Headgroup Order Research Articles

Charged Small Molecule Binding to Membranes in MD Simulations Evaluated against NMR Experiments.

Interactions of charged molecules with biomembranes regulate many of their biological activities, but their binding affinities to lipid bilayers are difficult to measure experimentally and model theoretically. Classical molecular dynamics (MD) simulations have the potential to capture the complex interactions determining how charged biomolecules interact with membranes, but systematic overbinding of sodium and calcium cations in standard MD simulations raises the question of how accurately force fields capture the interactions between lipid membranes and charged biomolecules. Here, we evaluate the binding of positively charged small molecules, etidocaine, and tetraphenylphosphonium to a phosphatidylcholine (POPC) lipid bilayer using the changes in lipid head-group order parameters. We observed that these molecules behave oppositely to calcium and sodium ions when binding to membranes: (i) their binding affinities are not overestimated by standard force field parameters, (ii) implicit inclusion of electronic polarizability increases their binding affinity, and (iii) they penetrate into the hydrophobic membrane core. Our results can be explained by distinct binding mechanisms of charged small molecules with hydrophobic moieties and monoatomic ions. The binding of the former is driven by hydrophobic effects, while the latter has direct electrostatic interactions with lipids. In addition to elucidating how different kinds of charged biomolecules bind to membranes, we deliver tools for further development of MD simulation parameters and methodology.

Ultrafast Dynamics at Lipid-Water Interfaces.

Lipid membranes are more than just barriers between cell compartments; they provide molecular environments with a finely tuned balance between hydrophilic and hydrophobic interactions that enable proteins to dynamically fold and self-assemble to regulate biological function. Characterizing dynamics at the lipid-water interface is essential to understanding molecular complexities from the thermodynamics of liquid-liquid phase separation down to picosecond-scale reorganization of interfacial hydrogen-bond networks.Ultrafast vibrational spectroscopy, including two-dimensional infrared (2D IR) and vibrational sum-frequency generation (VSFG) spectroscopies, is a powerful tool to examine picosecond interfacial dynamics. Two-dimensional IR spectroscopy provides a bond-centered view of dynamics with subpicosecond time resolutions, as vibrational frequencies are highly sensitive to the local environment. Recently, 2D IR spectroscopy has been applied to carbonyl and phosphate vibrations intrinsically located at the lipid-water interface. Interface-specific VSFG spectroscopy probes the water vibrational modes directly, accessing H-bond strength and water organization at lipid headgroup positions. Signals in VSFG arise from the interfacial dipole contributions, directly probing headgroup ordering and water orientation to provide a structural view of the interface.In this Account we discuss novel applications of ultrafast spectroscopy to lipid membranes, a field that has experienced significant growth over the past decade. In particular, ultrafast experiments now offer a molecular perspective on increasingly complex membranes. The powerful combination of ultrafast, interface-selective spectroscopy and simulations opens up new routes to understanding multicomponent membranes and their function. This Account highlights key prevailing views that have emerged from recent experiments: (1) Water dynamics at the lipid-water interface are slow compared to those of bulk water as a result of disrupted H-bond networks near the headgroups. (2) Peptides, ions, osmolytes, and cosolvents perturb interfacial dynamics, indicating that dynamics at the interface are affected by bulk solvent dynamics and vice versa. (3) The interfacial environment is generally dictated by the headgroup structure and orientation, but hydrophobic interactions within the acyl chains also modulate interfacial dynamics. Ultrafast spectroscopy has been essential to characterizing the biophysical chemistry of the lipid-water interface; however, challenges remain in interpreting congested spectra as well as designing appropriate model systems to capture the complexity of a membrane environment.

Improved Cation Binding to Lipid Bilayers with Negatively Charged POPS by Effective Inclusion of Electronic Polarization.

