reveal a percolation threshold, i.e., the point where rafts become connected and fluid domains disconnected, when 45-50% of the total membrane is converted to the l(o) phase. This happens between 20 and 25 mol % cholesterol in 1:1 bPC/bSM bilayers and between 25 and 30 mol % cholesterol in 2:1 bPC/bSM bilayers at room temperature, and at approximately 35 mol % cholesterol in 1:1 bPC/bSM bilayers at 37 degrees C. Area fractions of l(o) phase lipids obtained in multilamellar liposomes by a fluorescence resonance energy transfer method confirm and support the results obtained in planar lipid bilayers.     

Transmembrane peptides stabilize inverted cubic phases in a biphasic length-dependent manner: implications for protein-induced membrane fusion

WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan "anchors" at each end. They form membrane-spanning alpha-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) at concentrations up to 0.5 mol % peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (L(alpha)) phase first forms an inverted cubic (Q(II)) phase, and the inverted hexagonal (H(II)) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5 degrees C/h), WALP peptides were shown to decrease the temperatures of Q(II) and H(II) phase formation (T(Q) and T(H), respectively) as a function of peptide concentration. The shortest and longest peptides reduced T(Q) the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the L(alpha)/Q(II) phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion.     

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by ³¹P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.     

Thermodynamics of insertion and self-association of a transmembrane helix: a lipophobic interaction by phosphatidylethanolamine

Thermodynamic parameters for the insertion and self-association of transmembrane helices are important for understanding the folding of helical membrane proteins. The lipid composition of bilayers would significantly affect these fundamental processes, although how is not well understood. Experimental systems using model transmembrane helices and lipid bilayers are useful for measuring and interpreting thermodynamic parameters (ΔG, ΔH, ΔS, and ΔC(p)) for the processes. In this study, the effect of the charge, phase, acyl chain unsaturation, and lateral pressure profile of bilayers on the membrane partitioning of the transmembrane helix (AALALAA)(3) was examined. Furthermore, the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) on the thermodynamics for insertion and self-association of the helix in host membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was investigated in detail. Interbilayer transfer of the helix monomer from POPC to POPC/POPE (1/1) bilayers was unfavorable (ΔG = +4.5 ± 2.9 kJ mol(-1) at 35 °C) due to an increase in enthalpy (ΔH = +31.1 ± 2.1 kJ mol(-1)). On the other hand, antiparallel dimerization of the helices in POPC/POPE (1/1) bilayers was enhanced compared with that in POPC bilayers (ΔΔG = -4.9 ± 0.2 kJ mol(-1) at 35 °C) due to a decrease in enthalpy (ΔΔH = -33.2 ± 1.5 kJ mol(-1)). A greater thickness of POPC/POPE bilayers only partially explained the observed effects. The residual effects could be related to changes in other physical properties such as higher lateral pressure in the hydrocarbon core in the PE-containing membrane. The origin of the enthalpy-driven "lipophobic" force that modulates the insertion and association of transmembrane helices will be discussed.     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.