Small bowel rejection in isolated small bowel transplantation and in multivisceral transplantation: a comparative study in a large animal model

31P NMR lineshapes of multilamellar liposomes composed mostly of a bilayer-spanning tetraether lipid are consistent with rapid axially symmetric motion about the bilayer normal. The residual chemical shift anisotropy of 36 ppm is comparable to that seen for diacylphosphatidylglycerol systems and suggests comparable headgroup motion. The lateral diffusion rates for Thermoplasma acidophilum total polar lipids in mutilamellar liposomes was measured by two dimensional exchange NMR as a function of temperature. At 55 degrees C, near the growth temperature, the rate of lateral diffusion, DL, is comparable to that of diester phospholipids in the Lalpha liquid crystalline phase, having a value of 2 x 10(-8) cm2/s. DL decreases with temperature reaching a value of 8-6 x 10(-9) cm2/s at 30 degrees C. The activation energy Ea for lateral diffusion is estimated to be 10 kcal/mol (approximately 42 kJ/mol). The lateral diffusion rates indicate that the tetraether liposomes have a membrane viscosity at 30 degrees C which is considerably higher than that of diester phospholipids in the liquid crystalline phase.     

Bilayer packing characteristics of mixed chain phospholipid derivatives: Raman spectroscopic and differential scanning calorimetric studies of 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine (C(18):C(10)PC) and 1-stearoyl-2-capryl-sn-glycero-3-phospho-N-trimethylpropanolamine (C(18):C(10)TMPC)

Raman spectroscopy and high-sensitivity differential scanning calorimetry (DSC) were used to compare the effects of headgroup conformation on the acyl chain packing arrangements in two highly asymmetric phosphatidylcholine (PC) analogues, 1-stearoyl-2-capryl-sn-glycero-3-phosphocholine (C(18):C(10)PC) and a polar headgroup derivative of C(18):C(10)PC, 1-stearoyl-2-capryl-sn-glycero-3-phospho-N-trimethylpropanolami ne (C(18):C(10)TMPC), which contains an additional methylene group within the choline moiety; namely, -P-O-(CH2)3-N(CH3)3. The C(18):C(10)TMPC headgroup exhibits an extended trans conformation which is independent of bilayer phase. A comparison of gel phase spectral order parameters of the two lipid species indicates a mixed interdigitated state characteristic of three chains per headgroup for C(18): C(10)TMPC. A more intermolecularly ordered liquid crystalline phase is observed, however, for the C(18):C(10)TMPC bilayers. The phase transition cooperative unit size estimated for the C(18):C(10)PC bilayers (approximately 140 molecules per unit) is about 7-fold greater than that for the C(18):C(10)TMPC dispersions (approximately 20 molecules per unit). We suggest that the extended headgroup for C(18):C(10)TMPC induces a slight tilt in the gel phase packing arrangements for the acyl chains, which may persist in the partially interdigitated liquid crystalline phase bilayer. Macroscopically, tighter packed multilamellar dispersions of C(18):C(10)TMPC occur for systems prepared first in the presence of a higher ionic strength medium. The stacked bilayers may then be transferred to a lower ionic strength environment without loss of their more closely packed adjacent lamellae.     

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer

The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.     

Interfacial conformation of dipalmitoylglycerol and dipalmitoylphosphatidylcholine in phospholipid bilayers

Diacylglycerols are minor constituents of membrane lipids, yet are essential in the activation and membrane association of protein kinase C. Solid-state 13C NMR experiments have been used to characterize the orientation of the glycerol backbone of dipalmitoylglycerol (DPG) and dipalmitoylphosphatidylcholine (DPPC) in egg phosphatidylcholine (PC) bilayers. The 13C NMR spectra of both DPG and DPPC specifically 13C-labeled at the sn-2 chain carbonyl exhibit a single narrow resonance (approximately 2 ppm) in liquid-crystalline egg PC bilayers. In contrast, specific 13C-labeling of both the sn-1 and sn-2 chain carbonyls results in an additional broad component (24-32 ppm) with an axially symmetric line shape. These data reveal that DPG has a distinct motionally-averaged structure in PC bilayers that is similar to that of DPPC and is not significantly affected by the absence of the large polar PC headgroup. The NMR line shapes are roughly consistent with the results of previous FTIR and NMR studies that indicate the sn-1 chain extends from the C1 carbon of the glycerol backbone into the hydrophobic interior of the bilayer, while the sn-2 chain first extends parallel to the bilayer surface and incorporates a bend at the ester linkage in order to keep the sn-1 and sn-2 chains parallel. However, the data suggest that the time-averaged orientation of the glycerol backbone is tilted from the bilayer normal, in contrast to the nearly parallel orientation observed in the crystal structures of phosphatidylcholines and phosphatidylethanolamines or the perpendicular orientation observed in the crystal structures of diacylglycerols.     

