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.     

Interaction of nonachlazine and noradrenaline on a model phospholipid membrane

The binding of nonachlazine (NCL), imipramine (IMP), and noradrenaline (NA) with the model phospholipid membrane vesicles--liposomes--was studied. The binding was determined by the qunching effect of these substances on the fluorescence of 3-methoxybenzantrone (MBA) present in the membrane of the fluorescent probe. A method rendering possible calculation of the binding of the preparations under study with the membrane of the basis of the fluorescence changes was developed. The binding constant of the NCL, IMP, and NA interaction with the membrane was equal to (4.3 +/- 0.3)-10(3)M-1, (2.7 +/- 0.2)-10(3)M-1, and (0.7 +/- 0.15)-10(3)M-1, respectively. It was shown that NCL and IMP could compete with NA for the membrane binding centers. Such competitive interactions could be regarded as a probable mechanism of the block of the reverse NA transport through the synaptic and the vesicular membranes characteristic of NCL and IMP.     

Gramicidin channels in phospholipid bilayers with unsaturated acyl chains

In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.     

Bicelles: a model membrane system for all seasons?

OBJECTIVES: To establish population-based HIV survey data in selected populations, and to assess the validity of extrapolation from HIV sentinel surveillance amongst antenatal clinic attenders (ANC) to the general population. METHODS: In a population survey, adults aged > or = 15 years were selected by stratified random cluster sampling (n = 4195). The survey was carried out in catchment populations of clinics used for national HIV surveillance. The methodology allows detailed comparisons of HIV infection patterns to be made in two areas (urban and rural). Whereas the sentinel surveillance used serum-based HIV testing, the population survey used saliva (93.5% consented to provide a saliva sample). RESULTS: Surveillance of ANC tended to underestimate the overall HIV prevalence of the general population, but differences were not statistically significant. In the urban area, the adjusted overall HIV prevalence rate of ANC (aged 15-39 years) was 24.4% [95% confidence interval (CI), 20.9-28.0] compared with 26.0% (95% CI, 23.4-28.6) in the general population. The respective rural estimates were 12.5% (95% CI, 9.3-15.6) versus 16.4% (95% CI, 12.1-20.6). Age-specific prevalence rates showed ANC to overestimate infection in teenagers (aged 15-19 years), whereas in the reverse direction of those aged > or = 30 years. Teenagers analysed by single year of age revealed both ANC and women in the general population with about the same steep increase in prevalence by age, but the former at consistently higher rates. Extrapolations might be biased substantially due to the higher pregnancy rates amongst uninfected individuals. CONCLUSIONS: ANC-based data might draw a rather distorted picture of current dynamics of the HIV epidemic. Even though representing an obvious oversimplification, extrapolations of overall prevalence rates may correlate with that of the general population.     

Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains

The partition coefficient Kp was measured for a headgroup-labeled phospholipid (12:0,12:0)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PE (12-NBD-PE), equilibrated between LUV of a series of phosphatidylcholines (PC). Fluorescence resonance energy transfer between the 12-NBD-PE and a headgroup-rhodamine-labeled PE was used to find the equilibrium concentration of the 12-NBD-PE in the different LUV. Reliable equilibrium concentrations were obtained by monitoring the approach to equilibrium starting from a concentration below and from a concentration above the ultimate values. Using (16:0,18:1delta9)-PC as the reference lipid, Kp ranged from a high value of 1.65 favoring (16:0,18:1delta9)-PC over (16:1delta9,16:1delta9)-PC, to a low value of 0.90, favoring (22:1delta13,22:1delta13)-PC over (16:0,18:1delta9)-PC. The Kp values enabled calculation of the acyl chain contribution to the excess free energy of mixing for (12:0,12:0) acyl chains at infinite dilution in the L alpha phase of PC having acyl chains of (16:0,18:1delta9), (16:1delta9,16:1delta9), (18:1delta9,18:1delta9), (18:1delta6,18:1delta6), (20:1delta11,20:1delta11), and (22:1delta13,22:1delta13). (14:1delta9,14:1delta9)-PC was found to transfer so rapidly between LUV as to preclude reliable Kp measurement.     

Structural phase transitions involved in the interaction of phospholipid bilayers with octyl glucoside

