Effects of cannabinoids in membrane bilayers containing cholesterol.

The thermotropic and dynamic properties of the biologically active Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and its inactive congener O-methyl-Delta(8)-tetrahydrocannabinol (Me-Delta(8)-THC) in DPPC/cholesterol (CHOL) bilayers have been studied using a combination of DSC and solid-state NMR spectroscopy. The obtained results showed differential effects of the two cannabinoids under study. These are summarized as follows: (a) the presence of the active compound fluidizes more significantly the DPPC/CHOL bilayers than the inactive analog as it is revealed by DSC and NMR spectroscopy results; (b) cholesterol seems to play a significant role in the way cannabinoids act in membrane bilayers; (c) the observed additional peaks in (13)C/MAS-NMR spectra which were cannabinoid specific offer an evidence of their different dynamic properties in membranes. In particular, the aromatic part of the inactive cannabinoid appears more mobile than that of the active one. This finding is in agreement with previously obtained X-ray data which locate the inactive cannabinoid in the hydrophobic core of the bilayer while the active one in the polar region; and (d) the observed downfield shift of C-1 carbon in the preparation containing the active cannabinoid is a strong evidence that Delta(8)-THC resides nearby the polar region where also cholesterol is well known to locate itself. Such downfield shift is absent when Me-Delta(8)-THC is resided in the membrane bilayer. These differential effects of the two cannabinoids propose that the phospholipid/cholesterol core of the membrane may play an important role in the mode of cannabinoid action by regulating their thermotropic and dynamic properties.     

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.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Neutron diffraction from cannabinoids in phospholipid membranes

Neutron diffraction measurements have been utilized to study the effects of delta 9-tetrahydrocannabinol (delta 9-THC) and delta 8-tetrahydrocannabinol (delta 8-THC) incorporated in phospholipid membranes of dipalmitoylphosphatidylcholine (DPPC). Low-angle diffraction indicated that these cannabinoids induce increases in interlamellar spacing similar to those produced by cholesterol. Wide-angle diffraction indicated significant differences in how the intralamellar structure is affected by the inclusion of either cannabinoids or cholesterol. Similar weight percentages of cholesterol and cannabinoids in membranes yielded different thermal analysis profiles but the profiles for membranes with either delta 8 or delta 9-THC were similar. Since the neutron diffraction results for inclusions of delta 8 and delta 9-THC were also similar, this suggests that the difference in psychoactivity of delta 8 and delta 9-THC is probably due to interactions with membrane proteins rather than with phospholipids.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Pharmacological evaluation of water soluble cannabinoids and related analogs

The two water-soluble cannabinoids 1-[(4-morpholino) butyryloxy]-delta 8-tetrahydro-cannabinol (MB-delta 8-THC) and 5'-trimethylammonium (TMA)-delta 8-THC, as well as structurally similar compounds, were evaluated for cannabimimetic activity in the mouse (locomotor activity, tail-flick antinociception, rectal temperature, and ring-immobility) and dog (static-ataxia) procedures. MB-delta 8-THC possesses full cannabimimetic activity and is approximately equipotent to delta 8-THC. 5'-TMA-delta 8-THC only possesses partial cannabimimetic activity in that it is inactive in the ring-immobility and static-ataxia procedures. However, this analog is potent in other respects. All alterations at the 5' position do not necessarily produce this spectrum of effects, as evidenced by the pharmacological activity of 5'-bromo-delta 8-THC, 5'-OH-delta 8-THC acetate, and 5'-N-dimethyl-delta 8-THC.     