Effects of pH and cholesterol on DMPA membranes: a solid state 2H- and 31P-NMR study

The effect of pH and cholesterol on the dimyristoylphosphatidic acid (DMPA) model membrane system has been investigated by solid state 2H- and 31P-NMR. It has been shown that each of the three protonation states of the DMPA molecule corresponds to a 31P-NMR powder pattern with characteristic delta sigma values; this implies additionally that the proton exchange on the membrane surface is slow on the NMR time scale (millisecond range). Under these conditions, the 2H-labeled lipid chains sense only one magnetic environment, indicating that the three spectra detected by 31P-NMR are related to charge-dependent local dynamics or orientations of the phosphate headgroup or both. Chain ordering in the fluid phase is also found to depend weakly on the charge at the interface. In addition, it has also been found that the first pK of the DMPA membrane is modified by changes in the lipid lateral packing (gel or fluid phases or in the presence of cholesterol) in contrast to the second pK. The incorporation of 30 mol% cholesterol affects the phosphatidic acid bilayer in a way similar to what has been reported for phosphatidylcholine/cholesterol membranes, but to an extent comparable to 10-20 mol % sterol in phosphatidylcholines. However, the orientation and molecular order parameter of cholesterol in DMPA are similar to those found in dimyristoylphosphatidylcholine.     

Molecular dynamics investigation of the structure of a fully hydrated gel-phase dipalmitoylphosphatidylcholine bilayer

We report the results of a constant pressure and temperature molecular dynamics simulation of a gel-phase dipalmitoylphosphatidylcholine bilayer with nw = 11.8 water molecules/lipid at 19 degrees C. The results of the simulation were compared in detail with a variety of x-ray and neutron diffraction data. The average positions of specific carbon atoms along the bilayer normal and the interlamellar spacing and electron density profile were in very good agreement with neutron and x-ray diffraction results. The area per lipid and the details of the in-plane hydrocarbon chain structure were in excellent agreement with wide-angle x-ray diffraction results. The only significant deviation is that the chains met in a pleated arrangement at the bilayer center, although they should be parallel. Novel discoveries made in the present work include the observation of a bimodal headgroup orientational distribution. Furthermore, we found that there are a significant number of gauche conformations near the ends of the hydrocarbon chains and, in addition to verifying a previous suggestion that there is partial rotational ordering in the hydrocarbon chains, that the two chains in a given molecule are inequivalent with respect to rotations. Finally, we have investigated the lipid/water interface and found that the water penetrates beneath the headgroups, but not as far as the carbonyl groups, that the phosphates are strongly hydrated almost exclusively at the nonesterified oxygen atoms, and that the hydration of the ammonium groups is more diffuse, with some water molecules concentrated in the grooves between the methyl groups.     

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

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

Calorimetric and freeze fracture analysis of lipid phase transitions and lateral translational motion of intramembrane particles in mitochondrial membranes

Differential scanning calorimetry combined with freeze fracture electron microscopy reveals that thermotropic lipid phase transitions and lateral translational motion of intramembrane particles occur in both membranes of whole, intact rat liver mitochondria and in isolated inner and outer membranes. The onset temperature of the liquid crystalline to gel state lipid phase transition in whole mitochondria and in the isolated outer membrane fraction is biphasic with an initial transition exotherm occurring at 9 degrees C. The onset temperature of the transition exotherm of the isolated inner membrane occurs at -4 degrees C. The onset temperature of the lipid transition endotherm is -15 degrees C for whole mitochondria, the inner membrane, ane the outer membrane fractions. These calorimetric analyses reveal that the bilayer lipid in the inner, energy transducing membrane is more fluid than in the outer membrane. Mitochondrial membranes cooled to temperatures in the region of their transition exotherms and then frozen reveal striking lateral separations between smooth, intramembrane particle-free regions (rich in gel state lipid) and particle-dense regions (rich in integral proteins) in their hydrophobic fracture faces. Such thermotropic lipid-protein lateral separations are completely reversible. These freeze fracture observations suggest that both mitochondrial membranes are naturally fluid to the extent that the integrat membrane proteins can diffuse laterally in the bilayer lipid.     