Phosphatidylserine (PS) lipids are important signaling molecules and the most common negatively charged lipids in eukaryotic membranes. The signaling can be often regulated by calcium, but its interactions with PS headgroups are not fully understood. Classical molecular dynamics (MD) simulations can potentially give detailed description of lipid-ion interactions, but the results strongly depend on the used force field. Here, we apply the electronic continuum correction (ECC) to the Amber Lipid17 parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) lipid to improve its interactions with K+, Na+, and Ca2+ ions. The partial charges of the headgroup, glycerol backbone, and carbonyls of POPS, bearing a unit negative charge, were scaled with a factor of 0.75, derived for monovalent ions, and the Lennard-Jones σ parameters of the same segments were scaled with a factor of 0.89. The resulting ECC-POPS model gives more realistic interactions with Na+ and Ca2+ cations than the original Amber Lipid17 parameters when validated using headgroup order parameters and the "electrometer concept". In ECC-lipids simulations, populations of complexes of Ca2+ cations with more than two PS lipids are negligible, and interactions of Ca2+ cations with only carboxylate groups are twice more likely than with only phosphate groups, while interactions with carbonyls almost entirely involve other groups as well. Our results pave the way for more realistic MD simulations of biomolecular systems with anionic membranes, allowing signaling processes involving PS and Ca2+ to be elucidated.

Lipid Configurations from Molecular Dynamics Simulations

The extent to which current force fields faithfully reproduce conformational properties of lipids in bilayer membranes, and whether these reflect the structural principles established for phospholipids in bilayer crystals, are central to biomembrane simulations. We determine the distribution of dihedral angles in palmitoyl-oleoyl phosphatidylcholine from molecular dynamics simulations of hydrated fluid bilayer membranes. We compare results from the widely used lipid force field of Berger et al. with those from the most recent C36 release of the CHARMM force field for lipids. Only the CHARMM force field produces the chain inequivalence with sn-1 as leading chain that is characteristic of glycerolipid packing in fluid bilayers. The exposure and high partial charge of the backbone carbonyls in Berger lipids leads to artifactual binding of Na+ ions reported in the literature. Both force fields predict coupled, near-symmetrical distributions of headgroup dihedral angles, which is compatible with models of interconverting mirror-image conformations used originally to interpret NMR order parameters. The Berger force field produces rotamer populations that correspond to the headgroup conformation found in a phosphatidylcholine lipid bilayer crystal, whereas CHARMM36 rotamer populations are closer to the more relaxed crystal conformations of phosphatidylethanolamine and glycerophosphocholine. CHARMM36 alone predicts the correct relative signs of the time-average headgroup order parameters, and reasonably reproduces the full range of NMR data from the phosphate diester to the choline methyls. There is strong motivation to seek further experimental criteria for verifying predicted conformational distributions in the choline headgroup, including the 31P chemical shift anisotropy and 14N and CD3 NMR quadrupole splittings.

Effect of Mild Heating on Human Lens Epithelial Cells: A Possible Model of Lens Aging.

This study aims to investigate the effect of mild heating on lens epithelial cells and to explore its possibility as an in vitro model for lens aging. Human lens epithelial cells (LECs) were heated at 50 °C for a cellular lens aging study. Analysis of the head group order of lens membranes was performed using Laurdan labeling. Immunofluorescence was performed to detect changes in α-crystallin expression and its cellular distribution. The chaperone-like activity of α-crystallin was also assessed. After mild heating, α-crystallin in LECs showed a tendency towards accumulation around the nucleus. The membrane head group environment of lens epithelial cells became more fluid with increasing time of exposure to mild heating, as indicated by increased water penetration. Furthermore, the chaperone activity of α-crystallin decreased, and suggests a relatively lower protective effect on other functional proteins in LECs. Thus, compared to the mild heating model based on lens tissue, this cellular model could provide a more convenient and accurate method for studying lens aging in vitro, including changes in membrane head group order in each cell, the real-time observation of crystallin distribution, and the monitoring of functional changes in the chaperone activity of crystallins as a result of aging.

Monitoring phases and phase transitions in phosphatidylethanolamine monolayers using active interfacial microrheology.

