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)     

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.     

Anisodamine induces the hexagonal phase in phospholipid liposomes

The effects of anisodamine on the polymorphic phase behaviour of cardiolipin and dioleoylphosphatidylcholine liposomes have been investigated by freeze-fracture electron microscopy. Anisodamine induces the formation of lipidic particles in cardiolipin liposomes at pH 7.0 and hexagonal HII tubes at pH 8.8. When the molar ratio of anisodamine and dioleoylphosphatidylcholine is 4 to 1, lipidic particles can be observed in the fracture faces.     

The formation of an inverted hexagonal phase from thylakoid membranes upon heating

Barley thylakoid membranes were studied with FTIR and EPR spectroscopy. Thylakoids were exposed to elevated temperatures in order to induce structural changes. As temperatures increased through physiological to even higher levels, no features changed, but upon heating to above 45 degrees C, the fraction of lipid acyl chain segments with gauche-type vibration increased, accompanied by a sharp drop in the membranous spin probe component. These apparently conflicting observations in fact concur with the formation of an inverted hexagonal (H(II)) phase, supporting its putative role in protecting the photosynthetic machinery in thylakoid membranes against thermally-induced disassembly.     

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.     

Destabilization of phosphatidylethanolamine-containing liposomes: hexagonal phase and asymmetric membranes

We have measured the temperature of the L alpha-HII phase transition, TH, for several types of phosphatidylethanolamine (PE), their binary mixtures, and several PE/cholesteryl hemisuccinate (CHEMS) mixtures. We have shown for liposomes composed of pure PE and in mixtures with CHEMS that there is an aggregation-mediated destabilization which is greatly enhanced at and above TH. We now ask the question: How well can a dioleoylphosphatidylethanolamine/CHEMS liposome, for example, destabilize TPE (transesterified from egg phosphatidylcholine)/CHEMS liposome and vice versa? We use Ca2+ and H+ to induce aggregation and to provide different values of TH: the TH of the PE/CHEMS mixture is much lower at low pH than with Ca2+. We find that if the temperature is above the TH of one lipid mixture, e.g., A, and below the TH of the other lipid mixture, e.g., B, then the destabilization sequence [measured by the fluorescent 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylylenebis(pyridinium bromide) leakage assay] is AA greater than AB much greater than BB. That is, the bilayer of the lipid A (which on its own would end up in the HII phase) destabilizes itself better than it destabilizes the bilayer of lipid B (which on its own would remain in the L alpha phase). The BB contact is the least unstable. From these experiments, we conclude that the enhanced destabilization of membranes provided by the polymorphism accessible to these lipids above TH is effective even if only one of the apposed outer monolayers is HII phase competent. The surprising result is that if the temperature is above the TH of both lipid mixtures, then the destabilization sequence is AB greater than AA, BB. That is, the mixed bilayers are destabilized more by contact than either of the pure pairs. We believe that this is due to specific differences in the kinetics of aggregation or close approach of the membranes. Similar results were obtained with pure PE liposomes induced to aggregate by Ca2+ at pH 9.5. We also found that the kinetics of low-pH-induced leakage from PE/CHEMS liposomes were initially faster when the CHEMS on both sides of the bilayer is fully protonated. However, in a citrate buffer, which cannot cross intact membranes, the leakage was eventually faster. Flip-flop of the protonated CHEMS to the inner monolayer can explain this observation.     

Floundering about at cell membranes: a structural view of phospholipid signaling

Structures are now available for the majority of the enzyme families involved in the phosphorylation, dephosphorylation and hydrolysis of signaling phospholipids. Lipid kinase and phosphatase structures recapitulate catalytic motifs involved in protein phosphorylation and dephosphorylation, whereas cytosolic phospholipase A(2) manifests novel catalytic geometry. Structures have been determined for most known intracellular phospholipid 'receptor' domains, both those that bind membrane-embedded phospholipids and those that bind lipid monomers.     

External addition of gramicidin induces the hexagonal HII phase in dioleoylphosphatidylcholine model membranes

³¹P-NMR, small angle X-ray diffraction and freeze-fracture electron microscopy show that dioleoylphosphatidylcholine liposomes undergo a transition from the lamellar to the hexagonal H_II phase upon injection of an ethanolic solution of gramicidin in the aqueous medium, when the molar ratio of peptide to lipid is 1 to 20 or higher.     

