HII Phase Research Articles

Curvature properties of novel forms of phosphatidylcholine with branched acyl chains.

We studied the properties of a series of phosphatidylcholine molecules with branched acyl chains. These lipids have previously been shown to have marked stimulatory effects on the side-chain cleavage activity of cytochrome P450SCC (CYP11A1), an enzyme of the inner mitochondrial membrane. The synthetic lipids used were diacyl phosphatidylcholines with the decanoyl, dodecanoyl or tetradecanoyl chain having a hexyl, octyl or decyl straight chain aliphatic branch at the 2-position. All three lipids lowered the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine, the lipids with longer acyl chains being more effective in this regard. As pure lipids all of the forms were found by X-ray diffraction to be predominantly in the hexagonal phase (HII) over the entire temperature range of 7-75 degrees C. The properties of the HII phase were unusual with regard to the small size of the lattice spacings and the small temperature dependence of the spacings. We used tetradecane to relieve hydrocarbon packing constraints to determine the intrinsic radius of curvature of the lipid monolayer. The elastic bending modulus was measured in the presence of tetradecane by introducing an osmotic gradient across the hexagonal phase cylinders with aqueous solutions of poly(ethylene glycol). The elastic bending modulus was found to be higher than that observed with other lipids and to increase with temperature. Both the small intrinsic radius of curvature and the high elastic bending modulus indicate that the presence of these lipids in bilayer membranes will impose a high degree of negative curvature strain.

Tryptophan-anchored transmembrane peptides promote formation of nonlamellar phases in phosphatidylethanolamine model membranes in a mismatch-dependent manner.

To better understand the mutual interactions between lipids and membrane-spanning peptides, we investigated the effects of tryptophan-anchored hydrophobic peptides of various lengths on the phase behavior of 1,2-dielaidoylphosphatidylethanolamine (DEPE) dispersions, using (31)P nuclear magnetic resonance and small-angle X-ray diffraction. Designed alpha-helical transmembrane peptides (WALPn peptides, with n being the total number of amino acids) with a hydrophobic sequence of leucine and alanine of varying length, bordered at both ends by two tryptophan membrane anchors, were used as model peptides and were effective at low concentrations in DEPE. Incorporation of 2 mol % of relatively short peptides (WALP14-17) lowered the inverted hexagonal phase transition temperature (T(H)) of DEPE, with an efficiency that seemed to be independent of the extent of hydrophobic mismatch. However, the tube diameter of the H(II) phase induced by the peptides was clearly dependent on mismatch and decreased with shorter peptide length. Longer peptides (WALP19-27) induced a cubic phase, both below and above T(H). Incorporation of WALP27, which is significantly longer than the DEPE bilayer thickness, did not stabilize the bilayer. The longest peptide used, WALP31, hardly affected the lipid's phase behavior, and appeared not to incorporate into the bilayer. The consequences of hydrophobic mismatch between peptides and lipids are therefore more dramatic with shorter peptides. The data allow us to suggest a detailed molecular model of the mechanism by which these transmembrane peptides can affect lipid phase behavior.

The amphiphilic drug flufenamic acid can induce a hexagonal phase in DMPC: a solid state 31P- and 19F-NMR study

Established solid state 31P-NMR and novel 19F-NMR experiments are used in a complementary approach to describe the behaviour of a fluorinated drug, flufenamic acid (FFA), in phospholipid model membranes. The non-steroidal anti-inflammatory agent FFA was dissolved at 5% (w/w) in dimyristoylphosphatidylcholine (DMPC), and the system was investigated at low hydration (3 H2O per lipid) where morphological transitions of the lipid are strongly affected by additives. It is demonstrated that FFA induces a fluid HII phase in DMPC at ambient temperatures, i.e. much below its regular chain-melting transition which occurs around 50°C at low hydration. The guest molecules are preferentially accommodated in the hexagonal phase of the lipid, which coexists with the usual crystalline state of pure DMPC. The peculiar transition sequence LC→HII→Lα with increasing temperature is explained by a re-distribution of FFA in the lipid matrix and a concomitant phase separation under conditions of limiting hydration. Small-angle X-ray diffraction and freeze–fracture electron microscopy are used to confirm the existence of the hexagonal and bilayer phases, and to determine their respective dimensions. When the drug FFA dissolves in the bilayer, its structural effect on the surrounding lipid molecules may be related to its pharmacological activity in membranes. For example, FFA is known to modulate ion channel function, and it has been suggested that it inhibits phospholipase activity by accelerating the transbilayer flip-flop of lipids.