Active interfacial microrheology is a sensitive tool to detect phase transitions and headgroup order in phospholipid monolayers. The re-orientation of a magnetic nickel nanorod is used to explore changes in the surface rheology of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), which differ by two CH2 groups in their alkyl chains. Phosphatidylethanolamines such as DLPE and DMPE are a major component of cell membranes in bacteria and in the nervous system. At room temperature, DLPE has a liquid expanded (LE) phase for surface pressure, Π < ∼38 mN m(-1); DMPE has an LE phase for Π < ∼7 mN m(-1). In their respective LE phases, DLPE and DMPE show no measurable change in surface viscosity with Π, consistent with a surface viscosity <10(-9) N s m(-1), the resolution of our technique. However, there is a measurable, discontinuous change in the surface viscosity at the LE to liquid condensed (LC) transition for both DLPE and DMPE. This discontinuous change is correlated with a significant increase in the surface compressibility modulus (or isothermal two-dimensional bulk modulus). In the LC phase of DMPE there is an exponential increase in surface viscosity with Π consistent with a two-dimensional free area model. The second-order LC to solid (S) transition in DMPE is marked by an abrupt onset of surface elasticity; there is no measurable elasticity in the LC phase. A measurable surface elasticity in the S phase suggests a change in the molecular ordering or interactions of the DMPE headgroups that is not reflected in isotherms or in grazing incidence X-ray diffraction. This onset of measurable elasticity is also seen in DLPE, even though no indication of a LC-S transition is visible in the isotherms.

Test of Hofmeister-like series of anionic headgroups: clouding and micellar growth

Phenomenon of clouding in charged micellar solutions is a fairly recent addition to conventional phenomenon shown by aqueous nonionic micelles. In this paper, we have tested a Hofmeister-like ordering of charged headgroups in the context of cloud point (CP) and micellar growth. For this purpose, we have used various combinations of surfactant (sodium dodecyl sulfate, SDS; sodium dodecylbenzene sulfonate, SDBS; sodium salts of α-sulfonato myristic acid methyl ester, MES; and α-sulfonato palmitic acid methyl ester, PES) and tetra-n-butylammonium bromide (TBAB). Different surfactant concentrations and TBAB concentrations are used and CP measurements have been performed. CP values were found in the order SDBS < SDS < PES < MES for the same concentration of surfactant and TBAB. This order has been discussed in the light of water affinities of interacting ionic species (i.e., surfactant headgroup and TBA+ counterion). The ordering was found similar for the case of micellar growth studied by dynamic light scattering (DLS). A bimodal distribution of aggregate size was found that transforms to giant aggregates at CP. The micelles of roughly 10-nm size convert to aggregates of 1 μm. The study has a few novelties: (1) headgroup dependence of CP, (2) micellar growth on heating, and (3) confirmation of Hofmeister-like series of headgroup.

Quantitative Molecular Level Understanding of Ethoxysilane at Poly(dimethylsiloxane)/Polymer Interfaces

Because of the wide applications of silicone adhesives, it is important to study adhesion mechanisms of silicone elastomers to polymers. Adhesion properties are believed to be directly related to the molecular structures at the adhesive/substrate interfaces. To improve adhesion, adhesion promoters such as silanes are commonly used to modify the interfacial structures. It is difficult to study buried interfacial molecular structures between two materials in situ using conventional analytical techniques. In this study, sum frequency generation (SFG) vibrational spectroscopy was used to investigate molecular structures at buried silicone/poly(ethylene terephthalate) (PET) interfaces. Environmental-friendly epoxysilanes including (3-glycidoxypropyl)triethoxysilane (γ-GPES), (3-glycidoxypropyl)methyldiethoxysilane (γ-GPDES), and (3-glycidoxypropyl)dimethylethoxysilane (γ-GPDMES) and their mixtures with methylvinylsiloxanol (MVS) were used as adhesion promoters to modify silicone adhesion properties to PET. Various PET/silane, PET/uncured silicone, and PET/cured silicone interfaces were examined. The interfacial structures deduced from SFG spectra were correlated to adhesion testing results. It was found that silane headgroup order at the polymer interfaces is an important factor for improving adhesion. The decrease of silane headgroup order due to chemical reaction and disordering of such groups at the polymer interfaces can be associated with improved adhesion. The molecular level understanding on polymer/adhesive interfacial structures helps to design and develop adhesion promoters and polymer adhesives with improved performance.