The formation and annealing of structural defects in lipid bilayer vesicles

It is shown that sonication of phospholipid-water dispersions below the crystalline leads to liquid crystalline phase transition temperature (Tc) produces bilayer vesicles with structural defects within the bilayer membrane, which permit rapid permeation of ions and catalyze vesicle-vesicle fusion. These structural defects are annihilated simply by annealing the vesicle suspension above Tc. The rate of annealing was found to be slow, of the order of an hour for T = 3 degrees C above Tc, but annealing is complete within 10 min for T = 10 degrees C above Tc. It is proposed that these structural defects are fault-dislocations in the bilayer structure, which arise from a population defect in the distribution of the lipid molecules between the outer and inner monolayers, when small bilayer fragments reassemble to form the small bilayer vesicles during the sonication procedure. Such a population defect can only be remedied by lipid transport via the inside in equilibrium outside flip-flop mechanism, which would account for the slow kinetics of annealing observed even at 3 degrees C above the phase transition.     

Lipid membrane structure and interactions in dimethyl sulfoxide/water mixtures

In this paper we have investigated via x-ray diffraction the influence of dimethyl sulfoxide (DMSO), known for its biological and therapeutic properties, on the structure of lipid membranes of dipalmitoylphosphatidylcholine (DPPC) in excess of the solvent (DMSO/water) at mole DMSO fractions XDMSO in (0.1) and under equilibrium conditions. At small XDMSO 相似文献   

31P NMR studies of unsonicated aqueous dispersions of neutral and acidic phospholipids. Effects of phase transitions, p2H and divalent cations on the motion in the phosphate region of the polar headgroup

1. The 129 MHz (non-proton decoupled) and 36.4 MHz (proton decoupled) 31P NMR spectra arising from unsonicated aqueous dispersions of well defined species of phospholipid have been investigated. The phospholipids employed (and the parameters varied) include phosphatidylcholine (temperature), phosphatidylethanolamine (temperature), phosphatidic acid (temperature and p2H) and phosphatidylglycerol (temperature, p2H and Ca2+ (or Mg2+)) concentration. 2. At p2H = 7 the 31 P MNR spectra arising from saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol become progressively broader as the temperature is reduced below the phase transition, demonstrating reduced motion in the phosphate region of the polar headgroup. 3. In the liquid crystalline state at p2H = 7 the molecular dipolar order parameters obtained for saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol and very similar, and are independent of the acyl chain length for species derived from lauric and myristic acid. Thus the motion in the methylene-phosphate-methylene region is similar for these different liquid crystaline phospholipid species. 4. The 31 P NMR spectra of aqueous dispersions of 14:0/14:0 phosphatidic acid display anomalous temperature and p2H dependences. The effective chemical shift anistropy (delta v CSA EFF) at 5 degrees C varies from 71 ppm at p2H = 8.5 to 38 ppm at p2H = 2.5. Further, the motion in the phosphate region is relatively insensitive to the gel or liquid crystalline nature of the hydrocarbon chains. 5. The addition of 40 mol% Ca2+ (or Mg2+) to saturated species of phosphatidylglycerol causes an increase of approx. 20 degrees C in the hydrocarbon phase transition temperature as indicated by 31 P NMR. Equimolar concentrations of Ca2+ increase the transition temperature by approx. 70 degrees C, and no 31P NMR signal could be observed for the very condensed precipitate formed below this temperature. In the liquid crystalline state the motion in the phosphate region of the polar headgroup is not significantly affected by the presence of Ca+ or Mg2+. 6. The 31P NMR spectra obtained from 18 : 1c/18 : 1c phosphatidylethanolamine are consistent with a phase transition from a lamellar to an hexagonal (HII) phase in the region 10-15 degrees C. 7. The observed narrowing of the 31 P NMR spectra of aqueous dispersions of phospholipids as the temperature is raised toward the hydrocarbon transition temperature is discussed in terms of the "pretransition" observed in calorimetric studies.     

