Contribution of aqueous interphases to the permeability barrier of lipid bilayers for non-electrolytes

Equilibrium dialysis experiments are used to measure excluded volumes for the non-electrolyte permeant [U-¹⁴C] erythritol in lipid bilayer systems. The data indicate amounts of water associated with the lipid membranes which correspond with amounts calculated from calorimetric measurements.The membrane systems can be described as composite elements consisting of the lipid bilayers and adjacent water layers on both sides. The finding that the permeant is excluded indicates that the water layers contribute to the permeability barrier.The mean thickness of the water layers is about 6 Å for planar bilayers in multilayered liposomes and 10 Å for curved bilayers in sonicated vesicles. Next to the difference in thickness of the water layers differences in interfacial adsorption between the two systems are apparent.     

Constant surface tension molecular dynamics simulations of lipid bilayers with trehalose

Surface areas and fluctuations evaluated from 50 ns molecular dynamics simulations of fully hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers in a 1:2 trehalose:lipid ratio carried out at surface tensions 10, 17 and 25 dyn/cm/leaflet are compared with those of pure bilayers under the same conditions. Trehalose increases the surface area, as consistent with the surface tension lowering observed in simulations at constant area. The system bulk elastic modulus K _b  = 1.5 ± 0.3 × 10¹⁰ dyn/cm². It is independent of bilayer surface area and trehalose content within statistical error. In contrast, the area elastic modulus K _a shows a strong area dependence. At 64 Å²/lipid (the experimental surface area), K _a  = 138 ± 26 dyn/cm for a pure DPPC bilayer and 82 ± 10 dyn/cm for one with trehalose; i.e. trehalose increases fluidity of the bilayer surface at this area per lipid.     

Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis(phosphate) in water

The molecular organization of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis-(phosphate)/water systems is investigated over a wide range of lipid concentrations using X-ray diffraction, calorimetry, analytical ultracentrifugation, densitometry and viscometry.At high lipid concentrations, the lipid molecules are found to form a lamellar phase. The repeat distance increases from 60 to 120 Å with increasing water content to 70 wt% and the surface area per lipid molecule increases from 41.7 Ų to a limiting value of 100 Ų.On the other hand, at very low lipid concentrations the molecules are found to form not vesicles but micelles, the total molecular weight of which takes a value of 93 000.This finding revises the prevalent view that lipids containing two (or more) hydrocarbon chains form extended bilayers or vesicles, whereas single chained lipids form micelles. (Tanford, C.(1972) J. Phys. Chem. 76, 3020–3024).     

Preparation and comparison of model bilayer systems from chloroplasts thylakoid membrane lipids for 13C-NMR studies

Model bilayer systems from individual purified chloroplast thylakoid membrane lipids, from reconstituted mixtures of these purified lipids, and from leaf total polar lipid extracts have been prepared in water, and the longitudinal relaxation times (T's₁) of the individual carbon atoms of the fatty acyl chains measured by ¹³C-NMR spectroscopy. The T's₁ increasing distance of the carbon atoms from the polar headgroups in all cases, and as the results from each of the preparations are similar, all can be used as models of chloroplast membrane bilayers. Relaxation time measurements on intact chloroplast thylakoid membranes indicate the presence of chlorophyll resonances in the ¹³C-NMR spectrum of the membrane.     

Hydroperoxide and carboxyl groups preferential location in oxidized biomembranes experimentally determined by small angle X-ray scattering: Implications in membrane structure

We report small angle X-ray scattering (SAXS) data from large unilamellar vesicles as model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) and two oxidized species, namely its hydroperoxidized form POPC-OOH and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) lipid that has a carboxyl group at the end of its truncated sn-2 chain. The replacement of POPC by either POPC-OOH (POPC-OOH_xPOPC_1−x) or PazePC (PazePC_xPOPC_1−x), with oxidized lipid molar ratio x varying from 0.00 up to 1.00, permits to experimentally inspect changes in the membrane structural properties due to oxidation. The volume fraction distribution of each lipid chemical group along the bilayer is determined. The results quantify that 95% of the hydroperoxide group lies in the membrane polar moiety, near the carbonyl and phosphate groups, whereas just 5% of OOH group experiences the polar/apolar interface, for all values of x studied. In the case of PazePC up to x = 0.33, a bimodal distribution of the carboxyl group in the interior and polar regions of the lipid membrane is obtained, probably due to a dynamic movement of the shortened alkyl chain towards the water interface. The mean molecular area A gradually increases from 65.4 ± 0.4 Ų for POPC bilayers to 78 ± 2 Ų for pure POPC-OOH bilayers, whereas POPC-OOH membrane thickness resulted to be 20% thinner than the non-oxidized POPC membrane. For PazePC up to x = 0.33, A increases to 67 ± 2 Ų with 10% of membrane thinning. The SAXS results thus demonstrate how the lipid oxidation progress affects the membrane structural features, thus paving the way to better understand membrane damage under oxidative stress.     

