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.     

A mismatch between the length of gramicidin and the lipid acyl chains is a prerequisite for HII phase formation in phosphatidylcholine model membranes

Previously it was shown that gramicidin can induce HII phase formation in diacylphosphatidylcholine model membranes only when the lipid acyl chain length exceeds 16 carbon atoms (Van Echteld, C.J.A., De Kruijff, B., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1982) Biochim. Biophys. Acta 692, 126-138). Using 31P-NMR and small angle X-ray diffraction we now demonstrate that upon increasing the length of gramicidin, the peptide loses its ability to induce HII phase formation in di-C18:1c-PC but not in the longer chained di-C22:1c-PC. It is concluded that a mismatch in length between gramicidin and the lipid acyl chains, when the latter would provide excess bilayer thickness, is a prerequisite for HII phase formation in phosphatidylcholine model membranes.     

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)     

Phase separation and hexagonal HII phase formation by gramicidins A, B and C in dioleoylphosphatidylcholine model membranes. A study on the role of the tryptophan residues

The role of the tryptophan-residues in gramicidin-induced HII phase formation was investigated in dioleoylphosphatidylcholine (DOPC) model membranes. 31P-NMR and small angle X-ray diffraction measurements showed, that gramicidin A and C (in which tryptophan-11 is replaced by tyrosine) induce a similar extent of HII phase formation, whereas for gramicidin B and synthetic analogs in which one tryptophan, either at position 9 or 11 is replaced by phenylalanine, a dramatic decrease of the HII phase inducing activity can be observed. Modification of all four tryptophans by means of formylation of the indole NH group leads to a complete block of HII phase formation. Sucrose density centrifugation experiments on the various peptide/lipid samples showed a quantitative incorporation of the peptide into the lipid. For all samples in a 1/10 molar ratio of peptide to lipid distinct bands were found, indicative of a phase separation. For the gramicidin A'/DOPC mixture these bands were analyzed and the macroscopic organization was determined by 31P-NMR and small-angle X-ray diffraction. The results demonstrate that a quantitative phase separation had occurred between a lamellar phase with a gramicidin/lipid ratio of 1/15 and a hexagonal HII phase, which is highly enriched in gramicidin. A study on the hydration properties of tryptophan-N-formylated gramicidin in mixtures with DOPC showed that this analog has a similar dehydrating effect on the lipid headgroup as the unmodified gramicidin. In addition both the hydration study and sucrose density centrifugation experiments showed that, like gramicidin also its analogs have a tendency to aggregate, but with differences in aggregation behaviour which seemed related to their HII phase inducing activity. It is proposed that the main driving force for HII phase formation is the tendency of gramicidin molecules to self-associate and organize into tubular structures such as found in the HII phase and that whether gramicidin (analogs) form these or other types of aggregates depends on their tertiary structure, which is determined by intra- as well as intermolecular aromatic-aromatic stacking interactions.     

Solvent determined conformation of gramicidin affects the ability of the peptide to induce hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes

It is shown by 31P-NMR and small angle X-ray scattering that induction of an hexagonal HII phase in dioleoylphosphatidylcholine model membranes by external addition of gramicidin A' depends on the solvent which is used to solubilize the peptide. Addition of gramicidin from dimethylsulfoxide or trifluoroethanol solution leads to HII phase formation whereas addition of the peptide from ethanol does not. This solvent dependence is shown by circular dichroism to be correlated with the peptide conformation. The channel conformation appears to be responsible for HII phase formation by gramicidin.     

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.     