Physical properties of ceramides: effect of fatty acid hydroxylation

The structural and thermotropic properties of alpha-hydroxy fatty acid (HFA) and non-hydroxy fatty acid (NFA) ceramides (CER) have been studied using differential scanning calorimetry (DSC) and X-ray diffraction techniques. The DSC of anhydrous HFA-CER shows a single, sharp reversible transition at 95.6 degrees C (delta H = 15.3 kcal/mol). At intermediate hydrations HFA-CER exhibited more complex behavior but at maximum hydration only a single reversible transition is observed at 80.0 degrees C (delta H = 8.5 kcal/mol). X-ray diffraction of hydrated (74% water) HFA-CER at 20 degrees C shows a lamellar structure with a bilayer periodicity d = 60.7 Angstrum; a single wide angle reflection at 4.2 Angstrum is characteristic of hexagonal chain packing. Above the main transition temperature at 91 degrees C, a hexagonal (HII) phase is observed. In contrast, DSC of anhydrous NFA-CER demonstrates two thermal transitions at 81.3 degrees C (delta H = 6.8 kcal/mol) and 85.9 degrees C (delta H = 3.5 kcal/mol). With increasing hydration, both transitions shift towards lower temperatures; at maximum hydration, on heating, the endothermic transitions occur at 72.7 degrees C (delta H = 9.8 kcal/mol) and 81.1 degrees C (delta H = 4.0 kcal/mol). On cooling, there is hysteresis of both transitions. X-ray diffraction of NFA-CER (80% water) at 20 degrees C shows a well-ordered lamellar structure with a bilayer periodicity d = 58.6 Angstrum and three wide-angle reflections at 4.6 Angstrum, 4.2 Angstrum, and 3.8 Angstrum. At 77 degrees C (between the two transitions), again a lamellar structure exists with reduced bilayer periodicity d = 53.1 Angstrum and four wide-angle reflections at 4.6 Angstrum, 4.2 Angstrum, and 3.8 Angstrum are observed. Above the second transition, only a single low angle reflection at 30.0 Angstrum is observed; a diffuse reflection at 4.6 Angstrum is indicative of a melted chain phase. Thus, HFA-CER exhibits a simple phase behavior involving the reversible conversion of a gel phase to a hexagonal phase (L beta-->HII). However, NFA-CER shows a more complex polymorphic phase behavior involving two gel phases.     

Solid-state NMR studies of magnetically aligned phospholipid membranes: taming lanthanides for membrane protein studies

The addition of lanthanides (Tm3+, Yb3+, Er3+, or Eu3+) to a solution of long-chain phospholipids such as dimyristoylphosphatidylcholine (DMPC) and short-chain phospholipids such as dihexanoylphosphatidylcholine (DHPC) is known to result in a bilayer phase in which the average bilayer normal aligns parallel to an applied magnetic field. Lanthanide-doped bilayers have enormous potential for the study of membrane proteins by solid-state NMR, low-angle diffraction, and a variety of optical spectroscopic techniques. However, the addition of lanthanides poses certain challenges to the NMR spectroscopist: coexistence of an isotropic phase and hysteresis effects, direct binding of the paramagnetic ion to the peptide or protein of interest, and severe paramagnetic shifts and line broadening. Lower water concentrations and larger DMPC/DHPC ratios than those typically used in bicelles consistently yield a single oriented bilayer phase that is stable over a wide range of temperature (approximately 35-90 degrees C). Among the above choice of lanthanides, Yb3+ is found to give minimal paramagnetic shifts and line broadening at acceptably low concentrations necessary for alignment (i.e., Yb3+/DMPC mole ratios equal to or greater than 0.01). Finally, the addition of a phospholipid chelate, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine--diethylenetriaminepent aacetic acid, is observed to significantly reduce paramagnetic broadening and presumably prevent direct association of the peptide with the lanthanide ions.     

New look at force-frequency relationship of human skeletal muscle: effects of fatigue

16-Fluoropalmitic acid was synthesized from 16-hydroxypalmitic acid using diethylaminosulfur trifluoride. This monofluorinated fatty acid then was used to make 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) as a fluorinated analog of dipalmitoylphosphatidylcholine (DPPC). Surprisingly, we found that the phase transition temperature (Tm) of F-DPPC occurs near 50 degrees C, approximately 10 degrees C higher than its nonfluorinated counterpart, DPPC, as judged by both differential scanning calorimetry and infrared spectroscopy. The pretransition observed for DPPC is absent in F-DPPC. A combination of REDOR, rotational-echo double-resonance, and conventional solid-state NMR experiments demonstrates that F-DPPC forms a fully interdigitated bilayer in the gel phase. Electron paramagnetic resonance experiments show that below Tm, the hydrocarbon chains of F-DPPC are more motionally restricted than those of DPPC. X-ray scattering experiments confirm that the thickness and packing of gel phase F-DPPC is similar to that of heptanetriol-induced interdigitated DPPC. F-DPPC is the first phosphoglyceride containing sn-1 and sn-2 ester-linked fatty acyl chains of equal length that spontaneously forms interdigitated bilayers in the gel state in the absence of inducing agents such as alcohols.     

Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature

Molecular dynamics simulations of 500 ps were performed on a system consisting of a bilayer of 64 molecules of the lipid dipalmitoylphosphatidylcholine and 23 water molecules per lipid at an isotropic pressure of 1 atm and 50 degrees C. Special attention was devoted to reproduce the correct density of the lipid, because this quantity is known experimentally with a precision better than 1%. For this purpose, the Lennard-Jones parameters of the hydrocarbon chains were adjusted by simulating a system consisting of 128 pentadecane molecules and varying the Lennard-Jones parameters until the experimental density and heat of vaporization were obtained. With these parameters the lipid density resulted in perfect agreement with the experimental density. The orientational order parameter of the hydrocarbon chains agreed perfectly well with the experimental values, which, because of its correlation with the area per lipid, makes it possible to give a proper estimate of the area per lipid of 0.61 +/- 0.01 nm2.     

Modulation of lipid polymorphism by the feline leukemia virus fusion peptide: implications for the fusion mechanism

The structural effects of the fusion peptide of feline leukemia virus (FeLV) on lipid polymorphism were studied, using differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR), and time-resolved X-ray diffraction. This peptide lowers the bilayer to inverted hexagonal phase transition temperature, TH, of dipalmitoleoylphosphatidylethanolamine (DiPoPE) at peptide mole fractions of up to 1.5 x 10(-3) at pH 5.0 and at pH 7.4. The temperature at which isotropic 31P NMR signals for monomethyldioleoylphosphatidylethanolamine (MeDOPE) first occurred is lowered by the FeLV peptide. The amount of isotropic signal seen at 40 degrees C is directly correlated to the peptide:lipid molar ratio. In the peptide-containing samples, more lipid remains in the isotropic state over the whole recorded temperature range. Isotropic 31P NMR signals were observed for DiPoPE in the presence of the FeLV peptide for the entire recorded temperature range of 35-50 degrees C, while pure DiPoPE showed no significant amount of isotropic signal. X-ray studies of DiPoPE show the formation of a new lipid phase with peptide, which is not seen in the pure lipid samples. Disordering of the Lalpha phase is evidenced by broadening of the diffraction peaks, and the hexagonal cell parameter is decreased with peptide present. Our results suggest that the FeLV peptide is increasing the negative curvature of the lipid system, which is thought to be crucial to the formation of highly bent, high-energy structural fusion intermediates, such as the "stalk" model. Fusion activity for this putative fusogenic peptide was also demonstrated, using a resonance energy transfer (RET) lipid mixing assay. To our knowledge, this work provides the first published experimental evidence of both fusogenic activity and effects on lipid polymorphism for the FeLV fusion peptide.     

Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol

The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents.     

Determining ethanol distribution in phospholipid multilayers with MAS-NOESY spectra

The location of an ethanol molecule within a membrane, an issue of considerable controversy, was investigated directly by NMR with two-dimensional NOESY. Lipid and ethanol 1H NMR resonances of multilamellar liposomes were resolved by magic-angle spinning (MAS). We observed strong proton lipid-ethanol crosspeaks in dispersions of saturated dimyristoylphosphatidylcholine and monounsaturated stearoyloleoylphosphatidylcholine and in polyunsaturated stearoyldocosahexaenoylphosphatidylcholine. Crosspeak intensity has been interpreted in terms of an ethanol distribution function over the lipid bilayer. Ethanol resides with the highest probability at the lipid water interface near the lipid glycerol backbone and upper methylene segments of lipid hydrocarbon chains. Chain unsaturation has only a minor influence on the ethanol distribution function. In all cases, the ethanol concentration in the bilayer core is significantly lower. At ambient temperature all lipid-ethanol crosspeaks are positive. Crosspeak intensity decreases with increasing water content and increasing temperature most likely because of shorter correlation times of lipid and ethanol reorientation. This suggests a lifetime for specific lipid-ethanol contacts of about 1 ns. Lipid-ethanol and lipid-lipid crosspeaks reflect the high degree of motional disorder of lipids and incorporated ethanol in membranes and the rather arbitrary nature of the location of the lipid-water interface.     

Fast two dimensional computer simulation of bilayer hemifusion

Computer simulation of model membranes was used to evaluate the possible mechanism of lipid bilayer fusion. The simplified two dimensional model of the membrane cross section was used as an analog of three dimensional reality. Lipid molecules were represented by rod-like structures, and forces between them were limited to attraction/repulsion interactions described by a simple energy function with a minimum; 300-400 molecules were modeled in every simulation. Using the energy minimization procedure, it was possible to obtain stable linear or circular bilayer structures (two dimensional analogs of planar membranes and liposomes). In response to changes in attraction/repulsion equilibrium between molecules these bilayers were able to reorganize via cooperative process. By increasing the headgroup attraction parameter for contacting monolayers, it was possible to induce formation of a zone of hemifusion in the area of bilayer contact. The possible correlation between cooperative bilayer rearrangement in the model and in real bilayers is discussed.