Chemical exchange between lamellar and non-lamellar lipid phases. A one- and two-dimensional 31P-NMR study.

One- and two-dimensional 31P-exchange NMR has been used to investigate chemical exchange between coexisting lamellar (L alpha) and non-lamellar (hexagonal HII and cubic I2) lipid phases. Samples of DOPE, DOPE/DOPC (9:1 and 7:3), DOPE/cholesterol sulfate (9:1), DOPC/monoolein (MO) (3:7 and 1:1), and DOPC/DOPE/cholesterol (1:1:2) were macroscopically oriented on glass plates and studied at the 0 degree orientation (angle between the bilayer normal and the external magnetic field), where the L alpha, HII, and I2 resonances are resolved. A reversible L alpha to HII transition was observed for all of the samples except for the DOPC/MO mixtures, which displayed a reversible L alpha to I2 transition. Near-equilibrium mixtures of L alpha and either HII or I2 were obtained after prolonged incubation at a given temperature. Two-dimensional exchange experiments were performed on DOPE at 9-14 degrees C for mixing times ranging from 500 ms to 2 s. For all samples, one-dimensional exchange experiments were performed for mixing times ranging from 100 ms to 4 s, at temperatures ranging from 3 degrees C to 73 degrees C. No evidence of lipid exchange between lamellar and non-lamellar phases was observed, indicating that if such a process occurs it is either very slow on the seconds' timescale, or involves an undetectable quantity of lipid. The results place constraints on the stability or kinetic behaviour of proposed transition intermediates (Siegel, D.P. (1986) Biophys. J. 49, 1155-1170).     

2H-nuclear magnetic resonance investigations on phospholipid acyl chain order and dynamics in the gramicidin-induced hexagonal HII phase.

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.     

Molecular ordering of interfacially localized tryptophan analogs in ester- and ether-lipid bilayers studied by 2H-NMR.

Perdeuterated indole-d6 and N-methylated indole-d6 were solubilized in lamellar liquid crystalline phases composed of either 1,2-diacyl-glycero-3-phosphocholine (14:0)/water or 1,2-dialkyl-glycero-3-phosphocholine(14:0/water. The molecular ordering of the tryptophan analogs was determined from deuteron quadrupole splittings observed in 2H-NMR spectra on macroscopically aligned lipid bilayers. NMR spectra were recorded with the bilayers oriented perpendicular to or parallel with the external magnetic field, and the values of the splittings differed by a factor of 2 between these distinct orientations, indicating fast rotational motion of the molecules about an axis parallel to the bilayer normal. In all cases the splittings were found to decrease with increasing temperature. Relatively large splittings were observed in all systems, demonstrating that the tryptophans partition into a highly anisotropic environment. Solubilization most likely occurs at the lipid/water interface, as indicated by 1H-NMR chemical shift studies. The 2H-NMR spectra obtained for each analog were found to be rather similar in ester and ether lipids, but with smaller splittings in the ether lipid under similar conditions. The difference was slightly less for the indole molecule. Furthermore, in both lipid systems the positions of the splittings from indole were different from those of N-methyl indole. The results suggest that 1) the tryptophan analogs are solubilized in the interfacial region of the lipid bilayer, 2) the behavior may be modulated by hydrogen bonding in the case of indole, and 3) hydrogen bonding with the lipid carbonyl groups is not likely to play a major role in the solubilization of single indole molecules in the ester lipid bilayer interface.     

