A biophysical study of the interactions between the antimicrobial peptide indolicidin and lipid model systems

The naturally occurring peptide indolicidin from bovine neutrophils exhibits strong biological activity against a broad spectrum of microorganisms. This is believed to arise from selective interactions with the negatively charged cytoplasmic lipid membrane found in bacteria. We have investigated the peptide interaction with supported lipid model membranes using a combination of complementary surface sensitive techniques: neutron reflectometry (NR), atomic force microscopy (AFM), and quartz crystal microbalance with dissipation monitoring (QCM-D). The data are compared with small-angle X-ray scattering (SAXS) results obtained with lipid vesicle/peptide solutions. The peptide membrane interaction is shown to be significantly concentration dependent. At low concentrations, the peptide inserts at the outer leaflet in the interface between the headgroup and tail core. Insertion of the peptide results in a slight decrease in the lipid packing order of the bilayer, although not sufficient to cause membrane thinning. By increasing the indolicidin concentration well above the physiologically relevant conditions, a deeper penetration of the peptide into the bilayer and subsequent lipid removal take place, resulting in a slight membrane thinning. The results suggest that indolicidin induces lipid removal and that mixed indolicidin-lipid patches form on top of the supported lipid bilayers. Based on the work presented using model membranes, indolicidin seems to act through the interfacial activity model rather than through the formation of stable pores.     

Model-based approaches for the determination of lipid bilayer structure from small-angle neutron and X-ray scattering data

Some of our recent work has resulted in the detailed structures of fully hydrated, fluid phase phosphatidylcholine (PC) and phosphatidylglycerol (PG) bilayers. These structures were obtained from the joint refinement of small-angle neutron and X-ray data using the scattering density profile (SDP) models developed by Ku?erka et al. (Biophys J 95:2356–2367, 2008; J Phys Chem B 116:232–239, 2012). In this review, we first discuss models for the standalone analysis of neutron or X-ray scattering data from bilayers, and assess the strengths and weaknesses inherent to these models. In particular, it is recognized that standalone data do not contain enough information to fully resolve the structure of naturally disordered fluid bilayers, and therefore may not provide a robust determination of bilayer structure parameters, including the much-sought-after area per lipid. We then discuss the development of matter density-based models (including the SDP model) that allow for the joint refinement of different contrast neutron and X-ray data, as well as the implementation of local volume conservation within the unit cell (i.e., ideal packing). Such models provide natural definitions of bilayer thicknesses (most importantly the hydrophobic and Luzzati thicknesses) in terms of Gibbs dividing surfaces, and thus allow for the robust determination of lipid areas through equivalent slab relationships between bilayer thickness and lipid volume. In the final section of this review, we discuss some of the significant findings/features pertaining to structures of PC and PG bilayers as determined from SDP model analyses.     

Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that anionic lipid membranes can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40s presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40s strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases.     

X-ray diffraction studies of lipid phase transitions in cholesterol-rich membranes at sub-zero temperatures

In X-ray diffraction studies of hydrated (greater than 60%) cholesterol/dioleoylphosphatidylcholine mixtures the lipid packing band showed an abrupt transition from liquid crystal-type to gel-type position and definition at a temperature which decreased progressively to almost -50 degrees C as the proportion of cholesterol was increased to a saturation level of about 50 mol%. Plots of transition temperature against composition (mol% cholesterol) and of peak position against composition provided evidence of a significant change in phospholipid configuration at about 20 mol% cholesterol. However, the data overall suggested a uniform dispersion of the cholesterol molecules in the phospholipid bilayer at all concentrations up to the saturation point. Parallel studies of hydrated lipid extract of erythrocyte membranes and of several cholesterol-rich membrane preparations showed a similar overall change from liquid crystal-type packing at +20 degrees C to a gel-type packing at -30 degrees C to -40 degrees C but without displaying a defined transition temperature.     

The peculiar thermo-structural behavior of the anionic lipid DMPG

Aqueous dispersions of the anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG), around 100 mM ionic strength, are known to exhibit a thermal behavior similar to that of the largely studied lipid dimyristoyl phosphatidylcholine (DMPC), which undergoes a gel to liquid crystalline phase transition at 23 degrees C, well characterized by differential scanning calorimetry (DSC), and other methods. However, at low ionic strength, DMPG has been shown to present a large gel-fluid transition region, ranging from 18 to 35 degrees C. This intermediate phase is optically transparent and characterized by a continuous change in membrane packing. Structural properties of the DMPG gel-fluid transition region will be discussed, based on results obtained by several techniques: electron spin resonance (ESR) of spin labels at the membrane surface and intercalated at different depths in the bilayer; light scattering; DSC; small angle X-ray scattering (SAXS); and fluorescence spectroscopy of probes in the bilayer.     

Mechanism of alamethicin insertion into lipid bilayers.

Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the alamethicin insertion transition.     

Hemagglutinin fusion peptide mutants in model membranes: structural properties, membrane physical properties, and PEG-mediated fusion

While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events.     

riDOM,a cell penetrating peptide. Interaction with phospholipid bilayers

Melittin is an amphipathic peptide which has received much attention as a model peptide for peptide–membrane interactions. It is however not suited as a transfection agent due to its cytolytic and toxicological effects. Retro-inverso-melittin, when covalently linked to the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (riDOM), eliminates these shortcomings. The interaction of riDOM with phospholipid membranes was investigated with circular dichroism (CD) spectroscopy, dynamic light scattering, ζ-potential measurements, and high-sensitivity isothermal titration calorimetry. riDOM forms cationic nanoparticles with a diameter of ~ 13 nm which are well soluble in water and bind with high affinity to DNA and lipid membranes. When dissolved in bilayer membranes, riDOM nanoparticles dissociate and form transient pores. riDOM-induced membrane leakiness is however much reduced compared to that of authentic melittin. The secondary structure of the ri-melittin is not changed when riDOM is transferred from water to the membrane and displays a large fraction of β-structure. The ³¹P NMR spectrum of the nanoparticle is however transformed into a typical bilayer spectrum. The Gibbs free energy of riDOM binding to bilayer membranes is − 8.0 to − 10.0 kcal/mol which corresponds to the partition energy of just one fatty acyl chain. Half of the hydrophobic surface of the riDOM lipid extension with its 2 oleic acyl chains is therefore involved in a lipid–peptide interaction. This packing arrangement guarantees a good solubility of riDOM both in the aqueous and in the membrane phase. The membrane binding enthalpy is small and riDOM binding is thus entropy-driven.     

Synchrotron X-ray study of lung surfactant-specific protein SP-B in lipid monolayers.

This work reports the first x-ray scattering measurements to determine the effects of SP-B(1-25), the N-terminus peptide of lung surfactant-specific protein SP-B, on the structure of palmitic acid (PA) monolayers. In-plane diffraction shows that the peptide fluidizes a portion of the monolayer but does not affect the packing of the residual ordered phase. This implies that the peptide resides in the disordered phase, and that the ordered phase is essentially pure lipid, in agreement with fluorescence microscopy studies. X-ray reflectivity shows that the peptide is oriented in the lipid monolayer at an angle of approximately 56 degrees relative to the interface normal, with one end protruding past the hydrophilic region into the fluid subphase and the other end embedded in the hydrophobic region of the monolayer. The quantitative insights afforded by this study lead to a better understanding of the lipid/protein interactions found in lung surfactant systems.     

Propagating Subterahertz Longitudinal Phonon-like Modes in a Lipid Bilayer in Aqueous Medium

It has been revealed recently that the subterahertz/terahertz vibrational motions in enzymes and DNA immersed in aqueous solutions can be underdamped. Importantly, these motions are associated with coherent delocalized modes that control functional processes. Analogous propagating phonon-like modes have been found in free hydrated lipid bilayers. In the present work, subterahertz (frequencies of the order of tens and hundreds of gigahertz) longitudinal acoustic oscillations in a bilayer lipid membrane immersed in aqueous medium are investigated theoretically. We consider driven oscillations excited by tangential mechanical tensions at the bilayer surfaces and thermally induced phonon modes. The analysis is based on: (i) a generalized hydrodynamic model of two-dimensional lipid bilayer in aqueous medium; (ii) known estimates of frequencies and lifetimes of longitudinal acoustic phonons in free hydrated lipid bilayer and in water, which were obtained in the experiments on non-elastic X-ray scattering and the molecular dynamics simulations. We show that the membrane phonon-like excitations are underdamped for the typical values of the system parameters, and the contribution of aqueous medium to the membrane mode damping is small compared to the contribution of the lipid bilayer. The obtained results suggest the possibility of realization of thermally induced longitudinal membrane phonons in physiological conditions, as well as the possibility of resonance amplification of the impact of subnanosecond electric impulses and impulses of subterahertz electromagnetic radiation on membrane dynamics.     

Effect of incorporation of drugs,vitamins and peptides on the structure and dynamics of lipid assemblies

