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.     

Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.     

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.     

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.     

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.     

Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy.     

Correlation between acetylcholine receptor function and structural properties of membranes

Protein-lipid interactions were studied by using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into membranes containing various synthetic lipids and native lipids. AChR function was determined by measuring two activities at 4 degrees C: (1) low to high agonist affinity-state transition of AChR in the presence of an agonist (carbamylcholine) in either membrane fragments or sealed vesicles and (2) ion-gating activity of AChR-containing vesicles in response to carbamylcholine. Sixteen samples were examined, each containing different lipid compositions including phosphatidylcholine, cholesterol, phosphatidic acid, phosphatidylethanolamine, asolectin, neutral lipid depleted asolectin, native lipids, and cholesterol-depleted native lipids. Phosphatidylcholines with different configurations of fatty acyl chains were used. The dynamic structures of these membranes were probed by incorporating spin-labeled fatty acid into AChR-containing vesicles and measuring the order parameters. It was found that both aspects of AChR function were highly dependent on the lipid environment even though carbamylcholine binding itself was not affected. An appropriate membrane fluidity was necessarily required to allow the interconversion between the low and high affinity states of AChR. An optimal fluidity hypothesis is proposed to account for the conformational transition properties of membrane proteins. In addition, the conformational change was only a necessary, but not sufficient, condition for the AChR-mediated ion flux activity. Among membranes in which AChR manifested the affinity-state transition, only those containing both cholesterol and negatively charged phospholipids (such as phosphatidic acid) retained the ion-gating activity.     

Location and dynamics of basic peptides at the membrane interface: electron paramagnetic resonance spectroscopy of tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid-labeled peptides

The attractive interaction between basic protein domains and membranes containing acidic lipids is critical to the membrane attachment of many proteins involved in cell signaling. In this study, a series of charged model peptides containing lysine, phenylalanine, and the spin-labeled amino acid tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) were synthesized, and electron paramagnetic resonance (EPR) spectroscopy was used to determine their position on the membrane interface and free energy of binding. When membrane-bound, peptides containing only lysine and TOAC assume an equilibrium position within the aqueous double layer at a distance of approximately 5 A from the membrane interface, a result that is consistent with recent computational work. Substitution of two or more lysine residues by phenylalanine dramatically slows the backbone diffusion of these peptides and shifts their equilibrium position by 13-15 A so that the backbone lies several angstroms below the level of the lipid phosphate. These results are consistent with the hypothesis that the position and free energy of basic peptides when bound to membranes are determined by a long-range Coulombic attraction, the hydrophobic effect, and a short-range desolvation force. The differences in binding free energy within this set of charged peptides is not well accounted for by the simple addition of free energies based upon accepted side chain partition free energies, a result that appears to be in part due to differences in membrane localization of these peptides.     

Binding of β-Amyloid (1-42) Peptide to Negatively Charged Phospholipid Membranes in the Liquid-Ordered State: Modeling and Experimental Studies

To explore the initial stages of amyloid β peptide (Aβ42) deposition on membranes, we have studied the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L_o) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L_o state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5–5 mol %) than higher (10–20 mol %) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aβ42 binding than L_o ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of β-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aβ42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol % anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aβ42 and membrane.     

Membrane lipids have multiple effects on interfacial catalysis by a phosphatidic acid-preferring phospholipase A1 from bovine testis

We previously purified a cytosolic phospholipase A1 that could catalyze the preferential hydrolysis of phosphatidic acid in mixed-micelle assays. Here we studied the enzyme's interactions with unilamellar lipid membranes and examined effects of the lipids on enzyme binding, stability, and catalysis. A major finding was that membrane lipids could influence the stability, activity, and specificity of the enzyme under conditions where enzyme binding to the membranes was likely to be saturated. Thus, the enzyme was unstable at 37 degrees C in the absence of membranes but bound to membranes that contained anionic phosphoglycerides and could be stabilized by these membranes in the presence of albumin. The overall activity of the bound enzyme toward membrane phosphoglycerides, assayed in the presence of albumin, increased when phosphatidylethanolamine was substituted for phosphatidylcholine. Furthermore, the enzyme's catalytic preference for phosphatidic acid increased when cholesterol and diacylglycerol were included in the membranes, sn-1-stearoyl-2-arachidonoylphosphatidylethanolamine was substituted for sn-1-palmitoyl-2-oleoylphosphatidylethanolamine, and the concentration of phosphatidic acid was increased from 0 to 10 mol % of the total membrane phosphoglycerides. Finally, changes in the relative contents of phosphatidylcholine and phosphatidylserine in the membranes influenced the enzyme's catalytic preference for different molecular species of phosphatidic acid. These results provide the first available information about the enzyme's ability to interact with membranes and identify conditions that yield high enzyme activity toward membrane-associated phosphatidic acid.     

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, 1,2-dihexadecyl-sn-glycerol-3-phosphoric acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of the phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; 1,2-dimyristoyl-sn-glycerol-3-phosphoric acid, 1,3-dimyristoylglycerol-2-phosphoric acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoric acid. The phase transition temperatures (Tt) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90 degrees light scattering. The Tt 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 Tt 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 (pK1). The regions between the first and the second pK 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 Tt vs. pH diagram beyond pK2 becomes very steep. This is the result of reduced hydrocarbon interaction energy, which was demonstrated by differential scanning calorimetry (Blume, A. and Eibl, H., unpublished data).     

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.     

Lipid-protein interactions with cardiac phospholamban studied by spin-label electron spin resonance

Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane.     

