Pressure-induced changes in the molecular organization of a lipid-peptide complex. Polymyxin binding to phosphatidic acid membranes

The effect of 100 atm pressure on the organization of the lipid-peptide complex formed between polymyxin and dipalmitoyl phosphatidic acid has been investigated. Phase transition curves were obtained by electron paramagnetic resonance by measuring the partition coefficient of the spin label, 2, 2, 5, $\text{5-}\text{tetramethylpiperidine}\text{-N-}\text{oxyl}$. The three-step phase transition curve previously obtained with fluorescence polarization measurements was confirmed, demonstrating three distinct phosphatidic acid domains in the bilayer. Pressure increases binding of polymyxin to phosphatidic acid bilayers and alters the proportions of the two domains that differ in the mode of binding between phosphatidic acid and polymyxin. The binding curves of polymyxin to phosphatidic acid bilayers were determined and it was shown that application of pressure reduces the cooperativity of the binding curve.     

Polymyxin binding to charged lipid membranes. An example of cooperative lipid-protein interaction.

The binding of polymyxin-B to lipid bilayer vesicles of synthetic phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescence probes to study the lipid phase transition. The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (width deltaT = 5 degrees C) are observed. The upper transition (at Tu) is that of the pure lipid and the lower transition (at T1) concerns the lipid bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1 : 1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin. With both fluorescence methods the fraction of lipid bound to polymyxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction of Ca2+ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca2+, polymyxin) interaction. A model is proposed which explains the association of polymyxin within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

Calorimetric investigation of polymyxin binding to phosphatidic acid bilayers

The cooperative binding process between the antibiotic peptide polymyxin-B and negatively-charged phosphatidic acid bilayers was investigated by differential thermal analysis and completed by fluorescence polarization measurements. The sigmoidal binding curves were analyzed in terms of the interaction energy within a domain formed by polymyxin and phosphatidic acid molecules. The formation of such a heterogeneous domain structure was favoured by high concentration of external monovalent ions. The cooperativity of the binding increased while a charge-induced decrease in the phase transition temperature of the pure lipid phase was observed with increasing ion concentration at a given pH. The reduced lateral coupling within the lipid bilayer in the presence of salt ions, as demonstrated by an increase in the lipid phase transition enthalpy, was considered to facilitate the cooperative domain formation. Moreover, an increase in the cooperativity of the polymyxin binding could be observed if phosphatidic acids of smaller chain length and thus of a lowered phase transition temperature were used. By the use of chemically-modified polymyxin we were able to demonstrate the effect of electrostatic and hydrophobic interaction. Acetylated polymyxin with a reduced positive charge was used to demonstrate the pure hydrophobic effect of polymyxin binding leading to a decrease in the phosphatidic acid phase transition temperature by about 20 degrees C. The cooperativity of the binding was strongly reduced. Cleavage of the hydrophobic polymyxin tail yielded a colistinnonapeptide which caused an electrostatically-induced increase in the phosphatidic acid phase transition temperature. With unmodified polymyxin we observed the combined effects of electrostatic as well as hydrophobic interaction making this model system interesting for the understanding of lipid-protein interactions. Evidence is presented that the formation of the polymyxin-phosphatidic acid complex is a lateral phase separation phenomenon.     