Influence of the physical state of the membrane on the enzymatic activity and energy of activation of protein kinase C alpha.

The activation of protein kinase C alpha was studied by using a lipid system consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) (molar ratio 4:1) and different proportions of 1-palmitoyl-2-oleoyl-sn-glycerol (POG). The phase behavior of the lipidic system was characterized by using differential scanning calorimetry and 31P NMR, and a phase diagram was elaborated. The results suggested the formation of two diacylglycerol/phospholipid complexes, one at 15 mol % of POG and the second at 30 mol % of POG. These two complexes would define the three regions of the phase diagram: in the first region (concentrations of POG lower than 15 mol %) there is gel-gel immiscibility at temperatures below that of the phase transition between C1 and pure phospholipid, and a fluid lamellar phase above of the phase transition. In the second region (between 15 and 30 mol % of POG), gel-gel immiscibility between C1 and C2 with fluid-fluid immiscibility was observed, while inverted hexagonal HII and isotropic phases were detected by 31P NMR. In the third region (concentrations of POG higher than 30 mol %), gel-gel immiscibility seemed to occur between C2 and pure POG along with fluid-fluid immiscibility, while an isotropic phase was detected by 31P NMR. When PKC alpha activity was measured, as a function of POG concentration, maximum activity was found at POG concentrations as low as 5-10 mol %; the activity slightly decreased as POG concentration was increased to 45 mol % at 32 degrees C (above Tc) whereas activity did not change with increasing concentrations of POG at 5 degrees C (below Tc). When the activity was studied as a function of temperature, at different POG concentrations, and depicted as Arrhenius plots, it was found that the activity increased with increasing temperatures, showing a discontinuity at a temperature very close to the phase transition of the system and a lower activation energy at the upper slope of the graph, indicating that the physical state of the membrane affected the interaction of PKC alpha with the membrane.

The interaction of coenzyme Q with phosphatidylethanolamine membranes.

Coenzyme Q (CoQ) is a component of the mitochondrial respiratory chain which carries out additional membrane functions, such as acting as an antioxidant. The location of CoQ in the membrane and the interaction with the phospholipid bilayer is still a subject of debate. The interaction of CoQ in the oxidized (ubiquinone-10) and reduced (ubiquinol-10) state with membrane model systems of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (Ela2Gro-P-Etn) has been studied by means of differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (31P-NMR) and small angle X-ray diffraction (SAXD). Ubiquinone-10 did not visibly affect the lamellar gel to lamellar liquid-crystalline phase transition of Ela2Gro-P-Etn, but it clearly perturbed the multicomponent lamellar liquid-crystalline to lamellar gel phase transition of the phospholipid. The perturbation of both transitions was more effective in the presence of ubiquinol-10. A location of CoQ forming head to head aggregates in the center of the Ela2Gro-P-Etn bilayer with the polar rings protruding toward the phospholipid acyl chains is suggested. The formation of such aggregates are compatible with the strong hexagonal HII phase promotion ability found for CoQ. This ability was evidenced by the shifting of the lamellar to hexagonal HII phase transition to lower temperatures and by the appearance of the characteristic hexagonal HII 31P-NMR resonance and SAXD pattern at temperatures at which the pure Ela2Gro-P-Etn is still organized in extended bilayer structures. The influence of CoQ on the thermotropic properties and phase behavior of Ela2Gro-P-Etn is discussed in relation to the role of CoQ in the membrane.