Calcium-induced phase separation phenomena in multicomponent unsaturated lipid mixtures

The ability of calcium to induce phase separation in multicomponent lipid mixtures containing various unsaturated species of acidic and neutral phospholipids has been investigated by 31P NMR, 3H NMR, and small-angle X-ray diffraction techniques. It is shown that, in unsaturated (dioleoyl-) phosphatidylglycerol (PG)/phosphatidylethanolamine (PE) (1:1) and phosphatidic acid (PA)/phosphatidylcholine (PC) (1:1) mixtures, calcium is unable to induce lateral phase separation of the acidic and neutral lipids and that all the lipids adopt a hexagonal (HII) phase in the presence of calcium. In multicomponent mixtures containing one or more acidic species the presence of cholesterol either facilitates calcium-induced lamellar to hexagonal (HII) transitions for all the lipid components or, in systems already in a hexagonal (HII) phase, mitigates against calcium-induced lateral phase separations. Further, cholesterol is shown to exhibit no preferential interaction on the NMR time scale with either PC, PE, or phosphatidylserine (PS) when the lipids are in the liquid-crystal state. The ability of cholesterol to directly induce HII phase formation in PC/PE mixtures is also shown to be common to various other sterols including ergosterol, stigmasterol, coprostanol, epicoprostanol, and androstanol.     

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.     

Conformational analysis of gramicidin-gramicidin interactions at the air/water interface suggests that gramicidin aggregates into tube-like structures similar as found in the gramicidin-induced hexagonal HII phase

The energetics of interaction and the type of aggregate structure in lateral assemblies of up to five gramicidin molecules in the beta 6.3 helical conformation at the air/water interface was calculated using conformational analysis procedures. It was found that within the aggregate two types of gramicidin interaction occur. One leading to a linear organization with a mean interaction energy between monomers of -6 kcal/mol and one in a perpendicular direction leading to a circularly organization with a lower mean interaction energy of -10 kcal/mol. Extrapolation towards larger gramicidin assemblies predicts that gramicidin itself could form tubular structures similar to those found in the gramicidin-induced HII phase. The tryptophans appear to play an essential role in the tubular organization of the gramicidin aggregate, since they determine the cone shape of the monomer and contribute to the structure of the monomer and oligomer by stacking interactions. These results, which are discussed in the light of experimental observations of gramicidin self-association in model membranes and the importance of the tryptophans for HII phase formation, further support the view (Killian, J.A. and De Kruijff, B. (1986) Chem. Phys. Lipids 40, 259-284) that gramicidin is a first example of a new class of hydrophobic polypeptides which can form cylindrical structures within the hydrophobic core of the membrane.     

Gramicidin A induced fusion of large unilamellar dioleoylphosphatidylcholine vesicles and its relation to the induction of type II nonbilayer structures.

The fusogenic properties of gramicidin were investigated by using large unilamellar dioleoylphosphatidylcholine vesicles. It is shown that gramicidin induces aggregation and fusion of these vesicles at peptide to lipid molar ratios exceeding 1/100. Both intervesicle lipid mixing and mixing of aqueous contents were demonstrated. Furthermore, increased static and dynamic light scattering and a broadening of 31P NMR signals occurred concomitant with lipid mixing. Freeze-fracture electron microscopy revealed a moderate vesicle size increase. Lipid mixing is paralleled by changes in membrane permeability: small solutes like carboxyfluorescein and smaller dextrans, FD-4(Mr approximately 4000), rapidly (1-2 min) leak out of the vesicles. However, larger molecules like FD-10 and FD-17 (Mr approximately 9400 and 17,200) are retained in the vesicles for greater than 10 min after addition of gramicidin, thereby making detection of contents mixing during lipid mixing possible. At low lipid concentrations (5 microM), lipid mixing and leakage are time resolved: leakage of CF shows a lag phase of 1-3 min, whereas lipid mixing is immediate and almost reaches completion during this lag phase. It is therefore concluded that leakage, just as contents mixing, occurs subsequent to aggregation and lipid mixing. Although addition of gramicidin at a peptide/lipid molar ratio exceeding 1/50 eventually leads to hexagonal HII phase formation and a loss of vesicle contents, it is concluded that leakage during fusion (1-2 min) is not the result of HII phase formation but is due to local changes in lipid structure caused by precursors of this phase. By making use of gramicidin derivatives and different solvent conformations, it is shown that there is a close parallel between the ability of the peptide to induce the HII phase and its ability to induce intervesicle lipid mixing and leakage. It is suggested that gramicidin-induced fusion and HII phase formation share common intermediates.     

