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.     

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.     

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.     

Chemically induced lipid phase separation in model membranes containing charged lipids: a spin label study.

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca2+ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, Wex. By measuring Wex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithin-phosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers. In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8-10(-8) cm2/s at 59 degrees C. Addition of Ca2+ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca2+-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of Wex on the Ca2+ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca2+ concentration clusters containing about 30 mol % lecithin are formed. At high lecithin or Ca2+ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn2+ using ESR spectroscopy. Polylysine leads to the same strong increase in the lecithin segregation as Ca2+. The transition of the phosphatidic acid bound by the polypeptide is shifted from Tt = 47.5 degrees to Tt = 62 degrees C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

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.     

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.     

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.     

Chemically induced lipid phase separation in model membranes containing charged lipids: A spin label study

The lipid distribution in binary mixed membranes containing charged and uncharged lipids and the effect of Ca²⁺ and polylysine on the lipid organization was studied by the spin label technique. Dipalmitoyl phosphatidic acid was the charged, and spin labelled dipalmitoyl lecithin was the uncharged (zwitterionic) component. The ESR spectra were analyzed in terms of the spin exchange frequency, W_ex. By measuring W_ex as a function of the molar percentage of labelled lecithin a distinction between a random and a heterogeneous lipid distribution could be made. It is established that mixed lecithinphosphatidic acid membranes exhibit lipid segregation (or a miscibility gap) in the fluid state. Comparative experiments with bilayer and monolayer membranes strongly suggest a lateral lipid segregation. At low lecithin concentration, aggregates containing between 25% and 40% lecithin are formed in the fluid phosphatidic acid membrane. This phase separation in membranes containing charged lipids is understandable on the basis of the Gouy-Chapman theory of electric double layers.In dipalmitoyl lecithin and in dimyristoyl phosphatidylethanolamine membranes the labelled lecithin is randomly distributed above the phase transition and has a coefficient of lateral diffusion of D = 2.8·10^?8 cm²/s at 59°C.Addition of Ca²⁺ dramatically increases the extent of phase separation in lecithin-phosphatidic acid membranes. This chemically (and isothermally) induced phase separation is caused by the formation of crystalline patches of the Ca²⁺-bound phosphatidic acid. Lecithin is squeezed out from these patches of rigid lipid. The observed dependence of W_ex on the Ca²⁺ concentration could be interpreted quantitatively on the basis of a two-cluster model. At low lecithin and Ca²⁺ concentration clusters containing about 30 mol% lecithin are formed. At high lecithin or Ca²⁺ concentrations a second type of precipitation containing 100% lecithin starts to form in addition. A one-to-one binding of divalent ions and phosphatidic acid at pH 9 was assumed. Such a one-to-one binding at pH 9 was established for the case of Mn²⁺ using ESR spectroscopy.Polylysine leads to the same strong increase in the lecithin segregation as Ca²⁺. The transition of the phosphatidic acid bound by the polypeptide is shifted from T_t = 47.5° to T_t = 62°C. This finding suggests the possibility of cooperative conformational changes in the lipid matrix and in the surface proteins in biological membranes.     

Effects of lipid structure on peptide-lipid interactions. Complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon calcitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin- DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol . There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Mechanism of rat liver microsomal stearyl-CoA desaturase. Studies of the substrate specificity, enzyme-substrate interactions, and the function of lipid.

The three purified proteins which are required for microsomal stearyl-CoA desaturation, NADH-cytochrome b5 reductase, cytochrome b5, and desaturase, have been combined with egg lecithin or dimyristyl lecithin vesicles to reconstruct a functional electron transport system capable of utilizing NADH and O2 in the desaturation of stearyl-CoA. Such preparations appear to consist of phospholipid vesicles which contain the three proteins bound to the outer surface of the vesicles. Acyl-CoA derivatives containing 12 to 19 carbon fatty acyl chains are required for desaturase activity while derivatives containing 9 to 20 carbons are capable of binding to the enzyme. Shorter chain acyl-CoA derivatives, free CoA, and free fatty acids do not appear to bind to the enzyme. Inhibition and analog studies suggest that the methylene chain of stearyl-CoA assumes an eclipsed ("gauche") conformation at carbon atoms 9,10 in the enzyme-substrate complex. Furthermore, isotope rate effects obtained with deuterated stearyl-CoA derivatives indicate that hydrogen removal is the rate-limiting step of desaturation. Stearyl-CoA binds to pure liposomes and desaturase-containing liposomes, and it is this form of stearyl-CoA which appears to be the substrate for desaturase. The Arrhenius plots of desaturase activity obtained using desaturase bound to egg lecithin liposomes, in which the liquid crystalline to crystalline phase transition temperature is -5 degrees, was linear between 15 and 35 degrees, while that obtained using desaturase bound to dimyristyl lecithin liposomes showed a break at 24 degrees coinciding with the liquid crystalline to crystalline phase transition temperature for this lipid. The decrease observed in the deuterium isotope rate effect below the transition temperature indicates that a step in the reaction sequence other than hydrogen abstraction becomes rate-limiting when the lipid is in the crystalline state. In this system translational diffusion does not emerge as the rate-limiting step. The liposomes contained sufficient reductase and cytochrome b5 so that translational diffusion was not rate-limiting.     