Effects of cyclosporin A on model lipid membranes

Cyclosporin A (CSA) is a widely used immunosuppressant drug for transplant therapy, however its limitation is its toxicity. The effect of CSA on model membranes such as dimyristoyl phosphatidylcholine (DMPC) bilayers was studied using small-angle X-ray diffraction and differential scanning calorimetry (DSC). CSA abolishes the pretransition and affects the transition of DMPC model membranes in a concentration-related manner as is shown by DSC. CSA induces a second peak at the high temperature side of the main transition, which is interpreted as a phase separation between areas rich and poor in CSA concentration. Small angle X-ray diffraction shows that the repeat distance of the DMPC bilayers in the lamellar Lalpha state increases as a function of concentration up to 10 mol% and remains constant thereafter. Furthermore, CSA affects the fatty acyl chains of the bilayer, especially the part of the chain proximal to the head group. In conclusion, CSA, as both small-angle X-ray diffraction and DSC show, affects in a concentration-wise manner the DMPC model membranes and perturbs the bilayer, in particular the acyl chain region.     

Octyl-beta-D-glucopyranoside partitioning into lipid bilayers: thermodynamics of binding and structural changes of the bilayer.

The interaction of the nonionic detergent octyl-beta-D-glucopyranoside (OG) with lipid bilayers was studied with high-sensitivity isothermal titration calorimetry (ITC) and solid-state 2H-NMR spectroscopy. The transfer of OG from the aqueous phase to lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can be investigated by employing detergent at concentrations below the critical micellar concentration; it can be defined by a surface partition equilibrium with a partition coefficient of K = 120 +/- 10 M-1, a molar binding enthalpy of delta H degrees D = 1.3 +/- 0.15 kcal/mol, and a free energy of binding of delta G degrees D = -5.2 kcal/mol. The heat of transfer is temperature dependent, with a molar heat capacity of delta CP = -75 cal K-1 mol-1. The large heat capacity and the near-zero delta H are typical for a hydrophobic binding equilibrium. The partition constant K decreased to approximately 100 M-1 for POPC membranes mixed with either negatively charged lipids or cholesterol, but was independent of membrane curvature. In contrast, a much larger variation was observed in the partition enthalpy. delta H degrees D increased by about 50% for large vesicles and by 75% for membranes containing 50 mol% cholesterol. Structural changes in the lipid bilayer were investigated with solid-state 2H-NMR. POPC was selectively deuterated at the headgroup segments and at different positions of the fatty acyl chains, and the measurement of the quadrupolar splittings provided information on the conformation and the order of the bilayer membrane. Addition of OG had almost no influence on the lipid headgroup region, even at concentrations close to bilayer disruption. In contrast, the fluctuations of fatty acyl chain segments located in the inner part of the bilayer increased strongly with increasing OG concentration. The 2H-NMR results demonstrate that the headgroup region is the most stable structural element of the lipid membrane, remaining intact until the disordering of the chains reaches a critical limit. The perturbing effect of OG is thus different from that of another nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), which produces a general disordering at all levels of the lipid bilayer. The OG-POPC interaction was also investigated with POPC monolayers, using a Langmuir trough. In the absence of lipid, the measurement of the Gibbs adsorption isotherm for pure OG solutions yielded an OG surface area of AS = 51 +/- 3 A2. On the other hand, the insertion area AI of OG in a POPC monolayer was determined by a monolayer expansion technique as AI = 58 +/- 10 A2. The similar area requirements with AS approximately AI indicate an almost complete insertion of OG into the lipid monolayer. The OG partition constant for a POPC monolayer at 32 mN/m was Kp approximately 320 M-1 and thus was larger than that for a POPC bilayer.     

The effects of delta 9-tetrahydrocannabinol and other cannabinoids on cAMP accumulation in synaptosomes.

The effects of delta 9-THC and other cannabinoids on cAMP levels in synaptosomes from mouse brains were investigated in order to determine whether cannabinoids produced their behavioral effects through alterations in adenylate cyclase. delta 9-THC (0.01-10 microM) did not significantly alter basal cAMP levels, whereas delta 9-THC and other cannabinoids were able to alter forskolin-stimulated cAMP levels in synaptosomes. In general, three kinds of responses were observed. Some cannabinoids displayed a modest, concentration-dependent decrease in cAMP levels, producing significant inhibition between 1-10 microM. Other cannabinoids, including delta 9-THC and delta 8-THC, appeared to produce a biphasic effect in that inhibition of cAMP was observed only at a single concentration. Finally, some analogs were unable to significantly alter forskolin-stimulated cAMP. There was not a clear relationship between the ability of the cannabinoids to alter cAMP levels in synaptosomes and the behavioral effects observed in mice. However, it was demonstrated that the analogs which are the most potent in producing cannabimimetic effects in mice were the analogs which inhibited cAMP in a concentration-dependent manner. While cannabinoids were able to alter cAMP levels in synaptosomes, the ability to alter cAMP levels does not appear to be absolutely necessary for the production of cannabinoid effects in mice.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Cellular membrane composition defines A beta-lipid interactions.