Lyotropic Phase Behavior and Gel State Polymorphism of Phospholipids with Terminal Diene Groups: Infrared Measurements on Molecular Ordering in Lamellar and Hexagonal Phases

The phase behavior of the diene lipid 1,2-bis(11,13-tetradecadienoyl)-sn-glycero-3-phosphorylethanolamine (DTDPE) and the corresponding phosphatidylcholine analogue (DTDPC) has been studied as a function of temperature (T) and relative humidity (RH) by means of infrared (IR) dichroism measurements. Lamellar/nonlamellar and solid/fluid phase transitions of lipid films spread on an ATR crystal are determined with high resolution by means of spectral parameters. The nonlamellar inverse hexagonal (HII) phase has been found for DTDPE at T > 45 °C, a lamellar gel at T < 5 °C and the lamellar liquid−crystalline phase in between at RH > 90%. The ranges of the gel and the HII states can be extended into the range of moderate temperatures by dehydrating the lipid where a direct transformation between the gel and HII was found. The appearance of the HII phase is somewhat unexpected in view of the short effective length of the tetradecadienoyl chains. It can be attributed to the influence of the terminal diene groups which remains however unspecified. Similar to other PE lipids, DTDPE tends to transform from the gel into crystalline polymorphs. The PC analogue assembles into liquid−crystalline membranes at nearly all conditions studied. The hydration, conformation, and packing of the polar region of the lipid aggregates has been characterized by IR spectral parameters. This method has been complemented by 31P NMR to assign the nonlamellar phase and by DSC to yield thermodynamic information.

Tilt model of inverted amphiphilic mesophases

We present an alternative model of structure and energetics of the inverted amphiphilic mesophases. The previous studies of the inverted hexagonal, HII, and inverted micellar cubic, QII, phases considered the amphiphilic monolayers to be homogeneously bent. In contrast, we assume a unit cell of an inverted mesophase to consist of flat fragments of monolayer. Hence, the unit cells of the HII and QII phases are represented by a hexagonal rod and a polyhedron, respectively. Our model is motivated by Turner and Gruner's X-ray diffraction reconstruction of structure of the HII phase. The only deformation of the amphiphilic monolayers we consider is tilt of the hydrocarbon chains with respect to the monolayer surface, determined by the packing constraints imposed in the mesophases. Applying our recent model for the elastic energy of tilt in liquid membranes [#!ref23!#], we show that: i) tilt accounts in a natural way for the frustration energy of mesophases resulting from filling by the hydrocarbon chains the corners of the unit cells, ii) the energy of tilt variation along the membrane surface is analogous to the bending energy. We compute the energetics of the HII, QIIsc and QIIfcc phases and obtain a hypothetical phase diagram in terms of the elastic constants of monolayers. Moreover, we calculate the structural dimensions of the mesophases. We verify the model showing that the obtained phase diagram describes the recent data for the glycolipids/water systems; the predicted dimensions of the QII phase are in accord with the measured values; the model treats quantitatively the structural features observed for the HII phase.

Induction of a hexagonal phase in phospholipid-surfactant bilayers

The possibility of modifying the curvature of lipid bilayers by mixing them with additives is demonstrated and the evolution of geometrical parameters with composition is discussed. X-ray diffraction patterns of the POPC/C12EO2/2H2O system were observed as a function of the relative humidity. The formation of an unexpected hexagonal phase indicates peculiar behaviour in these mixtures. The cylinder radius of this phase is considerably smaller than previously observed in cubic phases. A discussion of the head group interactions is presented. We have also been able to show that the uptake of water by the Lβ gel phase is higher as a single phase than in the Lβ+HII two phase region. The water content is important for the stabilization of HII phase and determination of its characteristic dimensions. However, it is argued that the interaction between the surfactant and the lipid is the key factor for its formation and that the EO2 head groups displace water from the inner parts of the polar region of the mesogenic units.

The influence of cholesterol on phospholipid membrane curvature and bending elasticity

