Study of structure and orientation of mesentericin Y105, a bacteriocin from Gram-positive Leuconostoc mesenteroides, and its Trp-substituted analogues in phospholipid environments

Mesentericin Y105 (Mes-Y105) is a bacteriocin secreted by Leuconostoc mesenteroides which is particularly active on Listeria. It is constituted by 37 residues and reticulated by one disulfide bridge. It has two W residues, W18 and W37, which can be studied by fluorescence. Two single substituted W/F analogues were synthesized (Mes-Y105/W18 and Mes-Y105/W37) to differentiate the local environment around each W and to study their changes in the presence of lipid vesicles.Fluorescence experiments show that, for the pure Trp-analogues, W18 and W37 are fully exposed to solvent whatever pH and buffer conditions. In the presence of lipid vesicles, both became buried. Lipid affinities were estimated: they are weak for zwitterionic phospholipids but an order of magnitude higher for negatively charged phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids. On negatively charged PG lipids, Mes-Y105 and Mes-Y105/W37 display comparable lipid affinities. A decrease in lipid affinity is observed for Mes-Y105/W18 compared to Mes-Y105, which means that W37 would seem to be required for increased lipid selectivity. In the lipid-bound state W18 is strongly dehydrated, probably embedded into the acyl chains, while W37 stands more at the interface.Mes-Y105 was also studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS), alone and in various phospholipid environments, to obtain structural information and to assess lipid perturbations. At nanomolar concentrations close to those required for anti-Listeria activity, Mes-Y105 forms films at the air/water interface and inserts into negatively charged lipid monolayers. In situ infrared data show that Mes-Y105 binding only affects the polar head group vibrations while the lipid order of the acyl chains remains unaffected. The PMIRRAS show that Mes-Y105 folds into an N-terminal antiparallel β-sheet followed by an α-helix, both structures being tilted (40°) compared to the normal at the interface, which is in agreement with the thickness estimated by Brewster angle microscopy (BAM). All these data support the proposal of a new model for Mes-Y105 at the membrane interface.     

Partitioning of exchangeable fluorescent phospholipids and sphingolipids between different lipid bilayer environments

Exchangeable phospho- and sphingolipid probes (phosphatidylcholine, -ethanolamine, -serine, and -glycerol, phosphatidic acid, sphingomyelin, cerebroside, and sulfatide) have been synthesized in which one acyl chain is substituted with a fluorescent bimanyl, 7-(dimethylamino)coumarin-3-yl, or diphenyl-hexatrienyl group. The distribution of these probes between two different populations of lipid vesicles can be readily monitored by fluorescence intensity measurements, as described by Nichols and Pagano [Nichols, J. W., & Pagano, R. E. (1982) Biochemistry 21, 1720-1726], when one of the vesicle populations contains a low mole fraction of a nonexchangeable quencher, (12-DABS)-18-PC. The probes examined in this study exchange between phospholipid vesicles on a time scale of minutes, with kinetics indicating that the transfer process takes place by diffusion of probe monomers through the aqueous phase. As expected, lipid probes with different charges differ markedly in their equilibrium distributions between neutral and charged lipid vesicles. However, probes with different polar headgroups differ only modestly in their relative affinities for vesicles composed of "hydrogen-bonding" lipids (PE and PS) vs "non-hydrogen-bonding" lipids (PC and PG or O-methyl-PA). Probes with different headgroups also show modest, albeit reproducible, differences in their relative affinities for cholesterol-containing vs cholesterol-free PC/PG vesicles. Our results suggest that lipids with different headgroup structures may mix more nearly ideally in liquid-crystalline lipid bilayers than would be predicted from previous analyses of the phase diagrams for binary lipid mixtures.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3–11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events – large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.     

Minimal Effect of Lipid Charge on Membrane Miscibility Phase Behavior in Three Ternary Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.     

Analytic Approaches to Stochastic Gene Expression in Multicellular Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.     