Alzheimer's disease pathology has demonstrated amyloid plaque formation associated with plasma membranes and the presence of intracellular amyloid-beta (A beta) accumulation in specific vesicular compartments. This suggests that lipid composition in different compartments may play a role in A beta aggregation. To test this hypothesis, we have isolated cellular membranes from human brain to evaluate A beta 40/42-lipid interactions. Plasma, endosomal, lysosomal, and Golgi membranes were isolated using sucrose gradients. Electron microscopy demonstrated that A beta fibrillogenesis is accelerated in the presence of plasma and endosomal and lysosomal membranes with plasma membranes inducing an enhanced surface organization. Alternatively, interaction of A beta with Golgi membranes fails to progress to fibril formation, suggesting that A beta-Golgi head group interaction stabilizes A beta. Fluorescence spectroscopy using the environment-sensitive probes 1,6-diphenyl-1,3,5-hexatriene, laurdan, N-epsilon-dansyl-L-lysine, and merocyanine 540 demonstrated variations in the inherent lipid properties at the level of the fatty acyl chains, glycerol backbone, and head groups, respectively. Addition of A beta 40/42 to the plasma and endosomal and lysosomal membranes decreases the fluidity not only of the fatty acyl chains but also the head group space, consistent with A beta insertion into the bilayer. In contrast, the Golgi bilayer fluidity is increased by A beta 40/42 binding which appears to result from lipid head group interactions and the production of interfacial packing defects.     

Conformation of fatty acyl chains in alpha- and beta-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of alpha-phospholipids: PE, N-methyl-PE, N,N-dimethyl-PE, and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (beta-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The alpha-methylene group of the sn-1 and sn-2 fatty acyl chains of the alpha-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all alpha-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N-methyl-PE, and N,N-dimethyl-PE, but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the alpha-methylene protons. On the other hand, all of the beta-phospholipids (including beta-PE) were found to reflect the inside/outside packing differences in their alpha-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, beta-phospholipids showed one alpha-CH2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all alpha-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the beta-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.     

Deuterium NMR studies of cerebroside-phospholipid bilayers

2H-NMR was used to probe the interaction of non-hydroxy fatty acid cerebroside and 2-hydroxy fatty acid cerebroside with the polar head group and with the acyl chains of dipalmitoylphosphatidylcholine in unsonicated bilayers. It is shown that the interior of the bilayer exhibits uniformly increasing orientational order as the concentration of both types of cerebroside increases, whereas the surface of the bilayer, as reflected by the head group motion, becomes disordered. The extent of the disorder at the surface is dependent upon the type and concentration of the cerebroside. These results are discussed in terms of hydrogen-bonding interactions.     

The pH dependence of headgroup and acyl chain structure and dynamics of phosphatidylserine, studied by 2H-NMR

