Structural rearrangement of model membranes by the peptide antibiotic NK-2

We have developed a novel α-helical peptide antibiotic termed NK-2. It efficiently kills bacteria, but not human cells, by membrane destruction. This selectivity could be attributed to the different membrane lipid compositions of the target cells. To understand the mechanisms of selectivity and membrane destruction, we investigated the influence of NK-2 on the supramolecular aggregate structure, the phase transition behavior, the acyl chain fluidity, and the surface charges of phospholipids representative for the bacterial and the human cell cytoplasmic membranes. The cationic NK-2 binds to anionic phosphatidylglycerol liposomes, causing a thinning of the membrane and an increase in the phase transition temperature. However, this interaction is not solely of electrostatic but also of hydrophobic nature, indicated by an overcompensation of the Zeta potential. Whereas NK-2 has no effect on phosphatidylcholine liposomes, it enhances the fluidity of phosphatidylethanolamine acyl chains and lowers the phase transition enthalpy of the gel to liquid cristalline transition. The most dramatic effect, however, was observed for the lamellar/inverted hexagonal transition of phosphatidylethanolamine which was reduced by more than 10 °C. Thus, NK-2 promotes a negative membrane curvature which can lead to the collapse of the phosphatidylethanolamine-rich bacterial cytoplasmic membrane.     

Lipid-controlled peptide topology and interactions in bilayers: structural insights into the synergistic enhancement of the antimicrobial activities of PGLa and magainin 2

To gain further insight into the antimicrobial activities of cationic linear peptides, we investigated the topology of each of two peptides, PGLa and magainin 2, in oriented phospholipid bilayers in the presence and absence of the other peptide and as a function of the membrane lipid composition. Whereas proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that magainin 2 exhibits stable in-plane alignments under all conditions investigated, PGLa adopts a number of different membrane topologies with considerable variations in tilt angle. Hydrophobic thickness is an important parameter that modulates the alignment of PGLa. In equimolar mixtures of PGLa and magainin 2, the former adopts transmembrane orientations in dimyristoyl-, but not 1-palmitoyl-2-oleoyl-, phospholipid bilayers, whereas magainin 2 remains associated with the surface in all cases. These results have important consequences for the mechanistic models explaining synergistic activities of the peptide mixtures and will be discussed. The ensemble of data suggests that the thinning of the dimyristoyl membranes caused by magainin 2 tips the topological equilibrium of PGLa toward a membrane-inserted configuration. Therefore, lipid-mediated interactions play a fundamental role in determining the topology of membrane peptides and proteins and thereby, possibly, in regulating their activities as well.     

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.     

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, δ-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be ‘special cases’ within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Revisiting the bilayer structures of fluid phase phosphatidylglycerol lipids: Accounting for exchangeable hydrogens

We recently published two papers detailing the structures of fluid phase phosphatidylglycerol (PG) lipid bilayers (Ku?erka et al., 2012 J. Phys. Chem. B 116: 232–239; Pan et al., 2012 Biochim. Biophys. Acta Biomembr. 1818: 2135–2148), which were determined using the scattering density profile model. This hybrid experimental/computational technique utilizes molecular dynamics simulations to parse a lipid bilayer into components whose volume probabilities follow simple analytical functional forms. Given the appropriate scattering densities, these volume probabilities are then translated into neutron scattering length density (NSLD) and electron density (ED) profiles, which are used to jointly refine experimentally obtained small angle neutron and X-ray scattering data. However, accurate NSLD and ED profiles can only be obtained if the bilayer's chemical composition is known. Specifically, in the case of neutron scattering, the lipid's exchangeable hydrogens with aqueous D₂O must be accounted for, as they can have a measureable effect on the resultant lipid bilayer structures. This was not done in our above-mentioned papers. Here we report on the molecular structures of PG lipid bilayers by appropriately taking into account the exchangeable hydrogens. Analysis indicates that the temperature-averaged PG lipid areas decrease by 1.5 to 3.8 Å², depending on the lipid's acyl chain length and unsaturation, compared to PG areas when hydrogen exchange was not taken into account.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

Lipid Gymnastics: Evidence of Complete Acyl Chain Reversal in Oxidized Phospholipids from Molecular Simulations