The characteristics of vesicles formed from Dipalmitoyl Phosphatidyl Choline (DPPC) are sensitive to the presence of perturbing molecules such as drugs, peptides, hormones and vitamins. We have used ESR spin labeling and NMR techniques for studying interaction of such molecules with lipid bilayers. ESR spin labeling has been used to monitor thermotropic behaviour of model membranes. Different NMR probes such as¹H,³¹P,¹³C have been used to gather information regarding the mode of interaction. It has been observed that the model membrane systems respond differently depending upon the localization of the perturbing molecules in the lipid bilayer. Small molecules such as neurotransmitters epinephrine and norepinephrine decrease gel to liquid crystalline phase transition temperature significantly even when present in small amounts. Vitamine E acetate having a hydrophobic hydrocarbon tail orients parallel to the lipid molecule and thereby exhibits dynamics similar to palmitate chain. When the acetate group is replaced by hydroxyl group (-tocopherol), the phase transition becomes broad and the lipid molecules loose freedom of lateral diffusion. This can be attributed to formation of hydrogen bond between the hydroxyl group of -tocopherol and phosphate moiety of lipid. The conformation of antidepressants nitroxazepine and imipramine is significantly altered when embedded in lipid bilayer. Anaesthetic etomidate not only modifies thermotropic characteristics but also induces polymorphism. The normal bilayer arrangement of lipids gets transformed into hexagonal packing. Amino acid tryptophan induces cubic phases in the normal bilayer arrangement of DPPC dispersions. Peptide gonadoliberin shows a reduced internal motion due to the lipid peptide interaction.The major consequences of binding of lipids with externally added molecules are changes in the fluidity and permeability properties of membranes. It has been shown that permeability is effected by the presence of molecules such as propranolol, -tocopherol and its analogue, neurotransmitters, etc. The magnetic resonance methods have thus evolved as power techniques in the study of membrane structure and function.     

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Abeta(1-40) peptide in solid-supported lipid bilayers

We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.     

Lateral stress profile and fluorescent lipid probes. FRET pair of probes that introduces minimal distortions into lipid packing

The modern theory of lipid membrane structure incorporates the concept of lateral stress profile. The latter represents the forces that act on any solute inside the membrane. We used this concept to propose two lipid probes that introduce minimal distortions into the lipid bilayer packing: the surface pressure isotherms and volt-potentials of the pure and mixed (probe-containing) lipid monolayers are equal. The probes represent a FRET pair. They are applicable in lipid transfer and vesicle fusion experiments.     

The role played by lipids unsaturation upon the membrane interaction of the Helicobacter pylori HP(2–20) antimicrobial peptide analogue HPA3

The HPA3 peptide is an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein, able to interact with zwitterionic lipid membranes and generate pores. Herein we focused on the importance of the degree of unsaturation of lipid acyl chains on HPA3 peptide-membrane interactions. Electrophysiology experiments carried out in reconstituted lipid membranes formed from phosphatidylcholines with one (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine − POPC) and two monounsaturated acyl chains (1,2-dioleoyl-sn-glycero-3-phosphocholine − DOPC) demonstrate that the lesser degree of the packing density of membrane lipids encountered in DOPC-based planar membranes greatly enhances the electric activity of pores created by the HPA3 peptide. Data derived from fluorescence spectroscopy experiments demonstrate that upon interaction with the bilayer, the HPA3 peptide translocates to the trans-side of the membrane. From the same experiments, we demonstrate that in the case of DOPC-based planar membranes, the net amount of HPA3 peptide which passes across the membrane and re-dissolves in the trans solution is almost 22% greater than POPC-based membranes. Such data further emphasize the modulatory role played by lipid acyl chain in determining antimicrobial peptides-lipids interactions, and demonstrate that small differences in unsaturation degree can impose a sizeable influence on HPA3 peptide activity.     

Molecular dynamics simulation of lipid packing and mobility in bilayer membranes

A model of lipid bilayer membrane in water has been developed. Parameters have been selected that allow molecular dynamics simulation of lipid bilayers in the all-atom approximation. The calculated indices of packing and mobility of lipid molecules for the liquid crystalline state of the bilayer agree well with the experimental data. Based on the model of the liquid crystalline state of the membrane, a system in the gel-like state has been constructed. The gel-state model reproduces well the packing of lipids in real bilayers, whereas the mobility of molecules proves to be overestimated.     

Effects of topology, length, and charge on the activity of a kininogen-derived peptide on lipid membranes and bacteria

Effects of topology, length, and charge on peptide interactions with lipid bilayers was investigated for variants of the human kininogen-derived peptide HKH20 (HKHGHGHGKHKNKGKKNGKH) by ellipsometry, CD, fluorescence spectroscopy, and z-potential measurements. The peptides display primarily random coil conformation in buffer and at lipid bilayers, and their lipid interaction is dominated by electrostatics, the latter evidenced by higher peptide adsorption and resulting membrane rupture for an anionic than for a zwitterionic membrane, as well as by strongly reduced adsorption and membrane rupture at high ionic strength. At sufficiently high peptide charge density, however, electrostatic interactions contribute to reducing the peptide adsorption and membrane defect formation. Truncating HKH20 into overlapping 10 amino acid peptides resulted in essentially eliminated membrane rupture and in a reduced amount peptide charges pinned at the lipid bilayer. Finally, cyclic HKH20 was found to be less efficient than the linear peptide in causing liposome rupture, partly due to a lower adsorption. Analogous results were found regarding bactericidal effects.     

