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, 5-tetramethylpiperidine-N-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 wre determined and it was shown that application of pressure reduces the cooperativity of the binding curve.     

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     

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.     

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°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 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.     

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.     

The influence of charge on phosphatidic acid bilayer membranes

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, $\text{1,2-}\text{dihexadecyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of this phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, 1,3-dimyristoylglycerol-2-phosphoric acid and $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid.The phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90° light scattering. The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states.The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 ($\text{p}\text{K}{}_1$). The regions between the first and the second $\text{p}\text{K}$ of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H⁺ concentration results in an abrupt change of the transition temperature. The slope of the $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagram beyond $\text{p}\text{K}{}_2$ becomes very steep. This is the     

Asymmetric antagonistic effects of an inhalation anesthetic and high pressure on the phase transition temperature of dipalmitoyl phosphatidic acid bilayers

The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.     

Polymyxin B-phosphatidylglycerol interactions. A monolayer (π, ΔV) study

Through a monolayer investigation (π, ΔV), it is shown that the cationic antibiotic polymyxin B (or E) strongly interacts with films of acidic lipids, namely the didodecanoyl- and dihexadecanoylphosphatidylglycerol. The zwitterionic dihexadecanoylphosphatidylcholine was an unsuitable substrate. Interactions occurred at and above a polymyxin B concentration in the subphase of 2.5 · 10^?7 M, bringing about a considerable increase of both π and ΔV. These interactions proceeded in two steps, as revealed by a biphasic change of ΔV with time. They were independent of the film molecular packing (fluid or gel states) and of the initial film pressure.Since it was possible to monitor the relative number of polymyxin B and didodecanoyl- or dihexadecanoylphosphatidylglycerol molecules in the monolayer, it is demonstrated that, at saturation, one polymyxin B molecule is bound to five phosphatidylglycerol molecules, a result which accounts for an exact neutralization of the charges.From competition experiments, it is shown that Na⁺ is ineffective in removing polymyxin B from the interface. Ca²⁺ appeared to be a stronger competitor but no complete antibiotic desorption was observed even at a Ca²⁺ concentration of 100 mM.As a working hypothesis, the antibiotic/lipid ($\text{1}\text{5}$) system was assumed to constitute by itself one molecular species. The mixing of the polymyxin B/didodecanoylphosphatidylglycerol ($\text{1}\text{5}$) system with an excess of lipid molecules in the monolayer was found to be ideal both in terms of π and ΔV. With dihexadecanoylphosphatidylglycerol, a small condensing effect could be detected only at intermediate surface pressures, in a region where the lipid phase transition occurred.The molecular area of polymyxin B interacting with didodecanoylphosphatidylglycerol can be calculated to be 1.23 ± 0.05 nm². It is proposed that the whole antibiotic molecule penetrates the film, the five bound lipid molecules being distributed around the peptide structure, at given positions imposed by the five 2,4-diaminobutyric acid residues.     

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure

Differential polarized phase fluorometry was used to quantify the rotational rate ($\text{R}$) and limiting anisotropy ($\text{r}{}_{\mathrm{\infty }}$) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by $\text{r}{}_{\mathrm{\infty }}$. In addition to the well-known elevation of the transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with $\text{r}{}_{\mathrm{\infty }}$. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

Acyltransferases and the biosynthesis of pulmonary surfactant lipid in adenoma alveolar type II cells.

Acyltransferases are present in microsomes from alveolar type II cell adenomas (produced by urethan injections) that transfer palmitic acid in the presence of CoA, ATP, and Mg⁺⁺ to $\text{sn}$-glycerol-3-P to form phosphatidic acid, to dihydroxyacetone-P to form acyldihydroxyacetone-P, and to 1-acyl-$\text{sn}$-glycero-3-phosphocholine to form 3-$\text{sn}$-phosphatidylcholine. The data clearly demonstrate that the microsomal preparations can catalyze significant incorporation of palmitic acid into the 2-position of the disaturated species of 3-sn-phosphatidylcholine independently of phosphatidic acid formation as evidenced by the fact that $\text{sn}$-glycerol-3-P and calcium ions (which inhibit choline phosphotransferase) did not influence the incorporation of palmitic acid into the main surfactant lipid. Thus, a deacylation-acylation reaction involving 2-lysophosphatidylcholine appears to be an important pathway for the synthesis of surfactant lipid in alveolar type II cells; the control of acyl specificity at the 2-position is determined by the relative concentrations of the coparticipating substrates, l-palmitoyl-$\text{sn}$-glycero-3-phosphocholine and palmitoyl-CoA.     

Lipid phase transitions in cytoplasmic and outer membranes of Escherichia coli

The cytoplasmic and outer membranes containing either trans-Δ⁹-octadecenoate, trans-Δ⁹-hexadecenoate or cis-Δ⁹-octadecenoate as predominant unsaturated fatty acid residues in the phospholipids were prepared from a fatty acid auxotroph, Escherichia coli strain K1062. Order-disorder transitions of the phospholipids were revealed in both fractions of the cell envelope by fluorescent probing or wide angle X-ray diffraction. The mid-transition temperatures, $\text{T}\text{t}$, and the range of the transition, $\text{ΔT}$, are similar in the outer and cytoplasmic membrane. Relative to the corresponding extracted lipids, 60–80% of the hydrocarbon chains take part in the transition in the cytoplasmic membrane whereas in the outer membrane only 25–40% of the chains become ordered. The results suggest that in the outer membrane part of the lipids form fluid domains in the form of mono- and/or bilayers.     

Phase transition characteristics of diphosphatidylglycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers: Mono- and divalent metal ion effects

Synthesis and phase transition characteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>m</mtext>}}$) and the enthalpy of transition ($\text{ΔH}$) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric $\text{sn-3,3-}\text{and}\text{sn-1,1-}\text{phosphatidyldiacylglycerol}$ and for the diastereomeric, $\text{meso}\text{-sn-1,3-}\text{phosphatidyldiacylglycerol}$ are approximately the same. The molar enthalpy for the transition of cardiolipin-NH₄⁺ bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C_{1 6}-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e. the highest $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ is observed for Ca²⁺- cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca²⁺. The lowest $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ is observed for Rb⁺-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.     

The influence of protein-lipid interactions on the order-disorder conformational transitions of the hydrocarbon chain

The phases of simple systems involving one type of protein (lysozyme or cytochrome $\text{c}$) and one type of lipid (phosphatidic acid) have been characterized by X-ray crystallography, chemical analysis and spin-labeling technique as a function of temperature. They are of the lamellar type with alternative protein monolayers and lipid bilayers. According to the pH, two types of lamellar phases are obtained, one where the lipid-protein interactions are mainly hydrophobic, the other where they are electrostatic. In both cases, a phase transition occurs as temperature is lowered, between a high temperature phase, where all the lipids are in the liquid-like state, and another phase where some lipid chains are rigid. In the case of the phases with electrostatic interaction, it is shown that the onset of the order-disorder transition is shifted towards low temperature as compared with the homologous lipid-water phase and that the protein content of the phase decreases as the ratio of the liquid to rigid hydrocarbon chains decreases. This leads us to suggest that in the systems studied in this work the proteins interact only with lipid in the liquid-like state. In the case of the phases with hydrophobic interaction, it is shown that the extent of hydrophobic interaction between protein and lipid increases as the unsaturation of the hydrocarbon chains increases. The onset of the order-disorder transition shows a greater shift towards low temperture than the one observed in the case of the phase with electrostatic interaction.