In oxidative environments, biomembranes contain oxidized lipids with short, polar acyl chains. Two stable lipid oxidation products are PoxnoPC and PazePC. PoxnoPC has a carbonyl group, and PazePC has an anionic carboxyl group pendant at the end of the short, oxidized acyl chain. We have used MD simulations to explore the possibility of complete chain reversal in OXPLs in POPC-OXPL mixtures. The polar AZ chain of PazePC undergoes chain reversal without compromising the lipid bilayer integrity at concentrations up to 25% OXPL, and the carboxyl group points into the aqueous phase. Counterintuitively, the perturbation of overall membrane structural and dynamic properties is stronger for PoxnoPC than for PazePC. This is because of the overall condensing and ordering effect of sodium ions bound strongly to the lipids in the PazePC simulations. The reorientation of AZ chain is similar for two different lipid force fields. This work provides the first molecular evidence of the “extended lipid conformation” in phospholipid membranes. The chain reversal of PazePC lipids decorates the membrane interface with reactive, negatively charged functional groups. Such chain reversal is likely to exert a profound influence on the structure and dynamics of biological membranes, and on membrane-associated biological processes.     

Hydrophobic thickness, lipid surface area and polar region hydration in monounsaturated diacylphosphatidylcholine bilayers: SANS study of effects of cholesterol and β-sitosterol in unilamellar vesicles

The influence of a mammalian sterol cholesterol and a plant sterol β-sitosterol on the structural parameters and hydration of bilayers in unilamellar vesicles made of monounsaturated diacylphosphatidylcholines (diCn:1PC, n = 14-22 is the even number of acyl chain carbons) was studied at 30 °C using small-angle neutron scattering (SANS). Recently published advanced model of lipid bilayer as a three-strip structure was used with a triangular shape of polar head group probability distribution (Ku?erka et al., Models to analyze small-angle neutron scattering from unilamellar lipid vesicles, Physical Review E 69 (2004) Art. No. 051903). It was found that 33 mol% of both sterols increased the thickness of diCn:1PC bilayers with n = 18-22 similarly. β-sitosterol increased the thickness of diC14:1PC and diC16:1PC bilayers a little more than cholesterol. Both sterols increased the surface area per unit cell by cca 12 Å² and the number of water molecules located in the head group region by cca 4 molecules, irrespective to the acyl chain length of diCn:1PC. The structural difference in the side chain between cholesterol and β-sitosterol plays a negligible role in influencing the structural parameters of bilayers studied.     

Membrane Protein Frustration: Protein Incorporation into Hydrophobic Mismatched Binary Lipid Mixtures

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

Component volumes of unsaturated phosphatidylcholines in fluid bilayers: a densitometric study

The specific volumes of six 1,2-diacylphosphatidylcholines with monounsaturated acyl chains (diCn:1PC, n=14-24 is the even number of acyl chain carbons) in fluid bilayers in multilamellar vesicles dispersed in H(2)O were determined by the vibrating tube densitometry as a function of temperature. From the data obtained with diCn:1PC (n=14-22) vesicles in combination with the densitometric data from Tristram-Nagle et al. [Tristram-Nagle, S., Petrache, H.I., Nagle, J.F., 1998. Structure and interactions of fully hydrated dioleoylphosphatidylcholine bilayers. Biophys. J. 75, 917-925.] and Koenig and Gawrisch [Koenig, B.W., Gawrisch, K., 2005. Specific volumes of unsaturated phosphatidylcholines in the liquid crystalline lamellar phase. Biochim. Biophys. Acta 1715, 65-70.], the component volumes of phosphatidylcholines in fully hydrated fluid bilayers at 30 degrees C were obtained. The volume of the acyl chain CH and CH(2) group is V(CH)=22.30 A(3) and V(CH2) =A(3), respectively. The volume of the headgroup including the glyceryl and acyl carbonyls, V(H), and the ratio of acyl chain methyl and methylene group volumes, r=V(CH3):V(CH2) are linearly interdependent: V(H)=a-br, where a=434.41 A(3) and b=-55.36 A(3) at 30 degrees C. From the temperature dependencies of component volumes, their isobaric thermal expansivities (alpha(X)=V(X)(-1)(partial differential V(X)/ partial differential T) where X=CH(2), CH, or H were calculated: alpha(CH2)=118.4x10(-5)K(-1), alpha(CH)=71.0x10(-5)K(-1), alpha(H)=7.9x10(-5)K(-1) (for r=2) and alpha(H)=9.6x10(-5)K(-1) (for r=1.9). The specific volume of diC24:1PC changes at the main gel-fluid phase transition temperature, t(m)=26.7 degrees C, by 0.0621 ml/g, its specific volume is 0.9561 and 1.02634 ml/g at 20 and 30 degrees C, respectively, and its isobaric thermal expansivity alpha=68.7x10(-5) and 109.2x10(-5)K(-1) below and above t(m), respectively. The component volumes and thermal expansivities obtained can be used for the interpretation of X-ray and neutron scattering and diffraction experiments and for the guiding and testing molecular dynamics simulations of phosphatidylcholine bilayers in the fluid state.     