Examining the contributions of lipid shape and headgroup charge on bilayer behavior

To better understand bilayer property dependency on lipid electrostatics and headgroup size, we use atomistic molecular dynamics simulations to study negatively charged and neutral lipid membranes. We compare the negatively charged phosphatidic acid (PA), which at physiological pH and salt concentration has a negative spontaneous curvature, with the negatively charged phosphatidylglycerol (PG) and neutrally charged phosphatidylcholine (PC), both of which have zero spontaneous curvature. The PA lipids are simulated using two different sets of partial charges for the headgroup and the varied charge distribution between the two PA systems results in significantly different locations for the Na⁺ ions relative to the water/membrane interface. For one PA system, the Na⁺ ions are localized around the phosphate group. In the second PA system, the Na⁺ ions are located near the ester carbonyl atoms, which coincides with the preferred location site for the PG Na⁺ ions. We find that the Na⁺ ion location has a larger effect on bilayer fluidity properties than lipid headgroup size, where the A_lipid and acyl chain order parameter values are more similar between the PA and PG bilayers that have Na⁺ ions located near the ester groups than between the two PA bilayers.     

Secondary structure and orientation of the surfactant protein SP-B in a lipid environment. A Fourier transform infrared spectroscopy study.

Attenuated total reflection Fourier transform infrared spectroscopy was used to investigate the secondary structure of the surfactant protein SP-B. Nearly half of the polypeptide chain is folded in an alpha-helical conformation. No significant change of the secondary structure content was observed when the protein is associated to a lipid bilayer of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) or of dipalmitoylphosphatidylglycerol (DPPG). The parameters related to the gamma w(CH2) vibration of the saturated acyl chains reveal no modification of the conformation or orientation of the lipids in the presence of SP-B. A model of orientation of the protein at the lipid/water interface is proposed. In this model, electrostatic interactions between charged residues of SP-B and polar headgroups of PG, and the presence of small hydrophobic alpha-helical peptide stretches slightly inside the bilayers, would maintain SP-B at the membrane surface.     

Incorporation of dehydrodieugenol,a neolignan isolated from Nectandra leucantha (Lauraceae), in lipid Langmuir monolayers as biomembrane models

Dehydrodieugenol, a neolignan isolated from the Brazilian plant Nectandra leucantha (Lauraceae) with reported antiprotozoal and anticancer activity, was incorporated in Langmuir monolayers of selected lipids as cell membrane models, aiming to comprehend its action mechanism at the molecular level. The interaction of this compound with the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), and dipalmitoylphosphatidylglycerol (DPPG) was inferred through tensiometry, infrared spectroscopy, and Brewster angle microscopy. The interactions had different effects depending on the chemical nature of the lipid polar head, with expansion for DPPC monolayers, condensation for DPPE, and expansion (at low surface pressures) followed by the overlap of the isotherms (at high surface pressure values) for DPPS and DPPG. Effects caused by dehydrodieugenol in the negatively charged lipids were distinctive, which was also reflected in the hysteresis assays, surface potential-area isotherms, and rheological measurements. Infrared spectroscopy indicated that the drug interaction with the monolayer affects not only the polar groups, but also the acyl lipid chains for all lipids. These results pointed to the fact that the interaction of the drug with lipid monolayers at the air-water interface is modulated by the lipid composition, mainly considering the polar head of the lipids, as well as the hydrophobicity of the lipids and the drug. As negatively charged lipids pointed to distinctive interaction, we believe this can be related to the antiprotozoal and anticancer properties of the compound.     

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.     

Lipid mixing is mediated by the hydrophobic surfactant protein SP-B but not by SP-C.

Pulmonary surfactant contains two families of hydrophobic proteins, SP-B and SP-C. Both proteins are thought to promote the formation of the phospholipid monolayer at the air/fluid interface of the lung. The excimer/monomer ratio of pyrene-labeled PC fluorescence intensities was used to investigate the capacity of the hydrophobic surfactant proteins, SP-B and SP-C, to induce lipid mixing between protein-containing small unilamellar vesicles and pyrene-PC-labeled small unilamellar vesicles. At 37 degrees C SP-B induced lipid mixing between protein-containing vesicles and pyrene-PC-labeled vesicles. In the presence of negatively charged phospholipids (PG or PI) the SP-B-induced lipid mixing was enhanced, and dependent on the presence of (divalent) cations. The extent of lipid mixing was maximal at a protein concentration of 0.2 mol%. SP-C was not capable of inducing lipid mixing at 37 degrees C not even at protein concentrations of 1 mol%. The SP-B-induced lipid mixing may occur during the Ca(2+)-dependent transformation of lamellar bodies into tubular myelin and the subsequent formation of the phospholipid monolayer.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.     