Degradation of an Old Human Protein

Long-lived proteins exist in a number of tissues in the human body; however, little is known about the reactions involved in their degradation over time. Lens proteins, which do not turn over, provide a useful system to examine such processes. Using a combination of Western blotting and proteomic methodology, age-related changes to a major protein, γS-crystallin, were studied. By teenage years, insoluble intact γS-crystallin was detected, indicative of protein denaturation. This was not the only change, however, because blots revealed evidence of significant cross-linking as well as cleavage of γS-crystallin in all adult lenses. Cleavage at a serine residue near the C terminus was a major reaction that caused the release of a 12-residue peptide, SPAVQSFRRIVE, which bound tightly to lens cell membranes. Several other crystallin-derived peptides with double basic residues also lodged in the cell membrane fraction. Model studies showed that once cleaved from γS-crystallin, SPAVQSFRRIVE adopts a markedly different shape from that in the intact protein. Further, the acquired helical conformation may explain why the peptide seems to affect water permeability. This observation may help explain the changes to cell membranes known to be associated with aging in human lenses. Age-related cleavage of long-lived proteins may therefore yield peptides with untoward biological activity.

α- and β-Crystallins Modulate the Head Group Order of Human Lens Membranes during Aging

To examine the physical properties of human lens cell membranes as a function of age. The environment of the phospholipid head groups in fiber cell membranes from human lenses, aged 22 to 83 years, was assessed with Laurdan and two-photon confocal microscopy. The effect of mild thermal stress on head group order was studied with lens pairs in which one intact lens was incubated at 50 °C. Dihydrosphingomyelin vesicles were preloaded with Laurdan, α-, β-, or γ-crystallin was added, and surface fluidity was determined. The membrane head group environment became more fluid with age as indicated by increased water penetration. Furthermore, these changes could be replicated simply by exposing intact human lenses to mild thermal stress; conditions which decreased the concentration of soluble α- and β-crystallins. Vesicle binding experiments showed that α- and β-, but not γ-, crystallins markedly affected head group order. The physical properties of cell membranes in the lens nucleus change substantially with age, and α- and β-crystallins may modulate this effect. β-Crystallins may therefore play a role in lens cells, and cells of other tissues, apart from being simple structural proteins. Age-dependent loss of these crystallins may affect membrane integrity and contribute to the dysfunction of lenses in older people.

Fusion Peptide from Influenza Hemagglutinin Increases Membrane Surface Order: An Electron-Spin Resonance Study

A spin-labeling study of interactions of a fusion peptide from the hemagglutinin of the influenza virus, wt20, and a fusion-inactive mutant ΔG1 with dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoyl-phosphatdylcholine bilayers was performed. We found that upon binding of wt20, the ordering of headgroups and the ordering of acyl chains near the headgroup increased significantly, in a manner consistent with a cooperative phenomenon. However, changes in the order at the end of the acyl chains were negligible. The ordering effect of wt20 on the headgroup was much stronger at pH 5 than at pH 7. No effect of ΔG1 binding on the order of bilayers was evident. We also found that 1-palmitoyl-2-hydroxyl phosphatidylcholine, a membrane-fusion inhibitor, decreased the ordering of DMPC headgroups, whereas arachidonic acid, a membrane-fusion promoter, increased the ordering of DMPC headgroups. These results suggest that increases in headgroup ordering may be important for membrane fusion. We propose that upon binding of wt20, which is known to affect only the outer leaflet of the bilayer, this outer leaflet becomes more ordered, and thus more solid-like. Then the coupling between the hardened outer leaflet and the softer inner leaflet generates bending stresses in the bilayer, which tend to increase the negative curvature of the bilayer. We suggest that the increased ordering in the headgroup region enhances dipolar interactions and lowers electrostatic energy, which may provide an energy source for membrane fusion. Possible roles of bending stresses in promoting membrane fusion are discussed.