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.     

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.     

Distribution of decane within the unit cell of the inverted hexagonal (HII) phase of lipid-water-decane systems determined by neutron diffraction.

The addition of a free alkane such as decane to lipid-water systems is known to promote the formation of a low-temperature inverted hexagonal (HII) phase [Kirk, G. L., & Gruner, S. M. (1985) J. Phys. (Paris) 46, 761]. Kirk et al. [Kirk, G. L., Gruner, S. M., & Stein, D. E. (1984) Biochemistry 23, 1093] have discussed the hydrocarbon packing anisotropy in the HII unit cell and have suggested that free alkane will distribute in a way that reduces this packing anisotropy by allowing the lipid chain environment to become more uniform. By combining neutron and X-ray diffraction data to do a Fourier reconstruction of the HII phase of dioleoylphosphatidylethanolamine (DOPE) + water + deuterated decane, it was found that the decane preferentially partitions into the interstitial regions of the HII unit cell where it should be the most effective in alleviating the hydrocarbon chain packing stress, supporting the suggestion of Kirk et al. Using the distribution of decane within the unit cell, we have calculated the lipid length distribution for the situations with and without added alkane. With a suitable molecular model, this lipid length distribution may eventually be used to calculate the free energy change upon the addition of alkane. Such a measurement is important for a more realistic understanding of the interactions which lead to the formation of the HII phase.     

Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems

The transient membrane lipid diacylglycerol (DG) is known to modify and destabilize phospholipid bilayers and can lead to the formation of nonbilayer structures. Since cholesterol forms a major fraction of many plasma membranes, we have investigated how it modifies the structural effects of DG on bilayers of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE). We view these systems as modelling the behaviour of local, DG-containing sites in membranes. Using X-ray diffraction, we have characterized the lamellar (L alpha) and inverse hexagonal (HII) structures that these ternary lipid mixtures form in excess aqueous solution. As the DG level increases, the lipid progresses from a single L alpha structure to a mixture of L alpha and HII, and then to a pure HII structure. This allows determination of the DG levels at which the HII transition begins, which we interpret as those levels that destabilize bilayers. In both PC and PE bilayers, the presence of 30 mol% cholesterol reduces the amounts of DG required to destabilize the bilayer structure. The destabilization can be translated into the number of neighbouring lipid molecules that a DG molecule perturbs, and of bilayer areas that it affects. The data show that the presence of cholesterol greatly enhances the perturbing effects of DG. We examine the possible role of DG in enzyme activation and membrane fusion.     

Influence of vitamin E on phosphatidylethanolamine lipid polymorphism

The effect of vitamin E, in its major form alpha-tocopherol and its synthetic analog alpha-tocopheryl acetate, on phosphatidylethanolamine lipid polymorphism has been studied by mean of differential scanning calorimetry and 31P-nuclear magnetic resonance techniques. From the interaction of these tocopherols with dielaidoylphosphatidylethanolamine it is concluded that both molecules promote the formation of the hexagonal HII phase at temperatures lower than those of the pure phospholipid. When the tocopherols were incorporated in the saturated dimiristoylphosphatidylethanolamine, which has been shown not to undergo bilayer to hexagonal HII phase transition, up to 90 degrees C, they induce the phospholipid to partially organize in hexagonal HII phase. From our experiments it is shown that alpha-tocopherol is more effective than its analog in promoting HII phase in these systems. It is also shown that, while alpha-tocopheryl acetate does not significantly perturb the gel to liquid-crystalline phase transition of dimirystoylphosphatidylethanolamine, alpha-tocopherol does so and more than one peak appears in the calorimetric profile, indicating that lateral phase separations are taking place.     