Comparison of the orientational order of lipid chains in the L alpha and HII phases

The orientational order profile has been determined by using deuterium nuclear magnetic resonance (2H NMR) for POPE in the lamellar liquid-crystalline (L alpha) and the hexagonal (HII) phases and is shown to be sensitive to the symmetry of the lipid phase. In the HII phase, as compared to the L alpha phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. 2H NMR studies of POPE dispersions containing tetradecanol or decane, both of which can induce HII phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the L alpha to HII phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The L alpha to HII phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. This is consistent with a preferential partition of the decane molecules into a disordered environment such as the intercylinder spaces in the HII phase. The presence of decane in the HII phase structure does not modify the order of the lipid chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha-methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: (2)H-NMR quadrupole splittings from individual lipids in mixed bilayers

A Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with alpha-deuterated oleic acid. Phosphatidylglycerol (PG), the glucolipids monoglucosyldiacylglycerol (MGlcDAG), diglucosyldiacylglycerol (DGlcDAG) and monoacyldiglucosyldiacylglycerol, and the phosphoglucolipid glycerophosphoryldiglucosyldiacylglycerol (GPDGlcDAG) were purified, and the phase behaviour and molecular ordering for the individual lipids, as well as for mixtures of the lipids, were studied by (2)H-, (31)P-NMR and X-ray scattering methods. The chemical structure of all the A. laidlawii lipids, except PG, has been determined and verified previously; here also the chemical structure of PG was verified, utilising mass spectrometry and (1)H and (13)C high resolution NMR spectroscopy. For the first time, lipid dimers were found in the mass spectrometry measurements. The major findings in this work are: (1) addition of 50 mol% of PG to the non-lamellar-forming lipid MGlcDAG does not significantly alter the transition temperature between lamellar and non-lamellar phases; (2) the (2)H-NMR quadrupole splitting patterns obtained from the lamellar liquid crystalline phase are markedly different for PG on one hand, and DGlcDAG and GPDGlcDAG on the other hand; and (3) mixtures of PG and DGlcDAG or MGlcDAG give rise to (2)H-NMR spectra consisting of a superposition of splitting patterns of the individual lipids. These remarkable features show that the local ordering of the alpha-carbon of the acyl chains is different for PG than for MGlcDAG and DGlcDAG, and that this difference is preserved when PG is mixed with the glucolipids. The results obtained are interpreted in terms of differences in molecular shape and hydrophilicity of the different polar headgroups.     

Importance of hydration for gramicidin-induced hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes

The macroscopic organization, lipid head group conformation, and structural and dynamic properties of 2H2O were investigated in dioleoylphosphatidylcholine (DOPC) model systems of varying gramicidin and 2H2O (or H2O) content by means of small-angle X-ray diffraction and 31P and 2H NMR. At low stages of hydration, N less than 6 (N = 2H2O/DOPC molar ratio), a single lamellar phase is observed in which the gramicidin molecules become preferentially hydrated upon increasing N. For 6 less than N less than 12 phase separation occurs between a gramicidin-poor and a gramicidin-rich lamellar phase. This latter phase is characterized by a smaller repeat distance and decreased DOPC head group order. For N greater than 12, the gramicidin-rich lamellar phase converts to a hexagonal HII phase. Thus, hydration of gramicidin is a prerequisite for HII phase formation in the DOPC/gramicidin system. The HII phase is very rich in gramicidin and 2H2O (gramicidin:DOPC:H2O = 1:1.1:0.9 w/w/w). A model is proposed in which self-assembly of hydrated gramicidin molecules into domains of a specific structure plays a determinant role in the formation of the HII phase by gramicidin.     

Thermodynamic, motional, and structural aspects of gramicidin-induced hexagonal HII phase formation in phosphatidylethanolamine