Lateral Stress Profiles In Lipid Monolayers

We have used molecular dynamics simulations to study the lateral stress profiles in lipid monolayers at the air/water interface. From the calculations, we determined the “surface of tension” in the complex interfacial layer. We identified the factors for monolayer stability, which allows explaining the maximum surface pressure sustained by a selected lipid mixture (collapse pressure). This is relevant for understanding the function of biological interfaces, such as the surfactant-covered gas exchange interface in the lungs, and designing artificial/replacement surfactant mixtures.We calculated the stress distributions for lipid monolayers of different composition under varying surface pressure, including both liquid-expanded and liquid-condensed phases. The stress distribution in the hydrocarbon chain region is most affected by the surface pressure. In the liquid-expanded phase, the stress becomes negative at the chain/air interface. In the liquid-condensed phase, the negative stress in the chains is partially compensated by positive pressure due to increased density, and the profile is characterized by multiple peaks originating from chain and head group ordering. The simulations were performed with both atomistic and coarse-grained molecular models, which led to qualitatively similar results. To test the estimated collapse pressures, the coarse-grained model was used to simulate monolayer collapse upon lateral compression. To induce 2D-3D transformations that require long time scales, small defects were introduced, which provided nucleation sites for monolayer folding.

In vivotest of the vertical phase separation hypothesis: the display of major histocompatibility complex (MHC) class I molecules on membranes of B cells from mice fed high-fat diets

The membrane vertical phase separation hypothesis predicts that a decrease in plasma membrane acyl chain order will increase major histocompatibility complex (MHC) class I surface expression. The hypothesis is based on modification of plasma membrane acyl chain order in cell culture and has not been tested in vivo. In the present study, we isolated splenic B cells from C57/BL6 mice fed either a normal diet or high-fat diets enriched in SFA or MUFA and assayed for changes in plasma membrane acyl chain order and MHC class I surface expression. Plasma membranes of B cells from MUFA-fed mice had significantly decreased acyl chain order and increased headgroup order. The decrease in acyl chain order correlated with a significant increase in the acyl chain unsaturation of B cells from the MUFA-fed mice. MHC class I surface levels on B cells were not affected by the MUFA-rich diet. This study suggests that the membrane vertical phase separation hypothesis may have limited application in a physiologically relevant setting.

Restriction of lipid motion in membranes triggered by β-sheet aggregation of the anti-apoptotic BH4 domain

The regulative BH4 domain of human Bcl-2 protein exerts its anti-apoptic activity via the mitochondrion. In the present study, we investigated the molecular interactions of this domain with negatively charged liposomes mimicking the outer mitochondrial membrane. To model the overproduction of Bcl-2 found in cancer processes, we studied the impact of elevated concentrations of its regulative BH4 segment on these mitochondrial membranes from the peptide and lipid perspective. Combined solid state (2)H-NMR and differential scanning calorimetry revealed the coexistence of small sized fluid and rigid membrane domains over a large temperature range, which is confirmed by (31)P-NMR at 30 degrees C. The latter are stabilized, in a cholesterol-like manner, by the presence of a BH4 peptide. In the same time scale, the reduction of the headgroup order is seen in the static (14)N and (31)P-NMR spectra when BH4 inserts into the bilayers. Indeed, attenuated total reflection spectroscopy indicated a dominant aggregated beta-sheet secondary structure of BH4 with a 42 degrees tilt relative to the membrane surface. These results are discussed in terms of membrane stabilization versus apoptotic mechanisms at the outer mitochondrial membrane location.

Short-Range Structure of a GM3 Ganglioside Membrane: Comparison between Experimental WAXS and Computer Simulation Results

The local structure of a GM3 ganglioside bilayer, whose wide-angle X-ray spectrum is reconstructed from molecular dynamics simulations, is found to compare quantitatively well with the experimental one. By separating inter- and intramolecular contributions, correlations between distinct head groups are shown to contribute in a substantial way to the total scattering intensity. This finding supports the hypothesis of a strong local head group order as recently formulated on the basis of calorimetry and X-ray experimental data.

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.