The transitional stages induced by the interaction of the nonionic surfactant octyl glucoside (OcOse) on phosphatidylcholine liposomes were studied by means of transmission electron microscopy (TEM), light scattering and permeability changes. A linear correlation was observed between the effective surfactant/lipid molar ratio (Re; three-stage model proposed for liposome solubilization) and the OcOse concentration in the initial and final interaction stages, despite showing almost a constant value during bilayer saturation. The bilayer/aqueous phase partition coefficient (K) decreased in the subsolubilizing interaction steps and increased during solubilization. Thus, whereas a preferential distribution of surfactant monomers in the aqueous phase with respect to the lipid bilayers took place in the initial interaction steps, a larger association of OcOse molecules with these lipids in bilayers occurred during solubilization. The initial steps of bilayer saturation (50-70% permeability) were attained for a lower free surfactant (Sw) than that for its critical micellar concentration (cmc). When Sw reached the OcOse cmc, solubilization started to occur (Resat). Large unilamellar vesicles began to form as the OcOse exceeded 60 mol/100 mol, exhibiting for 65 mol/100 mol (50% permeability) vesicles of approximately 400 nm. TEM pictures for 100% permeability (72 mol/100 mol) and Resat still showed unilamellar vesicles, albeit that the Resat TEM picture showing traces of smaller structures. Exceeding surfactant amounts led to a decrease in static light scattering; the vesicle-size curve began to show a bimodal distribution. The TEM picture showed tubular structures together with bilayer fragments. Thereafter, the open structures were gradually affected by the surfactant and the scattered intensity gradually decreased to a constant low value.     

Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer

The effects of intravenous administration of flumazenil (n = 6) or bicuculline (n = 6) on the discharge of the phrenic nerve were studied following vagotomy in pentobarbital anesthetized mechanically ventilated rats. Morphine (0.4 mg.kg-1.min-1) was administrated until the respiratory rate decreased to about a half of the baseline respiratory rate. In this state, we first administered flumazenil (0.25 mg.kg-1) or bicuculline (0.4 mg.kg-1), intravenously and then administered naloxone (0.02 mg) intravenously in the two groups. The increase of inspiratory time from 0.7 +/- 0.1 to 2.0 +/- 0.5 s by morphine recovered to 0.8 +/- 0.2 s by bicuculline and to 0.6 +/- 0.1 s by naloxone. The increase of inspiratory time from 0.7 +/- 0.1 to 1.7 +/- 0.3 s by morphine, and to 2.1 +/- 0.5 s by flumazenil recovered to 0.6 +/- 0.1 s by naloxone. Expiratory time did not change during each drug administration in the two groups. The decrease of respiratory rate from 44 to 23 +/- 4 breaths.min-1 by morphine recovered to 37 +/- 5 breaths.min-1 by bicuculline and to 42 +/- 2 breaths.min-1 by naloxone. The decrease of respiratory rate from 45 +/- 3 to 22 +/- 6 breaths.min-1 by morphine, and to 18 +/- 4 breaths.min-1 by flumazenil recovered to 46 +/- 3 breaths.min-1 by naloxone. Amplitude of integrated phrenic nerve discharge increased to 125 +/- 42% by bicuculline and to 175 +/- 93% by naloxone compared to the baseline values. The decrease of amplitude to 54 +/- 18% by flumazenil recovered to 125 +/- 42% by naloxone. These results suggest that bicuculline not flumazenil antagonizes the respiratory depression of morphine by increasing the respiratory rate and respiratory movement.     

Thermally induced molecular disorder in human stratum corneum lipids compared with a model phospholipid system; FT-Raman spectroscopy

The molecular basis of lipid packing in human stratum corneum and a model phospholipid system has been studied as a function of temperature using Fourier Transform (FT) Raman spectroscopy. Thermally induced molecular rearrangements of the model lipid system, dipalmitoylphosphatidyl choline (DPPC), and stratum corneum were investigated using FT Raman spectroscopy coupled to a heating chamber. Spectra were recorded for a range of sample temperatures and the results for the two systems were compared, producing previously unreported information of the thermal behaviour for the different systems. Discrete thermal events were recorded for both systems by plotting band separation of the lipid v(CH2) symmetric and asymmetric stretching modes against temperature. The main thermal events observed for DPPC included a 'pre-melting' between 37 and 39 degrees C, the main transition observed between 41 and 42 degrees C, a 'post-transition' between 42 and 43 degrees C and three minor transitions at 58-60, 65-70 and 75-80 degrees C. No evidence was found for the pre-transition of DPPC, previously observed at 34-35 degrees C. The main transitions for human stratum corneum were observed at 35-45, 55, 72 and 83 degrees C, measured from lipid CH2 stretching and bending vibrations. The keratin thermal transition at about 100 degrees C exerted little effect on the lipid bands and no characterisable structural changes were reflected in the keratotic bands.     

Orientation of fusion-active synthetic peptides in phospholipid bilayers: determination by Fourier transform infrared spectroscopy

A group of synthetic peptides having an amino acid sequence related to the N-terminal region of the influenza virus hemagglutinin HA-2 chain can induce phospholipid membrane fusion in a pH-dependent manner. These peptides bind to membranes to form alpha-helices even at pH's where no fusion activity is seen. We determined the orientation of these alpha-helical peptides in lipid multibilayers using attenuated total reflection infrared spectroscopy and found that the peptide alpha-helices took a preferential orientation, the helix axis being about 70 degrees from the normal of the membrane plane, or in other words rather parallel to the membrane plane. The orientation was almost independent of pH and a modification of the N-terminal amino group which reduced the fusion activity of the peptides. The determination was carried out for peptides in lipid multibilayers in dry or hydrated (membranes equilibrated with D2O vapor) conditions. Although a slight decrease in the helix orientation angle from the membrane normal was noticed for a hydrated system, the difference between the results for dry and hydrated conditions was small.     

Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases

Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan.     

A compound-energy model of ordering in a phase with sites of different coordination numbers

When applied to a phase where all the sites do not have the same coordination number, the bond-energy model gives a term in u_AA − u_BB, which cannot be assigned a numerical value because the energy of different elements cannot be compared. As an alternative a compound-energy model is now developed from the well-known model for reciprocal systems. It gives a similar result but the u_AA − u_BB term does not appear. In addition, the new model takes into account two kinds of factors resulting in ordering, those which make different elements prefer different kinds of lattice sites and those which give a preference for unlike neighbors. The ordering in the sigma phase is considered in some detail.     

Evaluation of an extracorporeal membrane oxygenation system using a nonporous membrane oxygenator and a new method for heparin coating

A new heparin binding method was applied to a miniature extracorporeal membrane oxygenation (ECMO) system with a nonporous membrane oxygenator (the priming volume, 45 mL; the membrane surface area, 0.4 m2; maximal flow rate, 2 L/min) that is resistant to plasma leakage. The authors evaluated the stability of the immobilized heparin in vitro and the feasibility of this system in animals. Samples of hollow fibers and tubing were rinsed at 40 degrees C for 4 days in normal saline, Ringer's lactate, and 1 mol/L NaCl solution. Heparin activities on hollow fibers after rinsing were 99 +/- 2.3% (mean +/- SD), 96 +/- 3.9%, and 93 +/- 2.0% of the control in each solution, while those of the tubing were 87 +/- 4.1%, 86 +/- 3.1%, and 76 +/- 8.6%, respectively. Veno-arterial ECMO using this heparin-coated system was performed on five beagles (8 to 12 kg) for 10 hours. Neither major thrombus formation nor plasma leakage was detected during the procedure in spite of a low flow rate (300 mL/min) and a reduced activated clotting time (mean, 128 seconds). Platelets decreased to 52% of the control (P < .01) at 1 hour, but no progressive decrease was seen thereafter. Antithrombin-III decreased (P < .01) and thrombin/antithrombin III complex increased (P < .05 at 4 hours and P < .01 at 6, 8, and 10 hours) during bypass, but the changes of fibrinogen and fibrinopeptide A were not significant. Fibrinogen/fibrin degeneration products, fibrinopeptide B beta 15-42, and plasma-free hemoglobin levels did not rise significantly. O2 transfer of the oxygenators at a flow rate of 300 mL/min were 12.3 +/- 0.4 mL/min at 30 minutes, 14.3 +/- 1.2 mL/min at 5 hours, and 14.7 +/- 1.7 mL/min at 10 hours (no statistical difference). Histological examination of the brains and the kidneys showed no evidence of thromboembolic sequela in any of the animals. These results suggest that this new system is a promising device for long-term ECMO in which the amount of systemic heparinization can be reduced with the minimal possibility of plasma leakage.     

Protein-induced vertical lipid dislocation in a model membrane system: spin-label relaxation studies on avidin-biotinylphosphatidylethanolamine interactions

The change in vertical location of spin-labeled N-biotinyl phosphatidylethanolamine in fluid-phase dimyristoyl phosphatidylcholine bilayer membranes, on binding avidin to the biotinyl headgroup, has been investigated by progressive saturation electron spin resonance measurements. Spin-labeled phospholipids were present at a concentration of 1 mol%, relative to total membrane lipids. For avidin-bound N-biotinyl phosphatidylethanolamine spin-labeled on the 8 C atom of the sn-2 chain, the relaxation enhancement induced by 30 mM Ni2+ ions confined to the aqueous phase was 2.5 times that induced by saturating molecular oxygen, which is preferentially concentrated in the hydrophobic core of the membrane. For phosphatidylcholine also spin-labeled at the 8 position of the sn-2 chain, this ratio was reversed: the relaxation enhancement by Ni2+ ions was half that induced by molecular oxygen. In the absence of avidin, the enhancement by either relaxant was the same for both spin-labeled phospholipids. For a double-labeled system, in which both N-biotinyl phosphatidylethanolamine and phosphatidylcholine were spin-labeled on the 12 C atom of the sn-2 chain, the relaxation rate in the absence of avidin was greater than that predicted from linear additivity of the corresponding singly labeled systems, because of mutual spin-spin interactions between the two labeled lipid species. On binding of avidin to the N-biotinyl phosphatidylethanolamine, this relaxation enhancement by mutual spin-spin interaction was very much decreased. These results indicate that, on binding of avidin to the lipid headgroup, N-biotinyl phosphatidylethanolamine is lifted vertically within the membrane, relative to the phosphatidylcholine host lipids. The specific binding of avidin to N-biotinyl phosphatidylethanolamine parallels the liftase activity proposed for activator proteins associated with the action of certain gangliosidases.