Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores

Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.     

MSI-78, an analogue of the magainin antimicrobial peptides,disrupts lipid bilayer structure via positive curvature strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.     

Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37

LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.     

Effect of lipids with different spontaneous curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore

The channel activity of colicin E1 was studied in planar lipid bilayers and liposomes. Colicin E1 pore-forming activity was found to depend on the curvature of the lipid bilayer, as judged by the effect on channel activity of curvature-modulating agents. In particular, the colicin-induced trans-membrane current was augmented by lysophosphatidylcholine and reduced by oleic acid, agents promoting positive and negative membrane curvature, respectively. The data obtained imply direct involvement of lipids in the formation of colicin E1-induced pore walls. It is inferred that the toroidal pore model previously validated for small antimicrobial peptides is applicable to colicin E1, a large protein that contains ten alpha-helices in its pore-forming domain.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

Evidence for membrane affinity of the C-terminal domain of bovine milk PP3 component

Component PP3 is a phosphoglycoprotein isolated from bovine milk with unknown biological function, which displays in its C-terminal region a basic amphipathic alpha-helix, a feature often involved in membrane association. According to that, the behaviour of PP3 and of a synthetic peptide from the C-terminal domain (residues 113-135) was investigated in lipid environment. Conductance measurements indicated that the peptide was able to associate and form channels in planar lipid bilayers composed of neutral or charged phospholipids. Electrostatic interactions seemed to promote voltage-dependent channel formation but this was not absolutely required since the pore-forming ability of the 113-135 C-terminal peptide was also detected with the zwitterionic lipid bilayer. Additionally, a spectroscopic study using circular dichroism argues that the peptide adopts an alpha-helical conformation in interaction with neutral or charged micelles. Thus, the conducting aggregates in bilayers might be composed of a bundle of peptides in helical conformation. Besides, similar conductance measurements performed with the whole PP3 protein did not induce any channel fluctuations. However, with the latter, an early breakdown of the bilayers occurred, a finding that can be tentatively explained by a massive incorporation of PP3. In the light of the present results, it could be inferred that PP3 membrane attachment may be achieved by oligomerization of the C-terminal amphipathic helical region.     

Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles

Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100.     

Many-body effect of antimicrobial peptides: on the correlation between lipid's spontaneous curvature and pore formation

Recently we have shown that the free energy for pore formation induced by antimicrobial peptides contains a term representing peptide-peptide interactions mediated by membrane thinning. This many-body effect gives rise to the cooperative concentration dependence of peptide activities. Here we performed oriented circular dichroism and x-ray diffraction experiments to study the lipid dependence of this many-body effect. In particular we studied the correlation between lipid's spontaneous curvature and peptide's threshold concentration for pore formation by adding phosphatidylethanolamine and lysophosphocholine to phosphocholine bilayers. Previously it was argued that this correlation exhibited by magainin and melittin supported the toroidal model for the pores. Here we found similar correlations exhibited by melittin and alamethicin. We found that the main effect of varying the spontaneous curvature of lipid is to change the degree of membrane thinning, which in turn influences the threshold concentration for pore formation. We discuss how to interpret the lipid dependence of membrane thinning.     

Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores

The membrane-lytic peptide melittin has previously been shown to form pores in lipid bilayers that have been described in terms of two different structural models. In the "barrel stave" model the bilayer remains more or less flat, with the peptides penetrating across the bilayer hydrocarbon region and aggregating to form a pore, whereas in the "toroidal pore" melittin induces defects in the bilayer such that the bilayer bends sharply inward to form a pore lined by both peptides and lipid headgroups. Here we test these models by measuring both the free energy of melittin transfer (DeltaG degrees ) and melittin-induced leakage as a function of bilayer elastic (material) properties that determine the energetics of bilayer bending, including the area compressibility modulus (K(a)), bilayer bending modulus (k(c)), and monolayer spontaneous curvature (R(o)). The addition of cholesterol to phosphatidylcholine (PC) bilayers, which increases K(a) and k(c), decreases both DeltaG degrees and the melittin-induced vesicle leakage. In contrast, the addition to PC bilayers of molecules with either positive R(o), such as lysoPC, or negative R(o), such as dioleoylglycerol, has little effect on DeltaG degrees , but produces large changes in melittin-induced leakage, from 86% for 8:2 PC/lysoPC to 18% for 8:2 PC/dioleoylglycerol. We observe linear relationships between melittin-induced leakage and both K(a) and 1/R(o)(2). However, in contrast to what would be expected for a barrel stave model, there is no correlation between observed leakage and bilayer hydrocarbon thickness. All of these results demonstrate the importance of bilayer material properties on melittin-induced leakage and indicate that the melittin-induced pores are defects in the bilayer lined in part by lipid molecules.     

Sterol-dependent membrane association of the marine sponge-derived bicyclic peptide Theonellamide A as examined by 1H NMR

Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3β-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several ¹H NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2–9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the ¹H NMR spectra of TNM-A with sodium dodecyl sulfate-d₂₅ (SDS-d₂₅) micelles and small dimyristoylphosphatidylcholine (DMPC)-d₅₄/dihexanoylphosphatidylcholine (DHPC)-d₂₂ bicelles in the presence of a paramagnetic quencher Mn²⁺. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader ¹H signals of TNM-A were observed in 10 mol % cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn²⁺ to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid–water interface (close to the C2′ portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action.     