The behavior of dioleoylphosphatidylethanolamine (DOPE)/cholesterol/tetradecane and dioleoylphosphatidylcholine (DOPC)/cholesterol/tetradecane were examined using x-ray diffraction and the osmotic stress method. DOPE/tetradecane, with or without cholesterol, forms inverted hexagonal (HII) phases in excess water. DOPC/tetradecane forms lamellar phases without cholesterol at lower temperatures. With tetradecane, as little as 5 mol% cholesterol in DOPC induced the formation of HII phases of very large dimension. Increasing levels of cholesterol result in a systematic decrease in the HII lattice dimension for both DOPE and DOPC in excess water. Using osmotic pressure to control hydration, we applied a recent prescription to estimate the intrinsic curvature and bending modulus of the HII monolayers. The radii of the intrinsic curvature, RPO, at a pivotal plane of constant area within the monolayer were determined to be 29.4 A for DOPE/tetradecane at 22 degrees C, decreasing to 27 A at 30 mol% cholesterol. For DOPC/tetradecane at 32 degrees C, RPO decreased from 62.5 A to 40 A as its cholesterol content increased from 30 to 50 mol%. These data yielded an estimate of the intrinsic radius of curvature for pure DOPC of 87.3 A. The bending moduli kc of DOPE/tetradecane and DOPC/tetradecane, each with 30 mol% cholesterol, are 15 and 9 kT, respectively. Tetradecane itself was shown to have little effect on the bending modulus in the cases of DOPE and cholesterol/DOPE. Surprisingly, cholesterol effected only a modest increase in the kc of these monolayers, which is much smaller than estimated from its effect on the area compressibility modulus in bilayers. We discuss possible reasons for this difference.

Kinetics of glycolipid phase transitions: ms laser T‐jump synchrotron studies

AbstractThe kinetics of the lamellar gel (Lβ) to inverse hexagonal (HII) phase transition as well as the LC→Lα phase change of aqueous suspensions of glycolipids were characterized using ms laser T‐jump techniques in combination with synchrotron X‐ray diffraction. The glycolipids employed were 1,2‐di‐O‐alkyl‐3‐O‐β‐D‐glucosyl‐snglycerols with identical alkyl chains of 16 and 18 carbon atoms, respectively, and 1,2‐di‐O‐alkyl‐3‐O‐β‐D‐galactosyl‐sn‐glycerol with C16 chains. The time course of the LC→Lα phase transition was probed using 1,2‐di‐O‐hexadecyl‐3‐O‐β‐D‐lactosyl‐sn‐glycerol, whose head group consists of the disaccharide β‐D‐galactopyranosyl‐(1→4)‐β‐D‐glucopyranoside. Surprisingly the phase change from the lamellar to the inverse hexagonal topology is very fast despite the major structural changes involved in the lipid‐water interface. The overall phase change of both glucolipids can be quantitatively described by a sequential mechanism of the type \documentclass{article}\pagestyle{empty}\usepackage{amsmath}\begin{document}$ {\rm L}_\beta \xrightarrow{{k_1}}{\rm I}\xrightarrow{{k_2}}{{\rm H}}_{\rm II} $\end{document}$\end{document}. The intermediate I is thinner than both the Lβ and the HII phases. It has a relatively broad distribution of d‐spacings and relaxes into the HII phase with half times of approximately 125 ms (16‐1,2‐Glc) and 244 ms (18‐1,2‐Glc), respectively. Both rate constants, k1 and k2, are chain length dependent.The galactolipid transforms into the HII phase following a sequential mechanism of the type \documentclass{article}\pagestyle{empty}\usepackage{amsmath}\begin{document}$ {{\rm L}}_\beta \xrightarrow{{k_1}}{{\rm I'}}\xrightarrow{{k_2}}{{\rm I}}\xrightarrow{{k_3}}{{\rm H}}_{\rm II} $\end{document}. The additional intermediate I' can be assigned to a lamellar phase bearing similarity to a thin Lα phase. The specific features of the galactose head group compared to glucose as head group that have been previously observed in equilibrium studies are also evident in the kinetic mechanism in that comparative structural changes in the intermediates are slower in the galactolipid than in the chain‐homologous glucolipid. There is evidence from the X‐ray data that the general transition mechanism is identical for gluco‐ and galacto‐lipids. However, the first intermediate I' disappears too fast in the glucolipid phase change to be detectable with the present time resolution of the X‐ray measurements.The kinetics of the phase change of the lactolipid is characterized within the resolution of the X‐ray sequence (5 ms) by a strict two‐state mechanism \documentclass{article}\pagestyle{empty}\usepackage{amsmath}\begin{document}$ {{\rm L}}_{{\rm C}} \xrightarrow{k}{{\rm L}}_{\alpha} $\end{document} with transient coexistence of the initial and final phase during the kinetic phase transformation. The relaxation time has been found to be highly temperature dependent, being 57 ms at 79.1 ° and 14 ms at 81.1 °.It is worth noting that there appears to be a principal difference in the phase change mechanism between glycolipids with mono‐ or disaccharide head groups in that the latter (16‐1,2‐Lac) shows no sign of any kinetic intermediate state.