Interaction between lipid monolayers and poloxamer 188: an X-ray reflectivity and diffraction study

The mechanism by which poloxamer 188 (P188) seals a damaged cell membrane is examined using the lipid monolayer as a model system. X-ray reflectivity and grazing-incidence x-ray diffraction results show that at low nominal lipid density, P188, by physically occupying the available area and phase separating from the lipids, forces the lipid molecules to pack tightly and restore the barrier function of the membrane. Upon compression to bilayer equivalent pressure, P188 is squeezed out from the lipid monolayer, allowing a graceful exit of P188 when the membrane integrity is restored.     

Nonpolar interactions between trans-membrane helical EGF peptide and phosphatidylcholines, sphingomyelins and cholesterol. Molecular dynamics simulation studies.

A molecular dynamics simulation study of four lipid bilayers with inserted trans-membrane helical fragment of epithelial growth factor (EGF) receptor (EGF peptide) was performed. The lipid bilayers differ in their lipid composition and consist of (i) unsaturated phosphatidylcholine (palmitoyloleoylphosphatidylcholine, POPC), (ii) POPC and 20 mol% of cholesterol (Chol), (iii) sphingomyelin (SM) and 20 mol% of Chol, and (iv) SM and 50 mol% of Chol. Only 1 out of 26 residues in the EGF-peptide sequence is polar (Thr). The hydrophobic thickness of each bilayer is different but shorter than the length of the peptide and so, due to hydrophobic mismatch, the inserted peptide is tilted in each bilayer. Additionally, in the POPC bilayer, which is the thinnest, the peptide loses its helical structure in a short three-amino acid fragment. This facilitates bending of the peptide and burying all hydrophobic amino acids inside the membrane core (Figure 1(b)). Bilayer lipid composition affects interactions between the peptide and lipids in the membrane core. Chol increases packing of atoms relative to the peptide side chains, and thus increases van der Waals interactions. On average, the packing around the peptide is higher in SM-based bilayers than POPC-based bilayers but for certain amino acids, packing depends on their position relative to the bilayer center. In the bilayer center, packing is higher in POPC-based bilayers, while in regions closer to the interface packing is higher in SM-based bilayers. In general, amino acids with larger side chains interact strongly with lipids, and thus the peptide sequence is important for the pattern of interactions at different membrane depths. This pattern closely resembles the shape of recently published lateral pressure profiles [Ollila et alJ. Struct. Biol. DOI:10.1016/j.jsb.2007.01.012].     

Molecular dynamics simulations of local anesthetic articaine in a lipid bilayer

In order to investigate structural and dynamical properties of local anesthetic articaine in a model lipid bilayer, a series of molecular dynamics simulations have been performed. Simulations were carried out for neutral and charged (protonated) forms of articaine inserted in fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer. For comparison purpose, a fully hydrated DMPC bilayer without articaine was also simulated. The length of each simulation was 200 ns. Various properties of the lipid bilayer systems in the presence of both charged and uncharged forms of articaine taken at two different concentrations have been examined: membrane area per lipid, mass density distributions, order parameters, radial distribution functions, head group tilt, diffusion coefficients, electrostatic potential, etc, and compared with results of previous simulations of DMPC bilayer in the presence of lidocaine. It was shown that addition of both charged and neutral forms of articaine causes increase of the dipole electrostatic potential in the membrane interior.     

An AFM study of solid-phase bilayers of unsaturated PC lipids and the lateral distribution of the transmembrane model peptide WALP23 in these bilayers

An altered lipid packing can have a large influence on the properties of the membrane and the lateral distribution of proteins and/or peptides that are associated with the bilayer. Here, it is shown by contact-mode atomic force microscopy that the surface topography of solid-phase bilayers of PC lipids with an unsaturated cis bond in their acyl chains shows surfaces with a large number of line-type packing defects, in contrast to the much smoother surfaces observed for saturated PC lipids. Di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC (POPC) were used. Next, the influence of an altered lipid environment on the lateral distribution of the single α-helical model peptide WALP23 was studied by incorporating the peptide in the bilayers of di-n:1-PC (n = 20, 22, 24) and (16:0,18:1)-PC unsaturated lipids. The presence of WALP23 leads to an increase in the number of packing defects but does not lead to the formation of the striated domains that were previously observed in bilayers of saturated PC lipids and WALP. This is ascribed to the less efficient lateral lipid packing of the unsaturated lipids, while the increase in packing defects is probably an indirect effect of the peptide. Finally, the fact that an altered lipid packing affects the distribution of WALP23 is also confirmed in an additional experiment where the solvent TFE (2,2,2-trifluorethanol) is added to bilayers of di-16:0-PC/WALP23. At 3.5 vol% TFE, the previous striated ordering of the peptide is abolished and replaced by loose lines.