Effects of lipid structure on peptide-lipid interactions complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon cacitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin-DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol. There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.     

Structure of an apolipoprotein-phospholipid complex: apoC-III induced changes in the physical properties of dimyristoylphosphatidylcholine.

The effect of ApoC-III, a major apoprotein constituent of human very low density lipoproteins, on the physical properties of dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by magnetic resonance and fluorescence techniques. The sharp gel-liquid crystalline transition usually observed at 23 C in DMPC is both broadened and elevated when ApoC-III is bound as determined (a) from measurements of microscopic viscosity by pyrene excimer fluorescence, (b) from the distribution of di-tert-butyl nitroxide between the bulk aqueous phase and the fluid lipid phase, and (c) from the motion of fatty acyl chains of spin-labeled phosphatdylcholine. Experiments involving the translocation of ascorbate and charged nitroxide ions and the movement of paramagnetic Eu 3+ ions indicate that when ApoC-III binds to DMPC vesicles, it increases their permeability or destroys their original bilayer structure. These two possibilities were distinguishable by gel filtration of the DMPC-ApoC-III complex (approximately 34 mol mol) that indicated that the product particles were significantly smaller than the original vesicles. Taken together, the data indicate that ApoC-III binding to DMPC not only decreases the acyl chain motion of individual lipid molecules, but also induces break-down of bilamellar vesicular structure to give significantly smaller complexes.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Characteristics of the binding of tacrine to acidic phospholipids.

Tacrine (1,2,3,4-tetrahydro-9-acridinamine monohydrate) is an inhibitor of acetylcholinesterase currently used in the treatment of the symptoms of Alzheimer's disease. The present study demonstrates preferential binding of this drug to acidic phospholipids, as revealed by fluorescence polarization, penetration into lipid monolayers, and effects on the thermal phase behavior of dimyristoyl phosphatidic acid (DMPA). A fivefold enhancement in the polarization of tacrine emission is evident above the main phase transition temperature (T(m)) of DMPA vesicles, whereas below T(m) only a 0.75-fold increase is observed. In contrast, the binding of tacrine to another acidic phospholipid, dimyristoylphosphatidylglycerol, did not exhibit strong dependence on T(m). In accordance with the electrostatic nature of the membrane association of tacrine, the extent of binding was augmented with increasing contents of egg PG in phosphatidylcholine liposomes. Furthermore, [NaCl] > 50 mM dissociates tacrine (albeit incompletely) from the liposomes composed of acidic phospholipids. Inclusion of the cationic amphiphile sphingosine in egg PG vesicles decreased the membrane association of tacrine until at 1:1 sphingosine: egg PG stoichiometry binding was no longer evident. Tacrine also penetrated into egg PG but not into egg PC monolayers. Together with broadening of the main transition and causing a shoulder on its high temperature side, the binding of tacrine to DMPA liposomes results in a concentration-dependent reduction both in the combined enthalpy delta H of the above overlapping endotherms and the main transition temperature T(m). Interestingly, these changes in the thermal phase behavior of DMPA as a function of the content of the drug in vesicles were strongly nonlinear. More specifically, upon increasing [tacrine], T(m) exhibited stepwise decrements. Simultaneously, sharp minima in delta H were observed at drug:lipid stoichiometries of approximately 2:100 and 25:100, whereas a sharp maximum in delta H was evident at 18:100. The above results are in keeping with tacrine causing phase separation processes in the bilayer and may also relate to microscopic drug-induced ordering processes within the membrane.     

Hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, induced permeability change of phosphatidylcholine bilayers

Interactions of hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, with phosphatidylcholine vesicles were investigated to obtain information on its bioactive mechanism. The peptide induced the leakage of a fluorescent dye, calcein, entrapped in sonicated vesicles. The leakage rate depended on both the peptide and the lipid concentrations. Analysis of this dependency indicated that the leakage was due to the monomeric peptide and that the membrane-perturbing activity of the monomer was higher for solid distearoylphosphatidylcholine vesicles than for fluid egg yolk phosphatidylcholine vesicles. Hypelcin A also affected the gel to liquid-crystalline phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles. The transition was broadened with a reduced transition enthalpy, suggesting the peptide strongly binds the surrounding lipids to perturb the bilayer lipid packing. A circular dichroism study revealed that the helical content of hypelcin A increases upon membrane binding. We concluded that the monomeric peptide with an increased helical content, complexed with the lipids, perturbs the lipid organization and induces the increased permeability.