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.     

Virus replication inhibitory peptide inhibits the conversion of phospholipid bilayers to the hexagonal phase

Virus replication inhibitory peptide (carbobenzoxy-D-Phe-L-PheGly) was shown to be a potent specific inhibitor of the replication of paramyxovirus and myxovirus (Richardson, Scheid and Choppin (1980), Virology105, 205–222). This peptide inhibits the membrane fusing activity of a viral glycoprotein.Many agents which promote the formation of the hexagonal phase in membranes also accelerate membrane fusion. At a mole fraction of 0.1, viral replication inhibitory peptide can raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine by almost 10°. Two related peptides, carbobenzoxy-L-PheGly and carbobenzoxy-L-GlyPhe, are less potent in raising the bilayer to hexagonal phase transition temperature, with the latter peptide being the least effective of the three. This order of potency is the same as the order of potency in inhibiting viral replication. Substances which inhibit hexagonal phase formation of pure lipids may also inhibit membrane fusion.Abbreviations DEPE dielaidoylphosphatidyethanolamine - Z carbobenzoxy - DSC differential scanning calorimetry - VRIP virus replication inhibitory peptide (Z-D-Phe-L-PheGly)     

Myelin basic protein induces hexagonal phase formation in dispersions of diacylphosphatidic acid

31P nuclear magnetic resonance and low-angle X-ray diffraction measurements have shown that the basic protein of myelin caused diacylphosphatidic acid dispersions to change from a lamellar to a hexagonal lipid organisation. Several other basic proteins failed to effect a similar phase change, and had little influence on phospholipid headgroup structure and motion.     

Effects of proteins on the thermotropic phase transitions of phospholipid membranes

A variety of proteins have been studied for their ability to interact and alter the thermotropic properties of phospholipid bilayer membranes as detected by differential scanning calorimeter. The proteins studied included: basic myelin protein (A1 protein), cytochrome c, major apoprotein of myelin proteolipid (N-2 apoprotein), gramicidin A, polylysine, ribonuclease and hemoglobin. The lipids used for the interactions were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The interactions were grouped in three categories each having very different effects on the phospholipid phase transition from solid to liquid crystalline. The calorimetric studies were also correlated with data from vesicle permeability and monolayer expansion.Ribonuclease and polylysine which exemplify group 1 interactions, show strong dependence on electrostatic binding. Their effects on lipid bilayers include an increase in the enthalpy of transition (ΔH) accompanied by either an increase or no change in the temperature of transition (T_c). In addition, they show minimal effects on vesicle permeability and monolayer expansion. It was concluded that these interactions represent simple surface binding of the protein on the lipid bilayer without penetration into the hydrocarbon region.Cytochrome c and Al protein, which exemplify group 2 interactions, also show a strong dependence on the presence of net negative charges on the lipid bilayers for their binding. In contrast to the first group, however, they induce a drastic decrease in both T_c and ΔH of the lipid phase transition. Furthermore, they induce a large increase in the permeability of vesicles and a substantial expansion in area of closely packed monolayers at the air-water interface. It was concluded that group 2 interactions represent surface binding followed by partial penetration and/or deformation of the bilayer.Group 3 interactions, shown by proteolipid apoprotein and gramicidin A, were primarily non-polar in character, not requiring electrostatic charges and not inhibited by salt and pH changes. They had no appreciable effect on the T_c but did induce a linear decrease in the magnitude of the ΔH, proportional to the percentage of protein by weight. Membranes containing 50% proteolipid protein still exhibited a thermotropic transition with a ΔH one half that of the pure lipid, and only a small diminution of the size of the cooperative unit. It was concluded that in this case the protein was embedded within the bilayer, associating with a limited number of molecules via non-polar interactions, while the rest of the bilayer was largely unperturbed.     

Effects of proteins on thermotropic phase transitions of phospholipid membranes.

