Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions

Interactions of carbonyl 13C-enriched triacylglycerols (TG) with phospholipid bilayers [egg phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), and an ether-linked phosphatidylcholine] were studied by 13C NMR spectroscopy. Up to 3 mol % triolein (TO) or tripalmitin (TP) was incorporated into DPPC vesicles by cosonication of the TG and DPPC at approximately 50 degrees C. NMR studies were carried out in a temperature range (30-50 degrees C) in which pure TO is a liquid whereas pure TP is a solid. In spectra of DPPC vesicles with TG at 40-50 degrees C, both TO and TP had narrow carbonyl resonances, indicative of rapid motions, and chemical shifts indicative of H bonding of the TG carbonyls with solvent (H2O) at the aqueous interfaces of the vesicle bilayer. Below the phase transition temperature of the DPPC/TG vesicles (approximately 36 degrees C), most phospholipid peaks broadened markedly. In DPPC vesicles with TP, the TP carbonyl peaks broadened beyond detection below the transition, whereas in vesicles with TO, the TO carbonyl peaks showed little change in line width or chemical shift and no change in the integrated intensity. Thus, in the gel phase, TP solidified with DPPC, whereas TO was fluid and remained oriented at the aqueous interfaces. Egg PC vesicles incorporated up to 2 mol % TP at 35 degrees C; the TP carbonyl peaks had line-width and chemical shift values similar to those for TP (or TO) in liquid-crystalline DPPC. TO incorporated into ether-linked PC had properties very similar to TO in ester-linked PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A2: a 13C NMR study

A new approach to study phospholipase A2 mediated hydrolysis of phospholipid vesicles, using 13C NMR spectroscopy, is described. [13C]Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A2 (Crotalus adamanteus). The 13C-labeled carboxyl/carbonyl peaks from the products [lyso-1-palmitoylphosphatidylcholine (LPPC) and palmitic acid (PA)] were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was approximately 50% ionized at pH approximately 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of 13C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

Interactions of acyl carnitines with model membranes: a (13)C-NMR study

Esterification of fatty acids with the small polar molecule carnitine is a required step for the regulated flow of fatty acids into mitochondrial inner matrix. We have studied the interactions of acyl carnitines (ACs) with model membranes [egg yolk phosphatidylcholine (PC) vesicles] by (13)C-nuclear magnetic resonance (NMR) spectroscopy. Using AC with (13)C-enrichment of the carbonyl carbon of the acyl chain, we detected NMR signals from AC on the inside and outside leaflets of the bilayer of small unilamellar vesicles prepared by cosonication of PC and AC. However, when AC was added to the outside of pre-formed PC vesicles, only the signal for AC bound to the outer leaflet was observed, even after hours at equilibrium. The extremely slow transmembrane diffusion ("flip-flop") is consistent with the zwitterionic nature of the carnitine head group and the known requirement of transport proteins for movement of ACs through the mitochondrial membrane. The partitioning of ACs (8-18 carbons) between water and PC vesicles was studied by monitoring the [(13)C]carbonyl chemical shift of ACs as a function of pH and concentration of vesicles. Significant partitioning into the water phase was detected for ACs with chain lengths of 12 carbons or less. The effect of ACs on the integrity of the bilayer was examined in vesicles with up to 25 mol% myristoyl carnitine; no gross disruption of the bilayer was observed. We hypothesize that the effects of high levels of long-chain AC (as found in ischemia or in certain diseases) on cell membranes result from molecular effects on membrane functions rather than from gross disruption of the lipid bilayer.     

Dynorphin induced magnetic ordering in lipid bilayers as studied by P NMR spectroscopy

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T_m=24°C), as examined by ³¹P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T_m but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T_m. These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T_m in parallel with the bilayer surface, because a single ³¹P NMR signal appeared at the perpendicular position of the ³¹P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T_m, because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated ³¹P NMR lineshape.     