Comparative 2H- and 31P-NMR study on the properties of palmitoyllysophosphatidylcholine in bilayers with gramicidin, cholesterol and dipalmitoylphosphatidylcholine

The stoichiometric palmitoyllysophosphatidylcholine (lysoPC)/gramicidin (4:1, mol/mol) lamellar complex (Killian, J.A., De Kruijff, B., Van Echteld, C.J.A., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1983) Biochim. Biophys. Acta 728, 141-144) is a useful model system to investigate the various aspects of lipid protein interactions. To study the effect of gramicidin on local order and motion of 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC) we employed 31P and 2H nuclear magnetic resonance (NMR) using selectively deuterated lysoPC's and we compared the results to those obtained for lysoPC in bilayers with cholesterol (1:1, mol/mol) and dipalmitoylphosphatidylcholine (DPPC) (1:4, mol/mol). 2H-NMR experiments on acyl chain deuterated lysoPC showed similar quadrupole splittings in the liquid crystalline state for the lysoPC/DPPC and the lysoPC/gramicidin samples. In the lysoPC/cholesterol sample an increase of the quadrupole splitting was found. T1 measurements showed that gramicidin decreases the lysoPC acyl chain motion, especially at the C12 position. In the lysoPC/cholesterol sample an increase of motion was observed as compared to lysoPC in fluid bilayers of DPPC. 31P-NMR and 2-H-NMR measurements of lysoPC, deuterated at the alpha- and beta-position of the choline moiety, indicated an increase in headgroup flexibility in all samples as compared to the parent compound DPPC. In addition, a change in headgroup conformation was observed. The alpha- and beta-segments in all samples exhibited concerted motion. It was found that also in the polar headgroup gramicidin induces a decrease of the rate of motion.     

Sterol specific inactivation of gramicidin A induced membrane cation permeability.

Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.     

Bilayer-stabilizing properties of myelin basic protein in dioleoylphosphatidylethanolamine systems

31P-NMR and X-ray diffraction techniques are used to study the comparative ability of myelin basic protein (MBP) vs. other basic proteins to convert hexagonal (HII) phases to stable lamellar (L alpha) structures. Pure dioleoylphosphatidylethanolamine (DOPE) at pH 9 and 7, and mixtures of DOPE/phosphatidylserine (PS) (95:5 and 80:20% w/w) at pH 7 were employed for this investigation. The polymorphic behavior of the lipid suspensions was evaluated in the presence and absence of several basic proteins (MBP, calf thymus histone, lysozyme, melittin) and the cationic polypeptide, polylysine (PL). Each of the proteins and PL was capable of binding the pure DOPE HII phase at pH 9 but with varying morphological consequences, i.e., lamellar stabilization (MBP, histone, PL), formation of new protein-DOPE HII phases (lysozyme) or lipid disordering/vesiculation (melittin). Reduction to pH 7 resulted in the dissociation of protein from DOPE - with the exception of melittin - and the reformation of a pure lipid HII phase. Additions of PS to DOPE at pH 7 facilitated protein binding, but among the proteins examined, only MBP was capable of converting the lipid suspension into a stable multilamellar form. Differences in the lipid morphology produced by each protein are discussed in terms of protein physicochemical characteristics. In addition, a possible relationship between MBP-lipid interactions and the stability of myelin sheath lipid multilayers is inferred from the significant bilayer-stabilizing capacity of MBP.     

Relationship between gramicidin conformation dependent induction of phospholipid transbilayer movement and hexagonal HII phase formation in erythrocyte membranes

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal HII phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (beta 6.3) conformation of the peptide is essential for all three membrane perturbing effects.     

胆固醇对脂双层结构影响的SAXS和STM研究

用小角X射线散射（SAXS)和扫描隧道显微镜（STM）技术分别研究了模拟生物膜脂质体的结构以及胆固醇对生物膜双层结构的影响。结果表明,在扫描隧道显微镜照片中,磷脂分子在石墨表面形成规则的二维点状排列图像;磷脂胆固醇脂质体在石墨表面形成规则的二维波纹状排列图像。用小角X射线散射研究结果表明,DPPC脂质体是片层相结构,DPPC＋Chol脂质体是复相片层结构,DPPE＋Chol脂质体是片层立方相结构,DPPC＋DPPE＋Chol脂质体是立方六角形相结构。