Fourier transform infrared spectroscopic study of the interactions of a strongly antimicrobial but weakly hemolytic analogue of gramicidin S with lipid micelles and lipid bilayer membranes

Cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)), a synthetic tetradecameric ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S (GS), retains the strong antimicrobial activity of GS but is 15-20 times less hemolytic. To characterize its interaction with lipid membranes and to understand the molecular basis of its capacity to lyse bacterial cells, in preference to erythrocytes, we have investigated the interactions of GS14dK(4) with detergent micelles and with lipid bilayer model membranes by Fourier transform infrared spectroscopy and compared our results with those of a similar study of GS [Lewis, R. N. A. H., et al. (1999) Biochemistry 38, 15193-15203]. In both aqueous and organic solvent solutions, GS14dK(4) adopts a beta-sheet conformation that is somewhat distorted and more sensitive to the polarity of its environment than GS. Like GS, GS14dK(4) is completely or partially excluded from gel-state lipid bilayers but interacts strongly with liquid-crystalline lipid bilayers and detergent micelle, and interacts more strongly with more fluid liquid-crystalline lipid systems. However, its interactions are more strongly influenced by membrane lipid order and fluidity, and unlike GS, it is essentially excluded from cholesterol-containing phospholipid bilayers. Also, GS14dK(4) is excluded from cationic lipid bilayers, but partitions more strongly and/or penetrates more deeply into anionic lipid bilayers than into those composed of either zwitterionic or nonionic lipids. Anionic lipids also facilitate GS14dK(4) interactions with multicomponent lipid bilayers which are predominantly zwitterionic or nonionic. Although GS14dK(4) generally penetrates and/or partitions into zwitterionic or uncharged lipid bilayers less strongly than does GS, its greater size and altered distribution of positive charges make it intrinsically more perturbing with regard to membrane organization once associated with lipid bilayers. This fact, combined with its relatively strong interactions with anionic phospholipids, may explain why GS14dK(4) retains relatively high antimicrobial activity. However, its low hemolytic activity is probably largely attributable to its low propensity to penetrate and/or partition into cholesterol-containing zwitterionic lipid membranes.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic beta-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK(4) to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK(4) to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK(4) to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK(4) varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK(4) can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK(4) binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes

The binding of the amphiphilic, positively charged, cyclic beta-sheet antimicrobial decapeptide gramicidin S (GS) to various lipid bilayer model membrane systems was studied by isothermal titration calorimetry. Large unilamellar vesicles composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoylphosphatidylcholine or the anionic phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, or a binary mixture of the two, with or without cholesterol, were used to mimic the lipid compositions of the outer monolayers of the lipid bilayers of mammalian and bacterial membranes, respectively. Dynamic light scattering results suggest the absence of major alterations in vesicle size or appreciable vesicle fusion upon the binding of GS to the lipid vesicles under our experimental conditions. The binding isotherms can be reasonably well described by a one-site binding model. GS is found to bind with higher affinity to anionic phosphatidylglycerol than to zwitterionic phosphatidylcholine vesicles, indicating that electrostatic interactions in the former system facilitate peptide binding. However, the presence of cholesterol reduced binding only slightly, indicating that the binding of GS is not highly sensitive to the order of the phospholipid bilayer system. Similarly, the measured positive endothermic binding enthalpy (DeltaH) varies only modestly (2.6 to 4.4 kcal/mol), and the negative free energy of binding (DeltaG) also remains relatively constant (-10.9 to -12.1 kcal/mol). The relatively large but invariant positive binding entropy, reflected in relatively large TDeltaS values (13.4 to 16.4 kcal/mol), indicates that GS binding to phospholipid bilayers is primarily entropy driven. Finally, the relative binding affinities of GS for various phospholipid vesicles correlate relatively well with the relative lipid specificity for GS interactions with bacterial and erythrocyte membranes observed in vivo.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)+cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS)+cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAPmss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from +4 to +5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an alpha-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Interaction of phosphatidylserine synthase from E. coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies

The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy. The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films. Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (75:25, mol/mol). Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy. PS synthase exhibits a biphasic interaction with the bilayers. The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules. The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface. Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs. The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E. coli membranes.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and 31P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.     