Molecular dynamics simulations of palmitoyloleoylphosphatidylglycerol bilayers

Phosphatidylglycerol (PG) is one of the important components of biological membranes, but there is a paucity of experimental data to test the accuracy of molecular dynamics (MD) simulations. This work consists of testing the accuracy of the CHARMM36 (C36) lipid force field on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipid bilayers. MD simulations of POPG lipid bilayers are compared to recently available X-ray and neutron scattering and deuterium NMR measurements. Overall, the C36 lipid force field accurately represents the X-ray and neutron form factors, bilayer and hydrocarbon thicknesses and chain deuterium order parameters. The surface area per lipid from MD simulations with C36 (67.7 ± 0.2 Å²) is in excellent agreement with the experimentally determined value of 66.0 ± 1.3 Å². C36 outperforms the lipid force field developed by Berger et al. [15] and suggests that past studies with this force field may result in lateral areas that are too small. Moreover, our studies give some insight into the structural model used in experiments and suggest that the functional form for the head group may not be Gaussian-like. Based on our simulations, the POPG lipid in the C36 lipid force field is well parameterised and can be used for other PG lipids and membrane models with mixed lipids.     

The structure of lipid bilayers and the effects of general anaesthetics: An X-ray and neutron diffraction study

We have looked for the effects of three clinically used inhalational anaesthetics (nitrous oxide, halothane and cyclopropane) on the structure of lecithin/ cholesterol bilayers. The anaesthetics were delivered to the membranes in the gaseous phase, so that effects at clinical concentrations could be determined.High-resolution X-ray diffraction patterns were recorded out to 4 Å and analyzed using swelling experiments. Parallel neutron diffraction experiments were performed and analyzed using H₂O-²H₂O exchange. Methods were developed which enabled us to obtain confidence limits for the X-ray and neutron structure factors.The resultant X-ray and neutron scattering density profiles clearly define the positions of the principal molecular groups in the unperturbed bilayer. In particular, the high-resolution electron density profiles reveal features directly attributable to the cholesterol molecule. A comparison with the neutron scattering density profiles shows that cholesterol is anchored with its hydroxyl group at the water/hydrocarbon interface, aligned with the fatty acid ester groups of the lecithin molecule. We suggest that this positioning of the cholesterol molecule allows it to act as a thickness buffer for plasma membranes.In the presence of very high concentrations of general anaesthetics, the bilayers show increased disorder while maintaining constant membrane thickness. At surgical concentrations, however, there are no significant changes in bilayer structure at 95% confidence levels. We briefly review the literature previously used to support lipid bilayer hypotheses of general anaesthesia. We conclude that the lipid bilayer per se is not the primary site of action of general anaesthetics.     

Effect of glycophorin on lipid polymorphism: A 31P-NMR study

(1) The effect of glycophorin, a major intrinsic glycoprotein of the human erythrocyte membrane, on lipid polymorphism has been investigated by ³¹P-NMR (at 36.4 MHz) and by freeze-fracture electron microscopy. (2) Incorporation of glycophorin into vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) results in the formation of unilamellar vesicles (1000–5000 Å diameter) which exhibit ³¹P-NMR bilayer spectra over a wide range of temperature. A reduction in the chemical shift anisotropy (Δσ_csa^eff) and an increase in spectral linewidth in comparison to dioleoylphosphatidylcholine liposomes may suggest a decrease in phospholipid headgroup order. (3) 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), in the presence of excess water, undergoes a bilayer to hexagonal (H_II) phospholipid arrangement as the temperature is increased above 0°C. Incorporation of glycophorin into this system stabilizes the bilayer configuration, prohibiting the formation of the H_II phase. (4) Cosonication of glycophorin with DOPE in aqueous solution (pH 7.4) produces small, stable unilamellar vesicles (300–1000 Å diameter), unlike DOPE alone which is unstable and precipitates from solution. (5) The current study demonstrates the bilayer stabilizing capacity of an intrinsic membrane protein, glycophorin, most likely by means of a strong hydrophobic interaction between the membrane spanning portion of glycophorin and the hydrophobic region of the phospholipid.     

Fusion Peptide Interaction with Lipid Bilayer: Modeling with Monte Carlo Simulation and Continuum Electrostatics Calculation

Abstract

Conformation of 20-residue peptide E5, an analog of the fusion peptide of influenza virus hemagglutinin, was explored by Monte-Carlo technique starting with the fully buried in the membrane ideal α-helix. The lipid bilayer (of 30 Å width) together with surrounding water were modeled by the atomic solvation parameters. During the simulation, residues 2–18 of the peptide retained α-helical conformation, and the peptide was found to be partially immersed into the bilayer. In the resulting low-energy conformers, the N-terminus was buried inside the membrane, its position with respect to the bilayer surface (Z_NT) being varied from 2.5 to 7.5 Å, and the orientation of the helical axis relative to the membrane plane (Θ) – from 10 to 35°. The low-energy conformers (below -200kcal/mol) were clustered in the space (Z_NT, Θ) into 4 groups. To select low-energy states of the peptide compatible with NMR data, we calculated pKa values of E5 ionizable groups and compared them with the experimental values. It was shown that the best correlation coefficient (0.87) and rmsd (0.68 in pH units) were obtained for the group of states which is characterized by Θ = 15–19° and Z_NT = 3.5–4.5Å.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Structure of Fully Hydrated Fluid Phase Lipid Bilayers with Monounsaturated Chains