Molecular investigation of the interactions of trehalose with lipid bilayers of DPPC, DPPE and their mixture

Molecular dynamics simulations were performed to study structural and dynamic properties of fully hydrated pure and mixed bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) in the presence of trehalose (5 wt%). Simulations were performed for 50 ns at 350 K and 1 bar in the liquid-crystalline state of the lipid bilayers. At the concentration considered, the effect of trehalose on pure and mixed DPPC/DPPE structure are minimal, with the area per headgroup and lipid tail order parameter unchanged compared to systems without trehalose. Density profiles indicate a larger concentration of trehalose near the interface, suggesting preferential binding of trehalose with the bilayer. Hydrogen bond analysis between trehalose and the bilayers shows that the largest number of interactions occurs with DPPC lipids, whereas the fewest interactions occur in a mixed 1:1 DPPC/DPPE bilayer. The latter is a result of the inter and intramolecular binding of the amine group in DPPE, thus preventing trehalose from hydrogen bonding to the bilayer. For the pure DPPE bilayer, an excess of hydrogen-donors (amine groups) create a competitive hydrogen bonding environment that weakens lipid–lipid interactions and favors hydration of the amine groups, which enhances the binding of trehalose with the bilayer.

Effects of antidepressants on the conformation of phospholipid headgroups studied by solid-state NMR.

The effect of tricyclic antidepressants (TCA) on phospholipid bilayer structure and dynamics was studied to provide insight into the mechanism of TCA-induced intracellular accumulation of lipids (known as lipidosis). Specifically we asked if the lipid-TCA interaction was TCA or lipid specific and if such physical interactions could contribute to lipidosis. These interactions were probed in multilamellar vesicles and mechanically oriented bilayers of mixed phosphatidylcholine-phosphatidylglycerol (PC-PG) phospholipids using (31)P and (14)N solid-state NMR techniques. Changes in bilayer architecture in the presence of TCAs were observed to be dependent on the TCA's effective charge and steric constraints. The results further show that desipramine and imipramine evoke distinguishable changes on the membrane surface, particularly on the headgroup order, conformation and dynamics of phospholipids. Desipramine increases the disorder of the choline site at the phosphatidylcholine headgroup while leaving the conformation and dynamics of the phosphate region largely unchanged. Incorporation of imipramine changes both lipid headgroup conformation and dynamics. Our results suggest that a correlation between TCA-induced changes in bilayer architecture and the ability of these compounds to induce lipidosis is, however, not straightforward as imipramine was shown to induce more dramatic changes in bilayer conformation and dynamics than desipramine. The use of (14)N as a probe was instrumental in arriving at the presented conclusions.

Hydration, Structure, and Molecular Interactions in the Headgroup Region of Dioleoylphosphatidylcholine Bilayers: An Electron Spin Resonance Study

The relationship between bilayer hydration and the dynamic structure of headgroups and interbilayer water in multilamellar vesicles is investigated by electron spin resonance methods. Temperature variations of the order parameter of a headgroup spin label DPP-Tempo in DOPC in excess water and partially dehydrated (10 wt % water) show a cusp-like pattern around the main phase transition, T c. This pattern is similar to those of temperature variations of the quadrupolar splitting of interbilayer D 2O in PC and PE bilayers previously measured by 2H NMR, indicating that the ordering of the headgroup and the interbilayer water are correlated. The cusp-like pattern of these and other physical properties around T c are suggestive of quasicritical fluctuations. Also, an increase (a decrease) in ordering of DPP-Tempo is correlated with water moving out of (into) interbilayer region into (from) the bulk water phase near the freezing point, T f. Addition of cholesterol lowers T f, which remains the point of increasing headgroup ordering. Using the small water-soluble spin probe 4-PT, it is shown that the ordering of interbilayer water increases with bilayer dehydration. It is suggested that increased ordering in the interbilayer region, implying a lowering of entropy, will itself lead to further dehydration of the interbilayer region until its lowered pressure resists further flow, i.e., an osmotic phenomenon.

15N and 31P solid-state NMR investigations on the orientation of zervamicin II and alamethicin in phosphatidylcholine membranes.

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.