A variety of proteins have been studied for their ability to interact and alter the thermotropic properties of phospholipid bilayer membranes as detected by differential scanning calorimeter. The proteins studied included: basic myelin protein (A1 protein), cytochrome c, major apoprotein of myelin proteolipid (N-2 apoprotein), gramicidin A, polylysine, ribonuclease and hemoglobin. The lipids used for the interactions were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The interactions were grouped in three catagories each having very different effects on the phospholipid phase transition from solid to liquid crystalline. The calorimetric studies were also correlated with data from vesicle permeability and monolayer expansion. Ribonuclease and polylysine which exemplify group 1 interactions, show strong dependence on electrostatic binding. Their effects on lipid bilayers include an increase in the enthalpy of transition (deltaH) accompanied by either an increase or no change in the temperature of transition (Tc). In addition, they show minimal effects on vesicle permeability and monolayer expansion. It was concluded that these interactions represent simple surface binding of the protein on the lipid bilayer without penetration into the hydrocarbon region. Cytochrome c and A1 protein, which exemplify group 2 interactions, also show a strong dependence on the presence of net negative charges on the lipid bilayers for their binding. In contrast to the first group, however, they induce a drastic decrease in both Tc and deltaH of the lipid phase transition. Furthermore, they induce a large increase in the permeability of vesicles and a substantial expansion in area of closely packed monolayers at the air-water interface. It was concluded that group 2 interactions represent surface binding followed by partial penetration and/or deformation of the bilayer. Group 3 interactions, shown by proteolipid apoprotein and gramicidin A, were primarily non-polar in character, not requiring electrostatic charges and not inhibited by salt and pH changes. They had no appreciable effect on the Tc but did induce a linear decrease in the magnitude of the deltaH, proportional to the percentage of protein by weight. Membranes containing 50% proteolipid protein still exhibited a thermotropic transition with a deltaH one half that of the pure lipid, and only a small diminution of the size of the cooperative unit. It was concluded that in this case the protein was embedded within the bilayer, associating with a limited number of molecules via non-polar interactions, while the rest of the bilayer was largely unperturbed.     

Molecular species specificity of phospholipid breakdown in microsomal membranes of senescing carnation flowers

During senescence of cut carnation flowers, there is extensive breakdown of microsomal phospholipid. This is attributable, at least in part, to lipolytic activity associated directly with the microsomal membranes. Evidence indicating that one or more of the lipid-degrading enzymes in these membranes preferentially degrade phospholipid molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain has been obtained by using radiolabeled phosphatidylcholine substrates. 16:0^*/16:0^*, 16:0/18:2^*, and 18:1^*/18:1^* phosphatidylcholine were degraded only minimally over a 3 hour period by microsomes isolated from senescing flowers. By contrast, [U-¹⁴C]phosphatidylcholine, which comprises various molecular species including those containing polyunsaturated acyl chains, and 18:0/20:4^* phosphatidylcholine were extensively degraded. Under identical conditions, but in the absence of added radiolabeled substrate, endogenous 18:2/18:2, 18:1/18:3, and 18:2/18:3 phosphatidylcholine were selectively depleted from the membranes. During natural senescence of the flowers, there was a sharp decline in microsomal 16:0/18:1 and 18:1/18:2 phosphatidylcholine, whereas molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain remained unchanged or decreased only slightly. The data have been interpreted as indicating that provision of particular molecular species susceptible to lipase attack is a prerequisite to phospholipid catabolism in senescing membranes.     

Effects of pentanol isomers on the phase behavior of phospholipid bilayer membranes

Differential scanning calorimetry (DSC) was used to analyze the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of pentanol isomers. The concentration of each pentanol isomer needed to induce the interdigitated phase was determined by the appearance of a biphasic effect in the main transition temperatures, the onset of a hysteresis associated with the main transition from the gel-to-liquid crystalline phase, and the disappearance of the pretransition. Lower threshold concentrations were found to correlate with isomers of greater alkyl chain length while branching of the alkyl chain was found to increase biphasic behavior. The addition of a methyl group to butanol systems drastically decreased threshold concentrations. However, as demonstrated in the DPPC/neopentanol system, branching of the alkyl chain away from the -OH group lowers the threshold concentration while maintaining a biphasic effect.