Dioxygen transmembrane distributions and partitioning thermodynamics in lipid bilayers and micelles

Cellular respiration, mediated by the passive diffusion of oxygen across lipid membranes, is key to many basic cellular processes. In this work, we report the detailed distribution of oxygen across lipid bilayers and examine the thermodynamics of oxygen partitioning via NMR studies of lipids in a small unilamellar vesicle (SUV) morphology. Dissolved oxygen gives rise to paramagnetic chemical shift perturbations and relaxation rate enhancements, both of which report on local oxygen concentration. From SUVs containing the phospholipid sn-2-perdeuterio-1-myristelaidoyl, 2-myristoyl-sn-glycero-3-phosphocholine (MLMPC), an analogue of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), we deduced the complete trans-bilayer oxygen distribution by measuring (13)C paramagnetic chemical shifts perturbations for 18 different sites on MLMPC arising from oxygen at a partial pressure of 30 bar. The overall oxygen solubility at 45 °C spans a factor of 7 between the bulk water (23.7 mM) and the bilayer center (170 mM) and is lowest in the vicinity of the phosphocholine headgroup, suggesting that oxygen diffusion across the glycerol backbone should be the rate-limiting step in diffusion-mediated passive transport of oxygen across the lipid bilayer. Lowering of the temperature from 45 to 25 °C gave rise to a slight decrease of the oxygen solubility within the hydrocarbon interior of the membrane. An analysis of the temperature dependence of the oxygen solubility profile, as measured by (1)H paramagnetic relaxation rate enhancements, reveals that oxygen partitioning into the bilayer is entropically favored (ΔS° = 54 ± 3 J K(-1) mol(-1)) and must overcome an enthalpic barrier (ΔH° = 12.0 ± 0.9 kJ mol(-1)).     

A combined NMR and molecular dynamics study of the transmembrane solubility and diffusion rate profile of dioxygen in lipid bilayers

The transmembrane profile of oxygen solubility and diffusivity in a lipid bilayer was assessed by (13)C NMR of the resident lipids (sn-2-perdeuterio-1-myristelaidoyl-2-myristoyl-sn-glycero-3-phosphocholine) in combination with molecular dynamics (MD) simulations. At an oxygen partial pressure of 50 atm, distinct chemical shift perturbations of a paramagnetic origin were observed, spanning a factor of 3.2 within the sn-1 chain and an overall factor of 10 from the headgroup to the hydrophobic interior. The distinguishing feature of the (13)C NMR shift perturbation measurements, in comparison to ESR and fluorescence quenching measurements, is that the local accessibility of oxygen is achieved for nearly all carbon atoms in a single experiment with atomic resolution and without the use of a probe molecule. MD simulations of an oxygenated and hydrated lipid bilayer provided an immersion depth distribution of all carbon nuclei, in addition to the distribution of oxygen concentration and diffusivity with immersion depth. All oxygen-induced (13)C NMR chemical shift perturbations could be reasonably approximated by simply accounting for the MD-derived immersion depth distribution of oxygen in the bilayer, appropriately averaged according to the immersion depth distribution of the (13)C nuclei. Second-order effects in the paramagnetic shift are attributed to the collisionally accessible solid angle or to the propensity of the valence electrons in the vicinity of a given nuclear spin to be polarized or delocalized by oxygen. A method is presented to measure such effects. The excellent agreement between MD and NMR provides an important cross-validation of the two techniques.     