Calorimetric investigation of polymyxin binding to phosphatidic acid bilayers

The cooperative binding process between the antibiotic peptide polymyxin-B and negatively-charged phosphatidic acid bilayers was investigated by differential thermal analysis and completed by fluorescence polarization measurements. The sigmoidal binding curves were analyzed in terms of the interaction energy within a domain formed by polymyxin and phosphatidic acid molecules. The formation of such a heterogeneous domain structure was favoured by high concentration of external monovalent ions. The cooperativity of the binding increased while a charge-induced decrease in the phase transition temperature of the pure lipid phase was observed with increasing ion concentration at a given pH. The reduced lateral coupling within the lipid bilayer in the presence of salt ions, as demonstrated by an increase in the lipid phase transition enthalpy, was considered to facilitate the cooperative domain formation. Moreover, an increase in the cooperativity of the polymyxin binding could be observed if phosphatidic acids of smaller chain length and thus of a lowered phase transition temperature were used. By the use of chemically-modified polymyxin we were able to demonstrate the effect of electrostatic and hydrophobic interaction. Acetylated polymyxin with a reduced positive charge was used to demonstrate the pure hydrophobic effect of polymyxin binding leading to a decrease in the phosphatidic acid phase transition temperature by about 20°C. The cooperativity of the binding was strongly reduced. Cleavage of the hydrophobic polymyxin tail yielded a colistinnonapeptide which caused an electrostatically-induced increase in the phosphatidic acid phase transition temperature. With unmodified polymyxin we observed the combined effects of electrostatic as well as hydrophobic interaction making this model system interesting for the understanding of lipid-protein interactions. Evidence is presented that the formation of the polymyxin-phosphatidic acid complex is a lateral phase separation phenomenon.     

Polymyxin binding to charged lipid membranes an example of cooperative lipid-protein interaction

The binding of polymyxin-B to lipid bilayer vesicles of synthesis phosphatidic acid was studied using fluorescence, ESR spectroscopy and electron microscopy. 1,6-Diphenylhexatriene (which exhibits polarized fluorescence) and pyrene decanoic acid (which forms excimers) were used as fluorescene probes to study the lipid phase transition.The polymyxin binds strongly to negatively charged lipid layers. As a result of lipid/polymyxin chain-chain interactions, the transition temperature of the lipid. This can be explained in terms of a slight expansion of the crystalline lipid lattice (Lindeman's rule). Upon addition of polymyxin to phosphatidic acid vesicles two rather sharp phase transitions (with ΔT = 5°C) are observed. The upper transition (at T_u) is that of the pure lipid and the lower transition (at T₁) concerns the lipids bound to the peptide. The sharpness of these transitions strongly indicates that the bilayer is characterized by a heterogeneous lateral distribution of free and bound lipid regions, one in the crystalline and the other in the fluid state. Such a domain structure was directly observed by electron microscopy (freeze etching technique). In (1:1) mixtures of dipalmitoyl phosphatidic acid and egg lecithin, polymyxin induces the formation of domains of charged lipid within the fluid regions of egg lecithin.With both fluorescence methods the fraction of lipid bound to polymxin-B as a function of the peptide concentration was determined. S-shaped binding curves were obtained. The same type of binding curve is obtained for the interaction action of Ca²⁺ with phosphatidic acid lamellae, while the binding of polylysine to such membranes is characterized by a linear or Langmuir type binding curve. The S-shaped binding curve can be explained in terms of a cooperative lipid-ligand (Ca²⁺, polymyxin) interaction.A model is proposed which explains the association of polymyxing within the membrane plane in terms of elastic forces caused by the elastic distortion of the (liquid crystalline) lipid layer by this highly asymmetric peptide.     

Cooperative lipid-protein interaction. Effect of pH and ionic strength on polymyxin binding to phosphatidic acid membranes.

The binding of polymyxin-B to charged dipalmitoyl phosphatidic acid membranes has been studied as function of the external pH and of the ionic strength of the buffer solution. The phase transition curves were obtained by measuring the fluorescence depolarization of diphenyl hexatriene incorporated into the membrane with temperature. The molecular process of polymyxin binding was elucidated: 1. At an ionic strength of I greater than or equal to 0.1 mol/l a three step phase transition curve is found. A high-temperature step corresponds to the non-bound lipid. A lowered phase transition concerns to protein-bound lipid domains. This again is splitted into two steps. An inner core of the domain is characterized by a lipid-protein complex which is stabilized through hydrophobic and electrostatic interactions between polymyxin and the charged lipid. This core is surrounded by an outer belt of only hydrophobically bound molecules. This part shows a lower phase transition temperature than the inner core. 2. The binding curves of polymyxin to phosphatidic acid membranes depend strongly on the ionic strength of the water phase. The cooperativity of the binding process increases with increasing ionic strength and reaches a constant value at I greater than 0.2 mol/l. The maximum fraction of bound lipid decreases with increasing ionic strength. 3. The pH of the water phase strongly influences the cooperative binding process. At pH 6 a loss of cooperativity is observed at low ionic strength. Increasing the ion concentration to I = 0.3 mol/l recuperates the cooperativity of the binding process. At pH 3.0 no cooperative binding is obtained even at high ionic strength.     