Penetration of Lysozyme and Cytochrome C in Lipid Bilayer: Fluorescent Study

Lysozyme and cytochrome c (CytC) are well-investigated proteins. Their specific interactions with lipid membranes, however, keep surprising secrets. Lysozyme destroys bacterial membrane; CytC binds hydrophobically to alkyl chains of the membrane lipid tails, indicating that both proteins are able to interact directly with the inner membrane components, especially with the fatty acyl chains of membrane lipids. The degrees of integration, depth of localization in the hydrophobic interior of different types of model membranes, and the type of interaction of lysozyme and CytC with surrounding lipids were investigated by fluorescent spectroscopy. Three different fluorescent markers, located at approximately 6.5, 9, and 18 Å into the lipid bilayer, were used. In addition, liposomes were designed as electrically neutral or positively or negatively charged to unravel the importance of the net electrical charge for lipid/protein interaction. CytC penetrates deeper into the lipid bilayer in comparison with lysozyme, and data are discussed in the terms of Stern–Volmer quenching of fluorescence.     

Effect of magainin, class L, and class A amphipathic peptides on fatty acid spin labels in lipid bilayers

Magainins and other antimicrobial peptides increase ion flux across the membrane. They may do this by forming some type of pore or by perturbing lipid organization due to peptide lying on the bilayer surface. In order to determine if magainins perturb the lipid sufficiently to permeabilize the bilayer, their effect on the motion of fatty acid and lipid spin labels in phosphatidylcholine/phosphatidylglycerol (PC/PG) lipid vesicles was determined. Their effect was compared to two synthetic peptides, 18L and Ac-18A-NH(2), designed to mimic the naturally occurring classes of lytic (class L) and apolipoprotein (class A) amphipathic helices, respectively. We show that although magainins and 18L both had significant effects on lipid chain order, much greater than Ac-18A-NH(2), there was no correlation between these effects and the relative ability of these three peptide classes to permeabilize PC/PG vesicles in the order magainins=Ac-18A-NH(2) > 18L. This suggests that the perturbing effects of magainins on lipid chain order at permeabilizing concentrations are not directly responsible for the increased leakage of vesicle contents. The greater ability of the magainins to permeabilize PC/PG vesicles relative to 18L is thus more likely due to formation of some type of pore by magainins. The greater ability of Ac-18A-NH(2) relative to 18L to permeabilize PC/PG vesicles despite its lack of disordering effect must be due to its ability to cause membrane fragmentation. Effects of these peptides on other lipids indicated that the mechanism by which they permeabilize lipid bilayers depends both on the peptide and on the lipid composition of the vesicles.     

Binding of human normal and multiple sclerosis-derived myelin basic protein to phospholipid vesicles: effects on membrane head group and bilayer regions

The detailed interaction of human myelin basic protein (MBP) with charged lipids may be critical in organizing the myelin sheath into its biologically functional structure. Carbon-13 and phosphorus-31 nuclear magnetic resonance spectroscopy has been used to study this interaction by examining spectral consequences of additions of MBP to membrane preparations of the negatively charged lipid phosphatidylglycerol (PG). Lipid head group 13C and 31P linewidths were found to narrow upon addition of protein, while concomitant broadening was noted for bilayer carbon resonances. At intermediate MBP/PG ratios, two components in slow exchange on the NMR time scale (bulk PG and a protein-induced PG domain) were observed for the 13C resonance of the head group carbon atom adjacent to phosphate. These results, and other spectral evidence, suggested that head groups in free PG vesicles are motionally restricted by intermolecular interactions which are disrupted by competition with MBP Lys and Arg positively charged side chains. Titration of PG with the homopolypeptide poly-L-lysine produced comparable effects on PG 13C head group spectra, indicating that electrostatic attractions constitute the primary basis of the observed interactions. Vicinal and/or geminal 13C-31P coupling constants measured from the spectra of PG head group carbons were found to be essentially invariant for free PG in dimethyl sulfoxide solution, free PG vesicles, PG vesicles + MBP, and PG vesicles + poly-L-lysine. Comparison of the spectral effects induced in PG head group resonances by normal vs multiple sclerosis-derived MBP (MS-MBP) indicated that the MS-MBP is relatively less effective in converting PG to the protein-induced domain, a result which was attributed to increased protein self-aggregation arising from the reduced net positive character of the MS protein samples.     