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

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

The perturbation of model membranes by (-)-delta 9-tetrahydrocannabinol. Studies using solid-state 2H- and 13C-NMR

The effects of (-)-delta 9-tetrahydrocannabinol (delta 9-THC) on model phospholipid membranes were studied using solid-state 2H and 13C nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine with specific 2H- and 13C-labels as endogenous probes at the C7, methylene and the carbonyl groups, respectively, of the sn-2 chain were used to study the conformational and dynamic properties of the bilayer as a function of temperature and drug concentration. The drug molecule decreases the phase transition temperature of the bilayer in a concentration dependent manner up to 20 molar percent when full saturation has occurred. The 2H spectra show that delta 9-THC broadens the phase transition during which the spectra acquire a characteristic shape of a two-component system exchanging at an intermediate rate (approximately 10(6) s-1) with some liquid crystalline features. Such spectra provide information related to the melting of the phospholipid chains. At intermediate temperatures, the 13C spectra show a gel-like and a liquid-crystalline-like exchanging components and provide information about a conformational change at the phospholipid glycerol backbone occurring at or near the pretransition. The spectral composition and rate of exchange are both dependent on drug concentration. We have carried out computer simulations of the 13C spectra and obtained conformational information related to the phase transition process in the bilayer from gel to liquid crystal. Our studies show that delta 9-THC has a stronger effect on the sn-2 carbonyl near the bilayer interface than on the lipid chains and serve to describe the membrane perturbing effects of cannabinoids in molecular terms.     

Physical studies of the interaction between detergent-solubilized cholesterol and PC vesicles

The ¹³C NMR spectrum of [4-¹³C] cholesterol solubilized in aqueous solutions of sodium dodecyl sulfate indicates that the molecule has considerably more rotational freedom than when it is intercalated into phosphatidylcholine (PC) vesicles. The interaction between detergent-solubilized cholesterol and PC vesicles has been studied by both NMR and gel permeation chromatography. Our results indicate that greater that 90% of the cholesterol is slowly transferred into the PC bilayer when the detergent-solubilized cholesterol is mixed with PC vesicles.     

Influence of cholesterol on bilayers of ester- and ether-linked phospholipids. Permeability and 13C-nuclear magnetic resonance measurements

13C-NMR and permeability studies are described for sonicated vesicles of phosphatidylcholines bearing two 16-carbon saturated hydrocarbon chains with (a) one ether linkage at carbon 1 (3) or 2 of glycerol and one ester linkage at carbon 2 or 1 (3) of glycerol; (b) two ether linkages and (c) two ester linkages at carbons 1 (3) and 2 of glycerol. The results of 13C-NMR relaxation enhancement measurements using cholesterol enriched with 13C at the 4 position indicate that no significant relocation of the cholesterol molecules takes place in the bilayer when a methylene group is substituted for a carbonyl group in phosphatidylcholine. The 4-13C atom of cholesterol undergoes similar fast anisotropic motions in diester- and diether -phosphatidylcholine bilayers, as judged by spin-lattice relaxation time measurements in the liquid-crystalline phase; although the fast motions are unaltered, linewidth and spin-spin relaxation time measurements suggested some restriction of the slow motions of cholesterol molecules in bilayers from phosphatidylcholines containing an O-alkyl linkage at the sn-2 position instead of an acyl linkage. At temperatures above the gel to liquid-crystal phase transition, the kinetics of ionophore A23187-mediated 45Ca2+ efflux from vesicles prepared from each type of phosphatidylcholine molecule were the same; the kinetics of spontaneous carboxyfluorescein diffusion from diester- and diether -phosphatidylcholine vesicles were the same, whereas mixed ether/ester phosphatidylcholine molecules gave bilayers which are less permeable. The rate constants were reduced on cholesterol incorporation into the bilayers of each type of phosphatidylcholine molecule. The reductions were not statistically significant for 45Ca2+ release. The rate constants for carboxyfluorescein release were also reduced by cholesterol to the same extent in vesicles from diester-, diether -, and 1-ether, and 1-ether-2-ester-phosphatidylcholines; however, a smaller reduction was noted in bilayers from the 1-ester-2-ether analog. The results provide further evidence that there are no highly specific requirements for ester or ether linkages in phosphatidylcholine for cholesterol to reduce bilayer permeability. This is a reflection of the fact that in both diester- and diether -phosphatidylcholine bilayers, the 4-13C atom of cholesterol is located in the region of the acyl carboxyl group or the glyceryl ether oxygen atom.     