Polymerization of the Inverted Hexagonal Phase

The hydration of polar natural and synthetic lipids yields a variety of lipid phases including various inverted cubic phases and the inverted hexagonal (HII) phase. The HII phase can be considered as aqueous columns encased with a monolayer of lipids and arranged in a hexagonal pattern. The polar head groups are well-ordered at the water interface, whereas the lipid tails are disordered to fill the volume between the tubes of water. A particularly interesting characteristic of the HII phase is the large temperature effect on the basis vector length d of the hexagonal lattice. Previous studies indicate that polymerization of the lipid region of the HII phase might reduce the sensitivity of the basis vector to temperature. A phosphoethanolamine (PE) was designed and synthesized with dienoyl groups in each lipid tail in an attempt to cross-link the lipids around and along the water core of the HII phase. The synthesis of the the PE was accomplished by acylation of 3-(4-methoxybenzyl)-sn-glycerol with 2,4,13-...

Phase Behavior of Synthetic Phytanyl-Chained Glycolipid/Water Systems

We have synthesized 1,3-di-O-phytanyl-2-O-(glycosyl)glycerols possessing maltooligosaccharide headgroups, MalN(Phyt)2, as model archaebacterial glycolipids (the number of glucose units N = 1, 2, 3, and 5) and investigated the phase behavior of the lipid/water systems using small-angle X-ray scattering and differential scanning calorimetry. The hydrated-solid to liquid-crystalline phase transition temperatures of MalN(Phyt)2 are well below 0 °C, so that MalN(Phyt)2 are in a liquid crystalline state above 0 °C. The structures of the aqueous MalN(Phyt)2 are largely determined by N; a weakly birefringent mesophase for a glucolipid, Glc(Phyt)2, an HII phase for Mal2(Phyt)2, and an Lα phase for Mal3(Phyt)2 and Mal5(Phyt)2. This indicates an increasing tendency for the MalN(Phyt)2 monolayers to curve toward apolar regions with increasing N. The salient feature of the phytanyl chain lies in significantly larger cross section areas of the lipids at the hydrophobic part−headgroup interface (e.g., ∼1.0 nm2/lipid for...

Measured effects of diacylglycerol on structural and elastic properties of phospholipid membranes

Diacylglycerol, a biological membrane second messenger, is a strong perturber of phospholipid planar bilayers. It converts multibilayers to the reverse hexagonal phase (HII), composed of highly curved monolayers. We have used x-ray diffraction and osmotic stress of the HII phase to measure structural dimensions, spontaneous curvature, and bending moduli of dioleoylphosphatidylethanolamine (DOPE) monolayers doped with increasing amounts of dioleoylglycerol (DOG). The diameter of the HII phase cylinders equilibrated in excess water decreases significantly with increasing DOG content. Remarkably, however, all structural dimensions at any specific water/lipid ratio that is less than full hydration are insensitive to DOG. By plotting structural parameters of the HII phase with changing water content in a newly defined coordinate system, we show that the elastic deformation of the lipid monolayers can be described as bending around a pivotal plane of constant area. This dividing surface includes 30% of the lipid volume independent of the DOG content (polar heads and a small fraction of hydrocarbon chains). As the mole fraction of DOG increases to 0.3, the radius of spontaneous curvature defined for the pivotal surface decreases from 29 A to 19 A, and the bending modulus increases from approximately 11 to 14 (+/-0.5) kT. We derive the conversion factors and estimate the spontaneous curvatures and bending moduli for the neutral surface which, unlike the pivotal plane parameters, are intrinsic properties that apply to other deformations and geometries. The spontaneous curvature of the neutral surface differs from that of the pivotal plane by less than 10%, but the difference in the bending moduli is up to 40%. Our estimate shows that the neutral surface bending modulus is approximately 9kT and practically does not depend on the DOG content.