Phospholipid interactions with cytochrome P-450 in reconstituted vesicles Preference for negatively-charged phosphatidic acid

Cytochrome $\text{P-450}$ LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome $\text{P-450}$ were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome $\text{P-450}$ into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome $\text{P-450}$. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome $\text{P-450}$. For comparison, incorporation of cytochrome $\text{P-450}$ into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the     

Binding of cytochrome c to liposomes as revealed by the quenching of fluorescence from pyrene-labeled phospholipids

Resonance energy transfer from pyrene-fatty acid containing phospholipid derivatives to the heme of cytochrome c (cyt c) was used to observe the binding of this protein to liposomal membranes. Liposomes were formed of egg yolk phosphatidic acid (PA) and either egg yolk phosphatidylcholine or dipalmitoylphosphatidylcholine with 1 mol % of the fluorescent lipid. Binding of cyt c to liposomes was monitored by measuring the decrease either in the fluorescence intensity or in the lifetime of pyrene emission. The requirement for the presence of the acidic phospholipid in the membrane for the binding of cyt c could be reconfirmed. Below 5 mol % of phosphatidic acid in the membrane, no significant attachment of cyt c to liquid-crystalline bilayers was evident whereas upon increasing the concentration of PA further the association of cyt c progressively increased until a saturation was reached at about 30 mol % of phosphatidic acid. Addition of NaCl caused the fluorescence intensity and lifetimes to return to values observed in the absence of cyt c, thus revealing the dissociation of the protein from the membrane. The pyrene-labeled phosphatidic acid derivatives PPHPA and PPDPA were quenched more effectively than the corresponding phosphatidylcholines, apparently due to the direct involvement of the acidic head group in binding cyt c. When dipalmitoylphosphatidylcholine (DPPC) with 5 mol % of phosphatidic acid was used, no binding of cyt c to the liposomes above the phase transition temperature of the former lipid could be demonstrated whereas below the transition temperature (Tm) binding did take place.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of polylysine to charged bilayer membranes: molecular organization of a lipid.peptide complex.

The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule.     

Effect of polymyxin B on the conductivity of negatively charged bilayer lipid membranes

Effect of polymyxin B on the planar bilayer lipid membranes (BLM) formed from synthetic phosphatidic acid has been studied. The addition of cholesterol to phospholipid in molar ratio 1 : 2 was followed by an increase of BLM conductance from 2 x 10(-8) to 3 x 10(-7) Ohm-1 cm-2. It was suggested that the observed increase of conductance was due to the fluidity of the membrane matrix in the presence of cholesterol. It was shown that 10(-6)--10(-5) M polymyxin slightly affected the conductance of BLM from phosphatidic acid. It was found that polymyxin increased conductance of negatively charged BLM modified by palmitic acid from 10(-8) to 10(-6) Ohm-1 cm-2.     

Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces

Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid-peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms.     