The effect of gramicidin incorporation on the thermodynamic properties of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) dispersions was investigated by differential scanning calorimetry. The results show that incorporation of gramicidin in PC systems results in a decrease of the energy content of the gel to liquid-crystalline phase transition. When incorporated in PE systems, however, the peptide does not affect the properties of the gel to liquid-crystalline phase transition with the exception that at high gramicidin concentrations the onset of the melting process is shifted to a slightly lower temperature. We therefore assume that in the lamellar gel state of PE aggregation of the peptide occurs. To get more insight into the nature of the gramicidin-PE interaction, we studied the motional and structural details of HII phase formation in gramicidin/PE systems with the use of 31P and 13C nuclear magnetic resonance (NMR) and small-angle X-ray diffraction. In agreement with earlier results [Van Echteld, C. J. A., Van Stigt, R., de Kruijff, B., Leunissen-Bijvelt, J., Verkleij, A. J., & De Gier, J. (1981) Biochim. Biophys. Acta 648, 287-291] it was shown that gramicidin incorporation lowers and broadens the bilayer to hexagonal HII phase transition in PE systems. 31P NMR chemical shift anisotropy (CSA) measurements indicated that a phase separation occurs between a gramicidin-poor lamellar phase and a gramicidin-rich HII phase. From combined CSA and spin-lattice relaxation time (T1) measurements it was suggested that in the HII phase gramicidin decreases the molecular order and increases the rate of motion of the phosphate moiety of PE. In addition, 13C NMR line width measurements indicated that the acyl chains are more disordered in the HII phase than in the lamellar phase and that a similar disorder occurs in the HII phase of the pure PE as in the gramicidin-rich HII phase. This interpretation was supported by the X-ray diffraction data, which show similar first-order repeat distances in both types of HII phase. From saturation-transfer NMR experiments in PE and gramicidin-PE mixtures it was shown that no exchange occurs between the lamellar and the HII phases in the time scale of 1-2 s, suggesting a macroscopic phase separation. Finally, we discussed the gramicidin-lipid interaction and in particular the HII phase formation by gramicidin in PE and in PC systems. It is proposed that aggregation of the peptide plays a crucial role in HII phase formation.     

NMR study of the interactions of polymyxin B, gramicidin S, and valinomycin with dimyristoyllecithin bilayers

The interactions of three polypeptide antibiotics (polymyxin B, gramicidin S, and valinomycin) with artificial lecithin membranes were studied by nuclear magnetic resonance (NMR). Combination of 31P and 2H NMR allowed observation of perturbations of the bilayer membrane structure induced by each of the antibiotics in the regions of the polar headgroups and acyl side chains of the phospholipids. The comparative study of the effects of these membrane-active antibiotics and the lipid bilayer structure demonstrated distinct types of antibiotic-membrane interactions in each case. Thus, the results showed the absence of interaction of polymyxin B with the dimyristoyllecithin membranes. In contrast, gramicidin S exhibited strong interaction with the lipid above the gel to liquid-crystalline phase transition temperature: disordering of the acyl side chains was evident. Increasing the concentration of gramicidin S led to disintegration of the bilayer membrane structure. At a molar ratio of 1:16 of gramicidin S to lecithin, the results are consistent with coexistence of gel and liquid-crystalline phases of the phospholipids near the phase transition temperature. Valinomycin decreased the phase transition temperature of the lipids and increased the order parameters of the lipid side chains. Such behavior is consistent with penetration of the valinomycin molecule into the interior of the lipid bilayers.     

Influence of membrane-spanning alpha-helical peptides on the phase behavior of the dioleoylphosphatidylcholine/water system