A charge-modified general amber force field for phospholipids: improved structural properties in the tensionless ensemble

Accurately predicting the structural properties of phospholipid with a fully atomistic molecular model is critical for the study of pure phospholipid bilayers, mixed bilayer systems and bilayers containing proteins. The general amber force field (GAFF) has traditionally required the presence of a surface tension parameter to correctly model phospholipid bilayer properties such as area per lipid and order parameters. In this work, the GAFF partial charges for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphochiline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were re-parameterised utilising high-level ab initio calculations and the restrained electrostatic potential method. Simulations of pure POPA, POPC and POPG bilayers using the charge-modified GAFF and no applied surface tension are compared with available experimental data, the original GAFF model and the recent Lipid14 variant. The results indicate a significant improvement in the accuracy of the lipid model for reproducing experimental observables without the need for a surface tension parameter. The successful application of modifying the lipid charge distributions represents an alternative to the use of a surface tension parameter within GAFF, and highlights the importance of the partial charge calculations when modelling lipid bilayers.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 degrees C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 degrees C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Phospholipid Composition of Membranes Directs Prions Down Alternative Aggregation Pathways

Prion diseases are neurodegenerative disorders of the central nervous system that are associated with the misfolding of the prion protein (PrP). PrP is glycosylphosphatidylinositol-anchored, and therefore the hydrophobic membrane environment may influence the process of prion conversion. This study investigates how the morphology and mechanism of growth of prion aggregates on membranes are influenced by lipid composition. Atomic force microscopy is used to image the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS). Circular dichroism shows that PrP interactions with POPS membranes result in an increase in β-sheet structure, whereas interactions with POPC do not influence PrP structure. Prion aggregation is observed on both zwitterionic and anionic membranes, and the morphology of the aggregates formed is dependent on the anionic phospholipid content of the membrane. The aggregates that form on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger aggregates with a distinctive sponge-like morphology that are disruptive to membranes. These data provide detailed information on the aggregation mechanism of PrP on membranes, which can be described by classic models of growth.     

Peptide-lipid interactions of the beta-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a beta-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. (31)P and (2)H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other beta-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

Kinetics and thermodynamics of annexin A1 binding to solid-supported membranes: a QCM study

By means of the quartz crystal microbalance (QCM) technique, the interaction of annexin A1 with lipid membranes was quantified using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of a first octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer obtained from vesicle fusion. This experimental setup enabled us to determine for the first time rate constants and affinity constants of annexin A1 binding to phosphatidylserine-containing layers as a function of the calcium ion concentration in solution and the cholesterol content within the outer leaflet of the solid-supported bilayer. The results reveal that a decrease in Ca(2+) concentration from 1 mM to 100 microM significantly increases the rate of annexin A1 binding to the membrane independent of the cholesterol content. However, the presence of cholesterol in the membrane altered the affinity constants considerably. While the association constant decreases with decreasing Ca(2+) concentration in the case of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) membranes lacking cholesterol, it remains high in the presence of cholesterol.     

Peptide-lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. ³¹P and ²H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS) + cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAP^mss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

Peptide binding to lipid bilayers. Binding isotherms and zeta-potential of a cyclic somatostatin analogue

The binding of the cyclic somatostatin analogue SMS 201-995, (+)-D-Phe1-Cys2-Phe3-D-Trp4-(+)-Lys5-Thr6- Cys7-Thr(ol)8, to neutral and negatively charged lipids was investigated with a centrifugation assay and with electrophoretic and monolayer methods. Monolayers and bilayers were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), either in pure form or in a 75/25 (mol/mol) mixture. The expansion of monolayer films demonstrated the intercalation of the peptide between the lipid molecules with a surface area requirement of 135 A2 per peptide molecule, indicating a parallel alignment of the peptide long axis with the membrane surface. Above a limiting pressure of 32.5 mN/m for POPC and 38.5 mN/m for POPG, peptide penetration was no longer possible. The peptide binding isotherm could be measured for mixed POPC/POPG bilayers up to a peptide concentration of 0.5 mM. Due to electrostatic attraction, binding between the positively charged peptide and the negatively charged membrane surface was enhanced as compared to the binding to a neutral membrane. After correction for electrostatic effects by means of the Gouy-Chapman theory, the binding isotherm as well as the electrophoretic zeta-potential measurement could be described by the same partition equilibrium with a surface partition constant of Kp = 36 +/- 4 M-1 (at 0.1 M NaCl). About 60-70% of SMS 201-995 is probably embedded in the headgroup region with little penetration into the lipid core. The partition constant increases with increasing salt concentration or with decreasing lipid lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.