Analysis of the mechanism of histamine release induced by substance P

Substance P causes release of histamine from rat peritoneal mast cells; the structure-activity relationship shows that N-terminal residue is essential and the hydrophobic region of C-terminal plays an important role. Electrical conductivity of black lipid membrane containing phosphatidic acid was augmented by substance P. In this case, N-terminal residues and C-terminal hydrophobicity were also unavoidable. The partitioning of substance P into the organic phase increased in the presence of phosphatidic acid. The CD spectrum of substance P was changed from the unordered form to beta-form by coexistence of phosphatidic acid/PC liposomes in the medium. The addition of calcium or magnesium in the test solution is effective to prevent either of these phenomena. These findings indicate that substance P probably binds to negatively charged sites of membrane lipids, and subsequent penetration of C-terminal into the hydrophobic core of lipid bilayer may induce an increase of membrane permeability and the following histamine release.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies.

Lipid-peptide interactions with the 27-residue peptide of sequence KLEALYILMVLGFFGFFTLGIMLSYIR reconstituted as beta-sheet assemblies in dimyristoylphosphatidylcholine bilayers have been studied by electron spin resonance (ESR) spectroscopy with spin-labeled lipids. The peptide corresponds to residues 42-68 of the IsK voltage-gated K+ channel protein and contains the single putative transmembrane span of this protein. Lipid-peptide interactions give rise to a second component in the ESR spectra of lipids spin-labeled on the 14C atom of the chain that corresponds to restriction of the lipid mobility by direct interaction with the peptide assemblies. From the dependence on the lipid/peptide ratio, the stoichiometry of lipid interaction is found to be about two phospholipids/peptide monomer. The sequence of selectivity for lipid association with the peptide assemblies is in the order phosphatidic acid > stearic acid = phosphatidylserine > phosphatidylglycerol = phosphatidylcholine. Comparison with previous data for a corresponding 26-residue mutant peptide with a single deletion of the apolar residue Leu2 (Horvath et al., 1995. Biochemistry 34:3893-3898), indicates a very similar mode of membrane incorporation for native and mutant peptides, but a strongly modified pattern and degree of specificity for the interaction with negatively charged lipids. The latter is interpreted in terms of the relative orientations of the charged amino acid side chains in the beta-sheet assemblies of the native and deletion-mutant peptides.     

Molecular aspects of the bilayer stabilization induced by poly(L-lysines) of varying size in cardiolipin liposomes

The interaction between poly(L-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and 31P- and 13C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca2+ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca2+, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca2+ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca2+, the lipid was organized in the hexagonal HII phase in the presence of Ca2+. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes.     

Design and characterization of anchoring amphiphilic peptides and their interactions with lipid vesicles.

In an effort to develop a polymer/peptide assembly for the immobilization of lipid vesicles, we have made and characterized four water-soluble amphiphilic peptides designed to associate spontaneously and strongly with lipid vesicles without causing significant leakage from anchored vesicles. These peptides have a primary amphiphilic structure with the following sequences: AAAAAAAAAAAAWKKKKKK, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK, and KKAALLLAAAAAAAAAAAAAAAAAAAWKKKKKK and its reversed homologue KKKKKKWAAAAA AAAAAAAAAAAAAALLLAAKK. Two of the four peptides have their hydrophobic segments capped at both termini with basic residues to stabilize the transmembrane orientation and to increase the affinity for negatively charged vesicles. We have studied the secondary structure and the membrane affinity of the peptides as well as the effect of the different peptides on the membrane permeability. The influence of the hydrophobic length and the role of lysine residues were clearly established. First, a hydrophobic segment of 24 amino acids, corresponding approximately to the thickness of a lipid bilayer, improves considerably the affinity to zwitterionic lipids compared to the shorter one of 12 amino acids. The shorter peptide has a low membrane affinity since it may not be long enough to adopt a stable conformation. Second, the presence of lysine residues is essential since the binding is dominated by electrostatic interactions, as illustrated by the enhanced binding with anionic lipids. The charges at both ends, however, prevent the peptide from inserting spontaneously in the bilayer since it would involve the translocation of a charged end through the apolar core of the bilayer. The direction of the amino acid sequence of the peptide has no significant influence on its behavior. None of these peptides perturbs membrane permeability even at an incubation lipid to peptide molar ratio of 0.5. Among the four peptides, AALLLAAAAAAAAAAAAAAAAAAAWKKKKKK is identified as the most suitable anchor for the immobilization of lipid vesicles.     

A spectroscopic study of the membrane interaction of the antimicrobial peptide Pleurocidin

The cationic amphipathic alpha-helical antibiotic peptide, pleurocidin, from the winter flounder Pleuronectes americanus associates strongly with anionic membranes where it is able to translocate across the membrane, cause dye leakage from vesicles and induce pore like channel conductance. To investigate the mechanism of pleurocidin antibiotic activity in more detail we have applied a variety of spectroscopic techniques to study the interaction of pleurocidin with model membranes. At neutral pH the peptide inserts into membranes containing anionic lipids and, as shown by proton-decoupled 15N solid-state NMR spectroscopy of macroscopically oriented samples, is aligned parallel to the membrane surface. 2H solid-state NMR spectroscopy of chain deuterated phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) lipids in mixed membranes shows that pleurocidin interacts with both the zwitterionic PE and anionic PG but disrupts the lipid acyl chain order of the anionic PG lipids more effectively. At acidic pH the three histidine residues of pleurocidin become protonated and positively charged which does not alter the membrane disrupting effect nor the location of the peptide in the membrane. The results are interpreted in terms of a structural model for pleurocidin inserted into anionic lipid membranes and the implications of our data are discussed in terms of a general mechanism for the antibiotic activity.