Rate of phase transformations between mesophases of the 1:2 lecithin/fatty acid mixtures DMPC/MA and DPPC/PA ‐ a time‐resolved synchrotron X‐ray diffraction study

AbstractUsing the pressure‐jump relaxation technique in combination with synchrotron X‐ray diffraction we have investigated the rate and mechanism of phase transformations of mesophases of the lecithin/fatty acid mixtures dimyristoylphosphatidylcholine/myristic acid (DMPC/MA) and dipalmitoylphosphatidylcholine/palmitic acid (DPPC/PA) in the molar ratio 1:2. The investigations have been made as a function of the pressure‐jump amplitude and at two different levels of hydration, 50 and 75 wt% H2O. For the investigation of the structure of the lipid systems at elevated pressures as well as for the study of the kinetics of lipid phase transitions using the pressure‐jump technique, we built a high pressure X‐ray cell suitable for studies up to pressures of 2 kbar at temperatures ranging up to 140°C. In the first part of the paper, the temperature and pressure dependent structure and equilibrium phase behaviour of the two lecithin/fatty acid mixtures is discussed. In equilibrium, the chain melting transition of both mixtures occurs without forming a lamellar liquid‐crystalline Lα phase. In DMPC/MA (1:2) dispersions the phase sequence is L/Lc→Lc→Lβ→HII/Q/Q→HII, in DPPC/PA (1:2) LL/Lc→Lβ→HII with increasing temperature or decreasing pressure, respectively. A temperature‐pressure phase diagram has been established in the temperature range from 0‐100°C at pressures from ambient up to 1500 bar for both systems. Pressure‐jumps within the inverted hexagonal/Q cubic phase coexisting region of DMPC/MA (1:2) lead to a slight change of the composition of the mixture, and the structural relaxation process of the lattices proceeds in several steps. The rate‐determining part of the structural relaxation process is probably due to the restricted diffusion of water in and into the lattices and the relaxation of the hexagonal and cubic structures are found to be coupled to each other. Equilibrium values of the lattice constants are reached after about 30 s. Formation of further cubic lattices is possible, depending on the level of hydration. Experiments on the rate of the Lβ→HII/Q chain melting transition of DMPC/MA (1:2) exhibit the occurence of a long‐lived intermediate Lα phase, which is not observed under equilibrium conditions. The HII lattice starts to develop within the time‐resolution of this experiment, i.e. within the first 200 ms. First Bragg reflections of the cubic phase appear about 100 s after the pressure‐jump. Equilibrium values of the lattice constants are not reached before 30 min. The pressure‐jump amplitude has been found to have a significant influence on the kinetics of the phase transformation, the mechanism remaining. The increase of the pressure‐jump amplitude leads to a faster decay of the Lβ phase and the more rapid formation of the HII phase. The Lβ→HII transition of DPPC/PA (1:2) dispersion follows a simpler kinetics. No intermediate Lα phase is found. The transition is complete about 25 s after a pressure‐jump from 520 to 200 bar at 71 °C, and it seems to be a simple two‐state process to within the sensitivity limit of these experiments.

Detection of membrane packing defects by time-resolved fluorescence depolarization

Packing defects in lipid bilayer play a significant role in the biological activities of cell membranes. Time-resolved fluorescence depolarization has been used to detect and characterize the onset of packing defects in binary mixtures of dilinoleoylphosphatidylethanolamine/1-palmitoyl-2- oleoylphosphatidylcholine (PE/PC). These PE/PC mixtures exhibit mesoscopic packing defect state (D), as well as one-dimensional lambellar liquid crystalline (L alpha) and two-dimensional inverted hexagonal (HII) ordered phases. Based on previous electron microscopic investigations, this D state is characterized by the presence of interlamellar attachments and precursors of HII phase between the lipid layers. Using a rotational diffusion model for rod-shaped fluorophore in a curved matrix, rotational dynamics parameters, second rank order parameter, localized wobbling diffusion, and curvature-dependent rotational diffusion constants of dipyenylhexatriene (DPH)-labeled PC (DPH-PC) in the host PE/PC matrix were recovered from the measured fluorescence depolarization decays of DPH fluorescence. At approximately 60% PE, abrupt increases in these rotational dynamics parameters were observed, reflecting the onset of packing defects in the host PE/PC matrix. We have demonstrated that rotational dynamics parameters are very sensitive in detecting the onset of curvature-associating packing defects in lipid membranes. In addition, the presence of the D state can be characterized by the enhanced wobbling diffusional motion and order packing of lipid molecules, and by the presence of localized curvatures in the lipid layers.