The effect of solubilized hydrophobic peptides on the phase behavior of dioleoylphosphatidylcholine (DOPC)/water system was studied by 2H- and 31P-NMR spectroscopy and by x-ray diffraction, and partial phase diagrams were constructed. The utilized peptides were HCO-AWW(LA)5WWA-NHCH2CH2OH (WALP16), which is an artificial peptide designed to resemble a transmembrane part of a membrane protein; and VEYAGIALFFVAAVLTLWSMLQYLSAAR (Pgs peptide E), a peptide that is identical to one of the putative transmembrane segments of the membrane-associated protein phosphatidylglycerophosphate synthase (Pgs) in Escherichia coli. Circular dichroism spectroscopy suggests that both peptides are mostly alpha-helical in DOPC vesicles. The most striking features in the phase diagram of the WALP16/DOPC/water system are 1) a single lamellar liquid crystalline (L alpha) phase forms only at very low peptide concentrations. 2) At low water content and above a peptide/lipid molar ratio of approximately 1:75 a reversed hexagonal liquid crystalline (H[II]) phase coexists with an L alpha phase, while in excess water this phase forms at a peptide/lipid molar ratio of approximately 1:25. 3) At peptide/lipid ratios > or =1:6 a single H(II) phase is stable. Also, the Pgs peptide E strongly affects the phase behavior, and a single L alpha phase is only found at low peptide concentrations (peptide/lipid molar ratios <1:50), and water concentrations <45% (w/w). Higher peptide content results in coexistence of L alpha and isotropic phases. Generally, the fraction of the isotropic phase increases with increasing temperature and water concentration, and at 80% (w/w) water content only a single isotropic phase is stable at 55 degrees C. Thus, both peptides were found to be able to induce nonlamellar phases, although different in structure, in the DOPC/water system. The phase transitions, the extensions of the one-phase regions, and the phase structures observed for the two systems are discussed in terms of the molecular structure of the two peptides and the matching between the hydrophobic lengths of the peptides and the bilayer thickness of DOPC.     

NMR study of synthetic lecithin bilayers in the vicinity of the gel-liquid--crystal transition.

1H, 2H, and 31P NMR methods have been employed in the study of dimyristoyl lecithin bilayers hydrated with D2O in the gel (L beta'), intermediate (P beta') and liquid-crystalline (L alpha) phases. For D2O/lipid molar ratios, n, in the range 7 less than or equal to n less than or equal to 11 discontinuities are observed in the deuterium NMR splittings at both main and pretransitions. A partial phase diagram based on NMR and differential scanning calorimetry data is presented. 1H NMR dipolar splittings are observed for macroscopically oriented samples in all three phases. Changes in the 1H splittings are correlated with 2H and 31P data and interpreted to show that the chain tilt in the gel phase undergoes a discontinuous change on transition to the intermediate phase, which brings the chain axes closer to the bilayer normal. An estimate of chain tilt in the gel phase is made on the basis of NMR data and found to be approximately 23 degrees for a sample with n = 11 at 18 degrees C.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.     

Polymorphic phase behavior of lysopalmitoylphosphatidylcholine in poly(ethylene glycol)-water mixtures

The polymorphic phase behavior of 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine dispersions in excess water has been studied as a function of temperature and poly(ethylene glycol) (PEG) concentration, using proton dipolar-decoupled 31P NMR spectroscopy and turbidity measurements. The phase behavior was found to depend on both lipid concentration and PEG concentration, and most of the NMR experiments were conducted at a lipid concentration of 15 mg/mL. At low PEG concentrations (0-12 wt %), a thermotropic transition occurs at 3-5 degrees C with increasing temperature, from an interdigitated lamellar gel (L beta i) phase to a normal micellar phase. At intermediate PEG concentrations (12-20 wt %), thermotropic transitions take place with increasing temperature, first from the lamellar gel phase to a fluid cubic (Q alpha) phase and then at higher temperatures from the cubic phase to the micellar phase. At intermediate PEG concentrations above the former range (20-30 wt %), thermotropic transitions take place with increasing temperature, first from the lamellar gel phase to the cubic phase, then from the cubic phase to a normal hexagonal (HI) phase, and finally from the hexagonal phase to the micellar phase. At high PEG concentrations (greater than 30 wt %), a thermotropic transition takes place with increasing temperature from the lamellar gel phase directly to the fluid hexagonal phase. At these high PEG concentrations, the micellar phase is not attained within the accessible temperature range (less than or equal to 90 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of acyl chain composition on salt-induced lamellar to inverted hexagonal phase transitions in cardiolipin