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH₂) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Structure and mechanism of action of the antimicrobial peptide piscidin

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.     

Detergent-like properties of magainin antibiotic peptides: a 31P solid-state NMR spectroscopy study

(31)P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in (31)P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid (31)P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type (31)P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

Aggregation and porin-like channel activity of a beta sheet peptide

Beta sheet peptides (e.g., amyloid beta) are known to form ion channels in lipid bilayers possibly through aggregation, though the channel structure is not clear. We have recently reported that a short beta sheet peptide, (xSxG)(6), forms porin-like voltage-gated channels in lipid bilayers [Thundimadathil et al. (2005) Biochem. Biophys. Res. Commun. 330, 585-590]. To account for the porin-like activity, oligomerization of the peptide into a beta barrel-like structure was proposed. In this work, peptide aggregation in aqueous and membrane environments and a detailed study of channel properties were performed to gain insight into the mechanism of channel formation. The complex nature of the channel was revealed by kinetic analysis and the occurrence of interconverting multiple conductance states. Ion channels were inhibited by Congo red, suggesting that the peptide aggregates are the active channel species. Peptide aggregation and fibril formation in water were confirmed by electron microscopy (EM) and Congo red binding studies. Furthermore, oligomeric structures in association with lipid bilayers were detected. Circular dichroism of peptide-incorporated liposomes and peptide-lipid binding studies using EM suggest a lipid-induced beta sheet aggregation. Gel electrophoresis of peptide-incorporated liposomes showed dimeric and multimeric structures. Taken together, this work indicates insertion of (xSxG)(6) as oligomers into the lipid bilayer, followed by rearrangement into a beta barrel-like pore structure. A large peptide pore comprising several individual beta sheets or smaller beta sheet aggregates is expected to have a complex behavior in membranes. A dyad repeat sequence and the presence of glycine, serine, and hydrophobic residues in a repeated pattern in this peptide may be providing a favorable condition for the formation of a beta barrel-like structure in lipid bilayers.     

Effects of interfaces on aggregates of peptides derived from pancreatic islet amyloid polypeptide

Recent experimental studies indicate that oligomeric complexes of misfolded proteins and peptides are the primary agents of cytotoxicity in amyloid-related diseases. Given the prevalence of mixed-polarity interfaces in physiological environments, an understanding of the mechanisms of interactions between amorphous (pre-fibrillar) aggregates and surfaces is important for completing our knowledge of the behaviour of peptide aggregation phenomena. We have employed fully solvated molecular dynamics simulations to study the morphology, interactions and peptide conformations of disordered aggregates of the amyloidogenic NFGAIL (derived from human islet amyloid polypeptide) and non-amyloidogenic AGAIL peptides upon adsorption to vapour–water, decane–water, bilayer and solid–water interfaces. All of the interfaces studied promote elongation and surface-spreading of both peptide aggregates, with the liquid–liquid interface being particularly efficient at altering the gross morphology of disordered aggregates. NFGAIL aggregates are more effective at disrupting lipid bilayers compared to AGAIL. Additionally, the interfaces studied cause greater changes in peptide conformations within the NFGAIL aggregates compared to AGAIL. We propose that simulations may elucidate the capability of interfaces to effect changes in the behaviour of disordered peptide aggregates, which may also serve to provide measures of the intrinsic fibrillogenicity of a given peptide sequence.     

Effect of amphiphilic surfactant LDAO on the solubilization of DOPC vesicles and on the activity of Ca(2+)-ATPase reconstituted in DOPC vesicles

Solubilization of large unilamellar 1,2-dioleoylphosphatidylcholine (DOPC) vesicles by N-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was studied using turbidimetry. From turbidity data, the LDAO partition coefficient between the aqueous phase and DOPC bilayers was obtained. Using this partition coefficient, the LDAO:DOPC molar ratio in the bilayer was calculated and effects of LDAO on the bilayer stability, bilayer thickness and on the phosphohydrolase activity of sarcoplasmic reticulum Ca(2+) transporting ATPase (SERCA) reconstituted into DOPC were compared at the same LDAO:DOPC molar ratios in the bilayer. The sequence "bilayers in vesicles - bilayer fragments (flat mixed micelles) - tubular mixed micelles - globular mixed micelles" was suggested for the solubilization mechanism of DOPC vesicles from the combined turbidimetric and small-angle neutron scattering (SANS) results. The effective molecular packing parameter delta = 0.5, corresponding to the mixed bilayer - mixed tubular micelle transition, was calculated from fragmental DOPC and LDAO volumes at the molar ratio LDAO:DOPC = 2.00 in bilayers, in the middle of transition region observed earlier experimentally by small-angle neutron scattering (SANS). The bilayer thickness decrease induced by LDAO in DOPC observed by SANS did not result in the SERCA phosphohydrolase activity decrease and this indicates that some other factors compensated this bilayer effect of LDAO. The ATPase activity decrease at higher LDAO concentrations was caused by the bilayer deformation. This deformation resulted in the formation of non-bilayer aggregates in LDAO+DOPC system.     

Detergent-like properties of magainin antibiotic peptides: A P solid-state NMR spectroscopy study

³¹P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in ³¹P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid ³¹P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type ³¹P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

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

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