Characterization of the membrane binding mode of the C2 domain of PKC epsilon

PKCepsilon is a member of the group of novel PKCs that contain a C2 domain located in their N-terminal region. On the basis of recent structural studies, a series of mutants were prepared to increase our knowledge of the mechanism of the phospholipid binding site of this domain. The results revealed that this domain preferentially binds to phosphatidic acid- and phosphatidylserine-containing vesicles. Although the increase in affinity was linear in the case of phosphatidic acid, it became exponential when the vesicles contained increasing concentrations of phosphatidylserine. Site-directed mutagenesis studies showed that residues W23, R26, and R32 located in loop 1 and I89 and Y91 located in loop 3 are of critical importance when the binding is performed with phosphatidic acid-containing vesicles. Furthermore, when the same mutants were assayed with phosphatidylserine-containing vesicles, no binding was observed in any case, reflecting the smaller affinity of the C2 domain for phosphatidylserine-containing vesicles. A study of the ionic nature of the membrane interaction suggested that it is mainly driven by electrostatic interactions that are disrupted by very low salt concentrations. Differential scanning calorimetry experiments performed to ascertain whether this interaction affected the transition phase of the phosphatidic acid demonstrated that increasing concentrations of the protein lead to changes in the transition, with more than one peak appearing at lower temperatures, which suggests a weak interaction focused on the polar headgroup of the phospholids. In conclusion, a different membrane-binding mode from those previously described in other C2 domains has been found and is seemingly based on electrostatic, interfacial, and hydrophobic interactions without the participation of Ca(2+) ions.     

Carbachol stimulation of phosphatidic acid synthesis: competitive inhibition by pirenzepine in synaptosomes from rat cerebral cortex

Carbachol stimulated phosphatidic acid synthesis in cholinergically enriched synaptosomes from rat cerebral cortex. Increasing concentrations of pirenzepine (10-1000 nM) produced parallel concentration-response curves to carbachol which were shifted to the right. A pA2 value for pirenzepine of 8.4 +/- 0.3 was obtained from Schild analysis. We hypothesize that high affinity pirenzepine binding to M1 receptors is coupled to phosphatidic acid synthesis in the rat cerebral cortex.     

Interaction of polymyxin B nonapeptide with anionic phospholipids

The interaction of polymyxin B nonapeptide (PMBN) and polymyxin B (PMB) with the anionic phospholipids phosphatidylserine (PS), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), and 1:1 mixtures (w/w) of DPPA and distearoylphosphatidylcholine (DSPC) was studied by calorimetry, electron spin resonance, and fluorescence spectrometry, electron microscopy, and fusion and leakage assays. The phase transition temperatures of DPPA and DPPG were very similar when bound to PMB or PMBN, indicating that the lipids are in a similar state when bound to the cationic peptides. Both PMB and PMBN caused the interdigitation of DPPG bilayers, suggesting that the penetration of hydrophobic side chains from a peptide bound electrostatically on the surface is sufficient to induce this phenomenon. Stopped-flow experiments revealed that PMBN and PMB induced the fusion of small unilamellar PS and large unilamellar DPPA-DSPC vesicles. The aggregation of vesicles was found to be diffusion-controlled process; the subsequent fusion took place with a frequency of 10(2)-(5 X 10(2] s-1 for small vesicles and 1-100 s-1 for large vesicles. The freeze-fracture replicas of the PMB-treated vesicles displayed 12-50-nm depressions on several superimposed bilayers, indicating the formation of stable lipid-PMB domains. Since the incubation with PMBN produced similar depressions only if the specimens were fixed, PMBN-induced domain formation seems to be a reversible rapid process. The differences in the phospholipid-peptide interactions are correlated with the differences in the physiological action of the antibiotic PMB and the nonbactericidal PMBN on the cell envelope of Gram-negative bacteria.     

A study of the structure of polymyxin B-dipalmitoylphosphatidylglycerol complexes by vibrational spectroscopy

The effect of the antibiotic polymyxin B on dipalmitoylphosphatidylglycerol (DPPG) bilayers has been studied by Raman and infrared spectroscopies and small-angle X-ray diffraction. Each polymyxin B molecule binds five DPPG molecules at physiological pH and induces a macroscopic phase separation of the complex rather than a lateral phase separation. Below the phase transition of DPPG/polymyxin B bilayers, the results obtained show that the intermolecular vibrational coupling is high and suggest that the acyl chains of the bound lipid are interdigitated and that the hydrophobic tail of the antibiotic does not penetrate this tight assembly. On the other hand, the phase transition of DPPG is shifted down from 41 degrees C to 37 degrees C in the complexes and remains highly cooperative. Above the phase transition of the complexes, the conformation of the acyl chains of DPPG is slightly more disordered as a result of the penetration of the polymyxin chain, but the structure of the glycerol backbone of the lipid does not seem to be affected. However, the rotational rate of the lipid appears to be restricted by the peptide.     