Salt-induced fluid lamellar (L alpha) to inverted hexagonal (HII) phase transitions have been studied in diphosphatidylglycerols (cardiolipins) with different acyl chain compositions, using 31P nuclear magnetic resonance (NMR) spectroscopy. Cardiolipins with four myristoyl chains, tetramyristoyl cardiolipin (TMCL), and with four oleoyl chains, tetraoleoyl cardiolipin (TOCL), were synthesized chemically. TMCL was found to undergo a thermotropic lamellar gel to lamellar liquid-crystalline phase transition at 33-35 degrees C. This lipid exhibited an axially symmetric 31P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of TOCL, formation of an HII phase was induced by salt concentrations of 3.5 M NaCl or greater. These observations, taken together with earlier findings that bovine heart cardiolipin aqueous dispersions adopt an HII phase at salt concentrations of 1.5 M NaCl or greater, indicate that increasing unsaturation and length of the acyl chains favour formation of the HII phase in diphosphatidylglycerols.     

Order and dynamics in the lamellar L alpha and in the hexagonal HII phase. Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization.

Fluorescence depolarization techniques are used to determine the molecular order and reorientational dynamics of the probe molecule TMA-DPH embedded in the lamellar L alpha and the hexagonal HII phases of lipid/water mixtures. The thermotropically induced L alpha----HII phase transition of the lipid DOPE is used to obtain macroscopically aligned samples in the hexagonal HII phase at 45 degrees C from samples prepared in the lamellar L alpha phase at 7 degrees C. The interpretation of angle-resolved fluorescence depolarization experiments on these phases, within the framework of the rotational diffusion model, yields the order parameters (P2) and (P4), and the diffusion constants for the reorientational motions. The reorientational motion rates of the TMA-DPH molecules in the hexagonal HII phase are comparable with those in the lamellar L alpha phase. Furthermore, the lateral diffusion of the probe molecule on the surface of the lipid/water cylinder in the hexagonal phase is found to be considerably slower than the reorientational motion.     

Dehydration induces lateral expansion of polyunsaturated 18:0-22:6 phosphatidylcholine in a new lamellar phase.

To gain a better understanding of the biological role of polyunsaturated phospholipids, infrared (IR) linear dichroism, NMR, and x-ray diffraction studies have been conducted on the lyotropic phase behavior and bilayer dimensions of sn-1 chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35), a mixed-chain saturated (18:0)-polyunsaturated (22:6 omega 3) lipid. SDPC films were hydrated at definite values of temperature (T) and relative humidity (RH). In excess water, the lipid forms exclusively lamellar phases in the temperature range 0--50 degrees C. Upon dehydration the lipid undergoes the main phase transition between the liquid-crystalline (L(alpha)) and gel (L(beta)) phase at T < 15 degrees C. Both the saturated and polyunsaturated chains adopt a stretched conformation in the L(beta) phase, presumably the all-trans (stearoyl) and angle iron or helical (docosahexaenoyl) one. A new fluid lamellar phase (L(alpha)') was found in partially hydrated samples at T > 15 degrees C. SDPC membranes expand laterally and contract vertically in the L(alpha)' phase when water was removed. This tendency is in sharp contrast to typical dehydration-induced changes of membrane dimensions. The slope of the phase transition lines in the RH-T phase diagram reveal that the lyotropic L(alpha)'-L(alpha) and L(beta)-L(alpha) transitions are driven by enthalpy and entropy, respectively The possible molecular origin of the phase transitions is discussed. The properties of SDPC are compared with that of membranes of monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31).     

A comparison of spin probe ESR, 2H- and 31P-nuclear magnetic resonance for the study of hexagonal phase lipids