By varying the pH, the influence of the ionization degree on the structure and dynamics of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was studied, using 2H-NMR methods. For this purpose DOPS was synthesized with deuterium labels incorporated either stereospecifically at the beta-position of the serine headgroup ([2-2H]DOPS) or at the 11-position of both acyl chains ([11,11-2H2]DOPS), allowing the effects of pH on headgroup and acyl chains to be measured in parallel. A large scale synthesis procedure of stereospecific 1,2-dioleoyl-sn-glycero-3-phospho-[2-2H]-L- serine is described. The quadrupolar splitting (delta nu q) of [2-2H]DOPS is shown to be a sensitive sensor for the degree of protonation of the molecule. Whereas the delta nu q of [2-2H]DOPS decreases upon lowering the pH, that of [11,11-2H2]DOPS gradually increases, indicating an increase in acyl chain ordering. In the pH range below the pKa value, DOPS exhibits a temperature-dependent bilayer to hexagonal HII phase transition, apparent from the 31P-NMR spectra and the occurrence of a second component in the [11,11-2H2]DOPS 2H-NMR spectrum, with a much smaller delta nu q. The HII phase component in spectra from [2-2H]DOPS coincides with the isotropic position and has no defined delta nu q. In the bilayer organization delta nu q and spin-lattice relaxation time (T1) values for the acyl chain deuterated DOPS are similar to those obtained for other lipid systems. In contrast the PS headgroup region displays a relatively rigid structure as evidenced by a large delta nu q and very small T1 values. Upon adopting the HII phase the T1 values of the acyl chain deuterons are hardly affected. The uniqueness of the PS headgroup with respect to structure and motional properties is reinforced by the occurrence of a T1 minimum at 45 degrees C in the measurement of the temperature dependence of T1 for [2-2H]DOPS in the hexagonal HII configuration. Quantitative analysis yields a correlation time (tau c) for the motions determining T1 under these conditions, of 3.45 ns.     

Interactions of cholesterol with the membrane lipid matrix. A solid state NMR approach.

The effects of cholesterol on the structure and dynamics of dimyristoylphosphatidylcholine (DMPC) model membranes have been monitored as functions of temperature and cholesterol concentration in the membrane. The use of deuterium labels both on the cholesterol fused ring system and on the lipid chains in conjunction with solid state deuterium nuclear magnetic resonance (2H-NMR) afforded to monitor the degree of ordering of both molecules in a mixed system. The degree of ordering of the lipid head group was followed by phosphorus-31 (31P)-NMR. New findings on the effect of cholesterol on DMPC may be summarized as follows: i) cholesterol disorders the lipid chains below temperature of the DMPC gel-to-fluid transition (Tc) and orders them above; the effect is linear with cholesterol concentration at 0 and 60 degrees C but for intermediate temperatures, a saturation effect is observed at 20-30 mol %; ii) the ordering-disordering effects are perceived similarly by all chain segments with, however, a greater sensitivity for positions near the bilayer center; iii) below Tc, the lipid head group is considerably disordered by increasing amounts of cholesterol but slightly affected above; iv) the degree of ordering of cholesterol is quasi temperature independent for fractions greater than or equal to 30%; v) the average orientation of the cholesterol rigid body is perpendicular to the bilayer surface and exhibits little variations with temperature and cholesterol concentration. Variations in membrane dynamics are interpreted in terms of cholesterol-induced changes in bilayer thickness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depth-dependent photolabelling of membrane hydrophobic core with 9-diazofluorene-2-butyric acid

Hydrophobic photoactivable reagents, which readily partition into membranes, have proved very useful for studying membrane hydrophobic core. These reagents have been linked to fatty acids in order to obtain amphipathic photoactivable reagents which label membranes more effectively. By varying the length of these amphipathic reagents, an attempt to label membrane hydrophobic core at different depths can be made. We report here 9-diazofluorene-2-butyric acid as a new photoactivable reagent which labels the single bilayer vesicles prepared from egg phosphatidylcholine. The labelling site on the fatty acyl chains could be traced to be between the carbon atom 4 and 6. The new probe thus labels the membrane at a site which is proximal to what can be predicted from its length and transverse location in membranes.     

Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.     