Correlation between lipid plane curvature and lipid chain order

The 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE:POPC) system has been investigated by measuring, in the inverted hexagonal (HII) phase, the intercylinder spacings (using x-ray diffraction) and orientational order of the acyl chains (using 2H nuclear magnetic resonance). The presence of 20 wt% dodecane leads to the formation of a HII phase for the composition range from 0 to 39 mol% of POPC in POPE, as ascertained by x-ray diffraction and 2H nuclear magnetic resonance. The addition of the alkane induces a small decrease in chain order, consistent with less stretched chains. An increase in temperature or in POPE proportion leads to a reduction in the intercylinder spacing, primarily due to a decrease in the water core radius. A temperature increase also leads to a reduction in the orientational order of the lipid acyl chains, whereas the POPE proportion has little effect on chain order. A correlation is proposed to relate the radius of curvature of the cylinders in the inverted hexagonal phase to the chain order of the lipids adopting the HII phase. A simple geometrical model is proposed, taking into account the area occupied by the polar headgroup at the interface and the orientational order of the acyl chains reflecting the contribution of the apolar core. From these parameters, intercylinder spacings are calculated that agree well with the values determined experimentally by x-ray diffraction, for the variations of both temperature and POPE:POPC proportion. This model suggests that temperature increases the curvature of lipid layers, mainly by increasing the area subtended by the hydrophobic core through chain conformation disorder, whereas POPC content affects primarily the headgroup interface contribution. The frustration of lipid layer curvature is also shown to be reflected in the acyl chain order measured in the L alpha phase, in the absence of dodecane; for a given temperature, increased order is observed when the curling tendencies of the lipid plane are more pronounced.

Intrinsic curvature in normal and inverted lipid structures and in membranes

The intrinsic or spontaneous radius of curvature, R(o), of lipid monolayer assemblies is expressed in terms of a lipid molecular packing parameter, V/AI, for various geometries. It is shown that the equivalent lipid length, 1, in inverted hexagonal (HII) phases, defined by a cylindrical shell of equal total lipid volume, yields an expression for R o identical to that for inverted cylindrical micelles (or, equivalently, HII phases in the presence of excess hydrocarbon). This identity is used to obtain values of the effective packing parameter for various phosphatidylethanolamines. The temperature dependence of the intrinsic radius of curvature is predicted to be negative and to be considerably greater than that for the lipid length in nearly all cases. The thermal expansion coefficient is not constant but is found to vary, depending on the value of the lipid packing parameter. A possible addition rule is constructed for the intrinsic radius of curvature of lipid mixtures, based on the linear additivity of the effective molecular volumes, V, and molecular areas, A. This relation is found to hold for mixtures of dioleoyl phosphatidylcholine (DOPC) with dioleoyl phosphatidylethanolamine, and a value of R(o) of > or = 9 A (V/AI = 1.08) is obtained for DOPC. The energetics of the intrinsic curvature and lamellar-nonlamellar transitions are also discussed within the framework of the model.

Induction of nonbilayer structures in diacylphosphatidylcholine model membranes by transmembrane alpha-helical peptides: importance of hydrophobic mismatch and proposed role of tryptophans.