Phosphatidic acid synthesis in mitochondria. Topography of formation and transmembrane migration.

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80-100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A(2). The amount of phospholipase A(2)-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.     

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.     

Lipid-protein interactions at the nicotinic acetylcholine receptor. A functional coupling between nicotinic receptors and phosphatidic acid-containing lipid bilayers.

The structural and functional properties of reconstituted nicotinic acetylcholine receptor membranes composed of phosphatidyl choline either with or without cholesterol and/or phosphatidic acid have been examined to test the hypothesis that receptor conformational equilibria are modulated by the physical properties of the surrounding lipid environment. Spectroscopic and chemical labeling data indicate that the receptor in phosphatidylcholine alone is stabilized in a desensitized-like state, whereas the presence of either cholesterol or phosphatidic acid favors a resting-like conformation. Membranes that effectively stabilize a resting-like state exhibit a relatively large proportion of non-hydrogen-bonded lipid ester carbonyls, suggesting a relatively tight packing of the lipid head groups and thus a well ordered membrane. Functional reconstituted membranes also exhibit gel-to-liquid crystal phase transition temperatures that are higher than those of nonfunctional reconstituted membranes composed of phosphatidylcholine alone. Significantly, incorporation of the receptor into phosphatidic acid-containing membranes leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid crystal phase transition temperatures of the reconstituted lipid bilayers. These results suggest a functional link between the nicotinic acetylcholine receptor and the physical properties of phosphatidic acid-containing membranes that could underlie the mechanism by which this lipid preferentially enhances receptor function.     

Phase transitions and fatty acid spin label behavior in interdigitated lipid phases induced by glycerol and polymyxin

Glycerol and polymyxin have been shown by X-ray diffraction to induce interdigitated bilayers in phosphatidylcholine (PC) and phosphatidylglycerol (PG), respectively (McDaniel, R.V., et al. (1983) Biochim. Biophys. Acta 731, 97-108; Ranck, J.-L. and Tocanne, J.-F. (1982) FEBS Lett. 143, 175-178). In the present study we have investigated the phase behavior of PC and PG in the presence of glycerol and polymyxin by differential scanning calorimetry and the use of fatty acid spin labels. Interdigitation causes a large increase in the order parameter of a fatty acid spin labeled near the terminal methyl, 16-doxylstearate, so that it was similar to that of a fatty acid labeled much closer to the polar head group region, 5-doxylstearate. Thus interdigitation abolishes the fluidity gradient found in a non-interdigitated bilayer. 16-Doxylstearate may be useful in detecting interdigitation of lipid bilayers caused by other substances. The different samples all went through two transitions on heating or cooling, or both. However, use of the fatty acid spin label showed that the molecular events during these transitions varies for different samples. The results suggested that PC-glycerol freezes from the liquid-crystalline phase into a non-interdigitated gel phase. This subsequently becomes interdigitated upon lowering the temperature a few degrees, in a low enthalpy transition. PG-polymyxin shows a similar behavior except that the enthalpy of the non-interdigitated gel to interdigitated phase transition is greater and the transition is reversible on heating. Thus on heating PG-polymyxin first goes through a transition from the interdigitated phase to a non-interdigitated gel phase and then, in a separate transition, to the liquid-crystalline phase. This occurs because the fatty acid chains in the presence of polymyxin become too disordered with increase in temperature to maintain the interdigitated state. PG-glycerol goes into the interdigitated state less readily than the other mixtures. If cooled rapidly, PG-glycerol freezes into a metastable phase which is more disordered than the interdigitated phase. It goes into the interdigitated phase in an exothermic transition on heating. An increase in fatty acid chain length causes greater steric hindrance to interdigitation but also increases the stabilizing energy gained by interdigitation.