19F nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Orientational order in the presence of a series of positional isomers of cis-octadecenoic acid

The 19F nuclear magnetic resonance (NMR) spectra of membranes of Acholeplasma laidlawii B enriched with one of a series of positional isomers of cis-octadecenoic acid plus small amounts of one of a number of isomers of monofluoropalmitic acid were interpreted in terms of an orientational order parameter (Smol). The variation of Smol with the position of the fluorine label in the liquid-crystalline state yielded an "order profile" with characteristics similar to those obtained via 2H NMR and which was relatively invariant regardless of the site of cis unsaturation. In the gel state, values of Smol approached the theoretical maximum, and the order profiles in the presence of different isomeric cis-octadecenoic acids displayed distinct dissimilarities. When the cis double bond was located proximal to the methyl terminus of the fatty acyl chain, a gradient of order across the bilayer was still evident in the gel state. When the cis double bond was located near the carbonyl head group, values of Smol were approximately equal at all chain positions. These observations were interpreted as indicating that in the gel state the stringency of packing restrictions is still subject to variation across the width of the bilayer. Relative overall orientational order among all isomers examined (specifically, 18:1c delta 4, delta 5, delta 6, delta 7, delta 8, delta 9, delta 10, delta 11, delta 12, delta 13, delta 14, and delta 15) varied directly as a function of proximity to the lipid gel to liquid-crystalline phase transition (Tm) (determined via differential scanning calorimetry) when compared at a constant temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of electric dipoles with phospholipid head groups. A 2H and 31P NMR study of phloretin and phloretin analogues in phosphatidylcholine membranes.

Phloretin, 4-hydroxyvalerophenone, and 2-hydroxy-omega-phenylpropiophenone are lipophilic dipolar substances that modify ionic conductances of bilayer membranes. The structural changes at the level of the head groups and the hydrocarbon chains as induced by the incorporation of phloretin and its analogues were investigated with deuterium and phosphorus nuclear magnetic resonance. Membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were selectively deuterated at the choline head group and at the hydrocarbon chains, and 2H and 31P NMR spectra were recorded with varying concentrations of dipolar agents. Incorporation of phloretin leaves the bilayer structure intact, induces only a small disordering of the hydrocarbon chains, and has no significant effect on the head-group dynamics. On the other hand, quite distinct structural changes are observed for the phosphocholine head group. While the -P-N+ dipole is oriented approximately parallel to the membrane surface for pure POPC bilayers, addition of phloretin, and to a lesser extent 4-hydroxyvalerophenone and 2-hydroxy-omega-phenylpropiophenone, rotates the N+ end of the -P-N+ dipole closer to the hydrocarbon layer. The resulting normal component of the -P-N+ dipole partly compensates the electric field of the dipolar agents. In addition to this structural change, phloretin also modifies the hydration layer at the lipid-water interface. Much less 2H2O is adsorbed to the membrane surface when the bilayer contains phloretin, 4-hydroxyvalerophenone, or 2-hydroxy-omega-phenylpropiophenone. Moreover, a rather large change in the residual phosphorus chemical shielding anisotropy argues in favor of hydrogen-bond formation between the phosphate segment and the phloretin hydroxyl groups.     

Bilayer interfacial properties modulate the binding of amphipathic peptides

The free energy of transfer (DeltaG degrees ) from water to lipid bilayers was measured for two amphipathic peptides, the presequence of the mitochondrial peptide rhodanese (MPR) and melittin. Experiments were designed to determine the effects on peptide partitioning of the addition of lipids that produce structural modifications to the bilayer/water interface. In particular, the addition of cholesterol or the cholesterol analog 6-ketocholestanol increases the bilayer area compressibility modulus, indicating that these molecules modify lipid-lipid interactions in the plane of the bilayer. The addition of 6-ketocholestanol or lipids with attached polyethylene glycol chains (PEG-lipids) modify the effective thickness of the interfacial region; 6-ketocholestanol increases the width of hydrophilic headgroup region in the direction of the acyl chains whereas the protruding PEG chains of PEG-lipids increase the structural width of the headgroup region into the surrounding aqueous phase. The incorporation of PEG-lipids with PEG molecular weights of 2000 or 5000 had no appreciable effect on peptide partitioning that could not be accounted for by the presence of surface charge. However, for both MPR and melittin DeltaG degrees decreased linearly with increasing bilayer compressibility modulus, demonstrating the importance of bilayer mechanical properties in the binding of amphipathic peptides.