Lipid dynamics in fast-tumbling bicelles with varying bilayer thickness: Effect of model transmembrane peptides

The morphology of q = 0.5 fast-tumbling bicelles prepared with three different acyl chain lengths has been investigated by NMR. It is shown that bicelles prepared with DLPC (12 C) and DHPC are on average larger than those containing DMPC or DPPC (14 and 16 C) and DHPC, which may be due to a higher degree of mixing between DLPC and DHPC. The fast internal mobility of the lipids was determined from natural abundance carbon-13 relaxation. A similar dynamical behaviour of the phospholipids in the three different bicelles was observed, although the DPPC lipid acyl chain displayed a somewhat lower degree of mobility, as evidenced by higher generalized order parameters throughout the acyl chain. Carbon-13 relaxation was also used to determine the effect of different model transmembrane peptides, with flanking Lys residues, on the lipid dynamics in the three different bicelles. All peptides had the effect of increasing the order parameters for the DLPC lipid, while no effect was observed on the longer lipid chains. This effect may be explained by a mismatch between the hydrophobic length of the peptides and the DLPC lipid acyl chain.     

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm^-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm^-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.     

Subcellular localization and lysophospholipase/transacylation activities of human group IVC phospholipase A2 (cPLA2γ)

cPLA2γ was identified as an ortholog of cPLA2α, which is a key enzyme in eicosanoid production. cPLA2γ was reported to be located in endoplasmic reticulum (ER) and mitochondria and to have lysophospholipase activity beside phospholipase A2 (PLA2) activity. However, subcellular localization, mechanism of membrane binding, regulation and physiological function have not been fully established. In the present study, we examined the subcellular localization and enzymatic properties of cPLA2γ with C-terminal FLAG-tag. We found that cPLA2γ was located not only in ER but also mitochondria even in the absence of the prenylation. Purified recombinant cPLA2γ catalyzed an acyltransferase reaction from one molecule of lysophosphatidylcholine (LPC) to another, forming phosphatidylcholine (PC). LPC or lysophosphatidylethanolamine acted as acyl donor and acceptor, but lysophosphatidylserine, lysophosphatidylinositol and lysophosphatidic acid (LPA) did not. PC and phosphatidylethanolamine (PE) also acted as weak acyl donors. Reaction conditions changed the balance of lysophospholipase and transacylation activities, with addition of LPA/PA, pH > 8, and elevated temperature markedly increasing transacylation activity; this suggests that lysophospholipase/transacylation activities of cPLA2γ may be regulated by various factors. As lysophospholipids are known to accumulate in ischemia heart and to induce arryhthmia, the cPLA2γ that is abundant in heart may have a protective role through clearance of lysophospholipids by its transacylation activity.     

Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.     

Oxidized phospholipids as potential molecular targets for antimicrobial peptides

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.     

N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes

N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel → liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Membrane association and activity of 15/16-membered peptide antibiotics: Zervamicin IIB, ampullosporin A and antiamoebin I

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K⁺ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER > AMP >> ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC_16:1PC, diC_18:1PC, C_16:0/C_18:1PC, diC_20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.