We have investigated the effect of several hydrophobic polypeptides on the phase behavior of diacylphosphatidylcholines with different acyl chain length. The polypeptides are uncharged and consist of a sequence with variable length of alternating leucine and alanine, flanked on both sides by two tryptophans, and with the N- and C-termini blocked. First it was demonstrated by circular dichroism measurements that these peptides adopt an alpha-helical conformation with a transmembrane orientation in bilayers of dimyristoylphosphatidylcholine. Subsequent 31P NMR measurements showed that the peptides can affect lipid organization depending on the difference in hydrophobic length between the peptide and the lipid bilayer in the liquid-crystalline phase. When a 17 amino acid residue long peptide (WALP17) was incorporated in a 1/10 molar ratio of peptide to lipid, a bilayer was maintained in saturated phospholipids containing acyl chains of 12 and 14 C atoms, an isotropic phase was formed at 16 C atoms, and an inverted hexagonal (HII) phase at 18 and 20 C atoms. For a 19 amino acid residue long peptide (WALP19) similar changes in lipid phase behavior were observed, but at acyl chain lengths of 2 C-atoms longer. Also in several cis-unsaturated phosphatidylcholine model membranes it was found that these peptides and a shorter analog (WALP16) induce the formation of nonbilayer structures as a consequence of hydrophobic mismatch. It is proposed that this unique ability of the peptides to induce nonbilayer structures in phosphatidylcholine model membranes is due to the presence of two tryptophans at both sides of the membrane/water interface, which prevent the peptide from aggregating when the mismatch is increased. Comparison of the hydrophobic length of the bilayers with the length of the different peptides showed that it is the precise extent of mismatch that determines whether the preferred lipid organization is a bilayer, isotropic phase, or HII phase. The peptide-containing bilayer and HII phase were further characterized after sucrose density gradient centrifugation of mixtures of WALP16 and dioleoylphosphatidylcholine. 31P NMR measurements of the isolated fractions showed that a complete separation of both components was obtained. Chemical analysis of these fractions in samples with varying peptide concentration indicated that the HII phase is highly enriched in peptide (peptide/lipid molar ratio of 1/6), while the maximum solubility of the peptide in the lipid bilayer is about 1/24 (peptide/lipid, molar). A molecular model of the peptide-induced HII phase is presented that is consistent with the results obtained thus far.

Thermotropism and hydration properties of POPE and POPE-cholesterol systems as revealed by solid state2H and31P-NMR

The partial phase diagram and the hydration properties of the 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)-water system, in the absence and presence of 30 mol% cholesterol, have been investigated by solid state phosphorus NMR of the lipid and deuterium NMR of heavy water. The POPE-D2O phase diagram resembles other phosphatidylethanolamine (PE)-water systems: below water-to-lipid molar ratios (Ri) of 3 the lamellar gel (Lβ or Lc)-to-hexagonal type II (HII) phase sequence is observed on increasing the temperature. For Ri≥3 the thermotropic sequence (Lβ or Lc)-Lα-HII is detected. On increasing hydration from Ri=3, the HII phase is detected from 40°C to 85°C whereas the gel phase is observed from 40°C to 30°C. The limiting hydrations of the gel, Lα and HII phases are Ri ≈ 3, 17 and 20, respectively. The number of bound water molecules per lipid is ca. 8 in both the La and HII phases. The presence of cholesterol stabilizes the hexagonal phase 20°C below temperatures at which it is observed in its absence and reduces the limiting hydration of the fluid and hexagonal phases to Ri ≈ 9 and 14, respectively. The structure and/or dynamics of the water bound to the interface are markedly modified on going from the Lα to the HII phase.

The Temperature-Composition Phase Diagram and Mesophase Structure Characterization of the Monoolein/Water System

The temperature-composition phase diagram of monoolein in water was constructed using X-ray diffraction in the range of $0~^\circ{\rm C}$ to $104~^\circ{\rm C}$ and 0% (w/w) to 47% (w/w) water in the heating direction. The phases identified in this system include the lamellar crystalline (Lc) phase, the lamellar liquid crystalline (Lα) phase, the fluid isotropic (FI) phase, two inverted cubic phases (Q230, Ia3d; Q224, Pn3m), and the inverted hexagonal (HII) phase. The overall features of the monoolein/water phase diagram reported herein match those of existing phase diagrams for this system. There are some important differences, however. Accordingly, every effort has been taken to ensure that the current phase diagram represents equilibrium behavior and that the assorted phase boundaries have been determined accurately. The interpreted phase diagram is based on close to 400 discrete measurements in temperature-composition space recorded as a function of temperature in $5~^\circ{\rm C}$ increments ($3~^\circ{\rm C}$ in the HII phase) and of composition in 2% (w/w) water increments on average. The various mesophases have been characterized structurally as a function of temperature and hydration, and the corresponding thermal and composition expansion coefficients are reported. These and related data show that the average radius of water channels in the fully hydrated bicontinuous cubic Pn3m phase is remarkably sensitive to temperature and to monoacylglycerol chain identity.