We have investigated the feasibility of the various possible magnetic resonance probes of lipids which form non-bilayer phases. As a model system we have used equimolar mixtures of phosphatidylethanolamine (PE) and cholesterol, which exhibit a thermotropic transition from a bilayer to a hexagonal phase. Variable temperature electron spin resonance (ESR) spin probe spectra were obtained using random dispersion and oriented lipid systems. Simultations of the ESR spectra were performed in order to aid in the interpretation of the experimental results for the oriented system. ³¹P- and ²H-nuclear magnetic resonance (NMR) studies were carried out using a deuterated PE. The ESR spin probes in the random dispersions show essentially no effect attributable to the phase transition. However, there are large, reversible effects in the temperature-dependent behaviour for the oriented system. The orientation dependence of the spectra above the transition temperature indicate that the hexagonal phase lipids may spontaneously assume a macroscopic organization on a flat surface. We find, however, that such an organization cannot be unambiguously assigned from the ESR spin probe spectra, and point out a potential difficulty in the interpretation of spin probe spectra in oriented systems. In contrast, the ²H-NMR method provides a reliable monitor of the phase transformation. Taken together, the ²H and ³¹P data indicate that the structure of the headgroup in PE is quite similar in both the bilayer and hexagonal phase. ²H-NMR should be very useful in probing the structural and dynamic characteristics of lipids in non-bilayer phases.     

Gramicidin-induced hexagonal HII phase formation in erythrocyte membranes

Using 31P nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and freeze-fracture electron microscopic (FFEM) techniques, it is shown that gramicidin induces a hexagonal HII phase not only in liposomes prepared from total lipids extracted from human erythrocytes but also in isolated human erythrocyte membranes (white ghosts). A 37 degrees C, HII phase formation is detected at a gramicidin to phospholipid molar ratio exceeding 1:80. At a molar ratio of 1:5, about 30% of the phospholipid is organized in the HII phase. The gramicidin-induced HII phase exhibits a very small 31P chemical shift anisotropy [(CSA) approximately 10 +/- 1 ppm], indicating decreased head-group order, and it displays a temperature-dependent increase in tube diameter from 60.2 A at 4 degrees C to 64.2 A at 37 degrees C in ghosts and from 62.8 to 69.4 A at 37 degrees C in total lipid extracts, both in the presence of 1 mol of gramicidin/10 mol of phospholipid. This anomalous temperature-dependent behavior is probably due to the presence of cholesterol. 31P NMR data indicate that the HII phase formation by gramicidin is temperature dependent and show the gradual disappearance of the HII phase at low temperatures (less than 20 degrees C), resulting in a bilayer type of 31P NMR line shape at 4 degrees C, whereas SAXS and FFEM data suggest equal amounts of HII phases at all temperatures. This apparent discrepancy is probably the result of a decrease in the rate of lateral diffusion of the membrane phospholipids which leads to incomplete averaging of the 31P CSA in the HII phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of chromophores in model membranes by polarized light-absorption spectroscopy. The orientation and binding of tetracaine and procaine.

A method for studying the orientation and binding of chromophores in macroscopically aligned membranes by polarized light absorption spectroscopy is described. Here tetracaine and procaine solubilized in a lamellar phase of octanoyl-1-glyceride (monooctanoin) and water have been investigated. Tetracaine is found to be located in the lipid region with a preferential orientation of the molecular long axis parallel to the hydrocarbon chains. The orientation of procaine, mainly residing in the water region, is very small.     

Lipid-drug interaction: a structural analysis of pindolol effects on model membranes.

The ternary system constituted by distearoylphosphatidylcholine, pindolol (a vasodilator drug) and water has been investigated by using X-ray diffraction and calorimetric techniques. The structural modifications induced by the drug have been determined and a possible interaction model has been derived. In particular, the pindolol content-temperature dependent phase diagram shows the occurrence of two new phases: the first is an interdigitated gel, and the second is a lamellar structure presenting an unusual mixed disordered-ordered conformation of the hydrocarbon chains (L alpha beta). The comparative analysis of electron density profiles relative to the L alpha beta phase, reveals significant modifications in the paraffinic region of the lipid layer. In agreement with thermodynamic results, the structural data suggest that the drug induces a stiffening and a tightening of the hydrocarbon chains. Moreover, the hydrophilic properties of the membrane (particularly in P beta, and L alpha beta phases) present an evident dependence with the drug concentration.     

Characterization of the L lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.