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.     

Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects

The antibacterial peptide PGLa exerts its activity by permeabilizing bacterial membranes whereas eukaryotic membranes are not affected. To provide insight into the selectivity and the permeabilization mechanism, the binding of PGLa to neutral and negatively charged model membranes was studied with high-sensitivity isothermal titration calorimetry (ITC), circular dichroism (CD), and solid-state deuterium nuclear magnetic resonance ((2)H NMR). The binding of PGLa to negatively charged phosphatidylcholine (PC)/phosphatidylglycerol (PG) (3:1) vesicles was by a factor of approximately 50 larger than that to neutral PC vesicles. The negatively charged membrane accumulates the cationic peptide at the lipid-water interface, thus facilitating the binding to the membrane. However, if bulk concentrations are replaced by surface concentrations, very similar binding constants are obtained for neutral and charged membranes (K approximately 800-1500 M(-)(1)). Membrane selectivity is thus caused almost exclusively by electrostatic attraction to the membrane surface and not by hydrophobic insertion. Membrane insertion is driven by an exothermic enthalpy (DeltaH approximately -11 to -15 kcal/mol) but opposed by entropy. An important contribution to the binding process is the membrane-induced random coil --> alpha-helix transition of PGLa. The peptide is random coil in solution but adopts an approximately 80% alpha-helical conformation when bound to the membrane. Helix formation is an exothermic process, contributing approximately 70% to the binding enthalpy and approximately 30% to the free energy of binding. The (2)H NMR measurements with selectively deuterated lipids revealed small structural changes in the lipid headgroups and in the hydrocarbon interior upon peptide binding which were continuous over the whole concentration range. In contrast, isothermal titration calorimetry of PGLa solutions with PC/PG(3:1) vesicles gave rise to two processes: (i) an exothermic binding of PGLa to the membrane followed by (ii) a slower endothermic process. The latter is only detected at peptide-to-lipid ratios >17 mmol/mol and is paralleled by the induction of membrane leakiness. Dye efflux measurements are consistent with the critical limit derived from ITC measurements. The endothermic process is assigned to peptide pore formation and/or lipid perturbation. The enthalpy of pore formation is 9.7 kcal/mol of peptide. If the same excess enthalpy is assigned to the lipid phase, the lipid perturbation enthalpy is 180 cal/mol of lipid. The functional synergism between PGLa and magainin 2 amide could also be followed by ITC and dye release experiments and is traced back to an enhanced pore formation activity of a peptide mixture.     

Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.

The effects of negatively charged and neutral lipids on the function of the reconstituted nicotinic acetylcholine receptor from Torpedo californica were determined with two assays using acetylcholine receptor-containing vesicles: the ion flux response and the affinity-state transition. The receptor was reconstituted into three different lipid environments, with and without neutral lipids: (1) phosphatidylcholine/phosphatidylserine; (2) phosphatidylcholine/phosphatidic acid; and (3) phosphatidylcholine/cardiolipin. Analysis of the ion flux responses showed that: (1) all three negatively charged lipid environments gave fully functional acetylcholine receptor ion channels, provided neutral lipids were added; (2) in each lipid environment, the neutral lipids tested were functionally equivalent to cholesterol; and (3) the rate of receptor desensitization depends upon the type of neutral lipid and negatively charged phospholipid reconstituted with the receptor. The functional effects of neutral and negatively charged lipids on the acetylcholine receptor are discussed in terms of protein-lipid interactions and stabilization of protein structure by lipids.