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.     

Investigating the impact of cholesterol on magnetically aligned sphingomyelin/cholesterol multilamellar vesicles using static 31P NMR

The effect of cholesterol (5–40 mol%) on the magnetic induced orientation of sphingomyelin/cholesterol multilamellar vesicles (MLVs) was examined using static solid state ³¹P NMR spectroscopy. The orientation was modeled assuming an ellipsoidal deformation of the vesicles and was monitored as a function of cholesterol concentration and temperature. In addition, the static ³¹P chemical shift anisotropy (CSA) was used to assess the motional and dynamical changes occurring in the bilayer are reported. An exploration of the factors determining the magnetic orientation in sphingomyelin/cholesterol bilayers from the gel (s_o) to liquid crystalline (or liquid-ordered, l_o) phases is presented and discussed.     

Dynamics and orientation of transmembrane peptide from bacteriorhodopsin incorporated into lipid bilayer as revealed by solid state (31)P and (13)C NMR spectroscopy.

13C and (31)P NMR spectra of a transmembrane peptide, [1-(13)C]Ala(14)-labeled A(6-34), of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine (DMPC) bilayer were recorded to clarify its dynamics and orientation in the lipid bilayer. This peptide is shown to take an alpha-helical form both in liquid crystalline and gel phases, as viewed from the conformation dependent (13)C chemical shifts. In addition, this peptide undergoes rapid rigid-body rotation about the helical axis at ambient temperature as viewed from the axially symmetric (13)C chemical shift anisotropy, whereas this symmetric anisotropy is changed to an asymmetric pattern at temperatures below 10 degrees C. We further incorporated the peptide into the spontaneously aligned DMPC bilayer to applied magnetic field, induced by dynorphin (dynorphin:DMPC =1:10), a heptadeca-opioid peptide with very high affinity to opioid receptor, in order to gain insight into its orientation in the bilayer. This magnetically aligned system turned out to be persistent even at 0 degrees C as viewed from (31)P NMR spectra of the lipid bilayer, after this peptide was incorporated into this system [A(6-34): dynorphin: DMPC = 4:10:100]. It was found from the (13)C NMR spectra of [1-(13)C]Ala(14) A(6-34) that the helical axis of A(6-34) is oriented parallel to the bilayer normal irrespective of the presence or absence of reorientation motion about the helical axis at a temperature above the lowered gel to liquid crystalline phase transition.     

Assembly of the endoplasmic reticulum phospholipid bilayer: the phosphatidylcholine transporter

Phospholipids are synthesized and concomitantly inserted on the cytoplasmic surface of the endoplasmic reticulum. Assembly of the phospholipid bilayer requires translocation to the lumenal monolayer. The hypothesis that rat liver microsomes contain a protein transporter, or "flippase," for phosphatidylcholine was tested by measuring the transport of sn-1,2,-dibutyroylphosphatidylcholine (diC4PC). This homolog retains the polar head group, the portion of the phospholipid unable to undergo spontaneous transmembrane movement in vesicles, and its water solubility permits application of standard transport methods. DiC4PC entered the lumenal compartment of microsomal vesicles. Transport was saturable and was dependent on time, amount of microsomes, and an intact permeability barrier. DiC4PC transport was inhibited by structural analogs (but not by sn-2,3-diC4PC) and by treatment of microsomes with proteases, N-ethylmaleimide, and trinitrobenzenesulfonic acid. These data suggest that the transmicrosomal movement of diC4PC is protein mediated. DiC4PC was not transported across PC vesicles or red cell membranes, where PC translocation is slow.     

Investigating structural changes in the lipid bilayer upon insertion of the transmembrane domain of the membrane-bound protein phospholamban utilizing 31P and 2H solid-state NMR spectroscopy

Phospholamban (PLB) is a 52-amino acid integral membrane protein that regulates the flow of Ca(2+) ions in cardiac muscle cells. In the present study, the transmembrane domain of PLB (24-52) was incorporated into phospholipid bilayers prepared from 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC). Solid-state (31)P and (2)H NMR experiments were carried out to study the behavior of POPC bilayers in the presence of the hydrophobic peptide PLB at temperatures ranging from 30 degrees C to 60 degrees C. The PLB peptide concentration varied from 0 mol % to 6 mol % with respect to POPC. Solid-state (31)P NMR spectroscopy is a valuable technique to study the different phases formed by phospholipid membranes. (31)P NMR results suggest that the transmembrane protein phospholamban is incorporated successfully into the bilayer and the effects are observed in the lipid lamellar phase. Simulations of the (31)P NMR spectra were carried out to reveal the formation of different vesicle sizes upon PLB insertion. The bilayer vesicles fragmented into smaller sizes by increasing the concentration of PLB with respect to POPC. Finally, molecular order parameters (S(CD)) were calculated by performing (2)H solid-state NMR studies on deuterated POPC (sn-1 chain) phospholipid bilayers when the PLB peptide was inserted into the membrane.     

Molecular dynamics of the local anesthetic tetracaine in phospholipid vesicles

Upon introduction into phosphatidylcholine vesicles, the 13C magnetic resonance peaks of the aromatic resonances of tetracaine are broadened while the T1 relaxation times show little change. Addition of tetracaine to vesicles containing 30% cholesterol produces a similar broadening in the 13C NMR spectrum of tetracaine. Nuclear magnetic resonance parameters of phosphatidylcholine in vesicles which are unchanged by the addition of equimolar tetracaine include 13C T1 relaxation time and 31P linewidth, T1 relaxation time, and nuclear Overhauser effect enhancement. These results are interpreted as indicating a hydrophobic interaction between hydrocarbon portions of the anesthetic and phospholipid bilayer. The rotational correlation time of tetracaine about its long axis in the vesicles has been calculated from the 13C NMR spin lattice relaxation times to be about 10(-10.3) s and is unchanged by incorporation into the phospholipid bilayer. The positively charged ammonium group of tetracaine interacts with the negatively charged phosphate group of the vesicle lipids. Using shift reagents and 31P NMR, tetracaine has been shown to displace cations from the bilayer surface, and does not undergo fast flip-flop across the vesicle bilayer.     

Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure

By use of neutron diffraction, the structural parameters of oriented multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine with deuteriocarbon chains/cholesterol (molar ratio 70:30), multilamellar lipid vesicles composed of pure lipids and lipid/cholesterol mixtures, and crystalline purple membrane patches from Halobacterium halobium have been measured at pressures up to 2 kbar. Pressurization of the oriented 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/cholesterol multilayers results in an in-plane compression with the mean deuteriocarbon chain spacing of 4.44 A obtained under ambient conditions decreasing by 3-7% at 1.9 kbar. The thickness for this bilayer increases by approximately equal to 1.5 A, but the net bilayer volume decreases and the isothermal compressibility is estimated to be in the range (-0.1 to -0.6) X 10(-4)/bar at 19.0 degrees C. The d spacings for multilamellar vesicles composed of lipids in the liquid crystalline state and lipid/cholesterol mixtures increase linearly as a function of pressure, suggesting that these bilayers are also compressed in the membrane plane. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine MLVs in the gel state, the d spacing decreases with pressure. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, the hexagonally packed chains are anisotropically compressed in the bilayer plane, resulting in a pseudohexagonal chain packing at 1.9 kbar. The bilayer compressibility is (-0.4 or -0.5) X 10(-4)/bar depending on whether the chain tilt increases with pressure or terminal methyl groups of apposing lipid monolayers approach each other.(ABSTRACT TRUNCATED AT 250 WORDS)