Quantitative structures are obtained at 30°C for the fully hydrated fluid phases of palmitoyloleoylphosphatidylcholine (POPC), with a double bond on the sn-2 hydrocarbon chain, and for dierucoylphosphatidylcholine (di22:1PC), with a double bond on each hydrocarbon chain. The form factors F(q _z ) for both lipids are obtained using a combination of three methods. (1) Volumetric measurements provide F(0). (2) X-ray scattering from extruded unilamellar vesicles provides ΙF(q _z )Ι for low q _z . (3) Diffuse X-ray scattering from oriented stacks of bilayers provides ΙF(q _z )Ι for high q _z . Also, data using method (2) are added to our recent data for dioleoylphosphatidylcholine (DOPC) using methods (1) and (3); the new DOPC data agree very well with the recent data and with (4) our older data obtained using a liquid crystallographic X-ray method. We used hybrid electron density models to obtain structural results from these form factors. The result for area per lipid (A) for DOPC 72.4 ± 0.5 Ų agrees well with our earlier publications, and we find A = 69.3 ± 0.5 Ų for di22:1PC and A = 68.3 ± 1.5 Ų for POPC. We obtain the values for five different average thicknesses: hydrophobic, steric, head-head, phosphate-phosphate and Luzzati. Comparison of the results for these three lipids and for our recent dimyristoylphosphatidylcholine (DMPC) determination provides quantitative measures of the effect of unsaturation on bilayer structure. Our results suggest that lipids with one monounsaturated chain have quantitative bilayer structures closer to lipids with two monounsaturated chains than to lipids with two completely saturated chains.     

Partitioning, dynamics, and orientation of lung surfactant peptide KL4 in phospholipid bilayers

Lung surfactant protein B (SP-B) is a lipophilic protein critical to lung function at ambient pressure. KL₄ is a 21-residue peptide which has successfully replaced SP-B in clinical trials of synthetic lung surfactants. CD and FTIR measurements indicate KL₄ is helical in a lipid bilayer environment, but its exact secondary structure and orientation within the bilayer remain controversial. To investigate the partitioning and dynamics of KL₄ in phospholipid bilayers, we introduced CD₃-enriched leucines at four positions along the peptide to serve as probes of side chain dynamics via ²H solid-state NMR. The chosen labels allow distinction between models of helical secondary structure as well as between a transmembrane orientation or partitioning in the plane of the lipid leaflets. Leucine side chains are also sensitive to helix packing interactions in peptides that oligomerize. The partitioning and orientation of KL₄ in DPPC/POPG and POPC/POPG phospholipid bilayers, as inferred from the leucine side chain dynamics, is consistent with monomeric KL₄ lying in the plane of the bilayers and adopting an unusual helical structure which confers amphipathicity and allows partitioning into the lipid hydrophobic interior. At physiologic temperatures, the partitioning depth and dynamics of the peptide are dependent on the degree of saturation present in the lipids. The deeper partitioning of KL₄ relative to antimicrobial amphipathic α-helices leads to negative membrane curvature strain as evidenced by the formation of hexagonal phase structures in a POPE/POPG phospholipid mixture on addition of KL₄. The unusual secondary structure of KL₄ and its ability to differentially partition into lipid lamellae containing varying levels of saturation suggest a mechanism for its role in restoring lung compliance.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the ³¹P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. ²H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. ³¹P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, ³¹P and ²H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Solid-State H NMR Shows Equivalence of Dehydration and Osmotic Pressures in Lipid Membrane Deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S_CD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10−100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Water state and diffusion through lipid bilayers: Effect of hydration degree

The dependences of adsorbed water state (obtained from the variations in ¹H NMR spectra with the angle between the bilayer normal and magnetic field direction) and water diffusion along the bilayer normal (measured using pulsed field gradient ¹H NMR) on hydration degree have been studied in macroscopically oriented bilayers of dioleoylphosphatidylcholine. The angle dependences of the shape of NMR spectrum are qualitatively different only for water concentrations higher and lower than that achieved by hydration from saturated vapors (χ_eq, about 23%). At concentrations lower than χ_eq, all water in the sample either makes the hydration shells of the lipid polar heads or is in fast exchange with the shell water, so the spin-echo signal from water is detected only within a narrow range of angles close to the magic angle, 54.7°. At concentration exceeding χ_eq, the spin-echo signal from water is retained at all orientations, suggesting that a portion of water between bilayers (quasi-free water) slowly exchanges with water bound to the polar heads. There is an inverse dependence of the coefficient of water self-diffusion through the bilayer system on the hydration degree, which is described in the Tanner model with account of water self-diffusion in the hydrophobic part of the bilayer. Bilayer permeability, distribution coefficient of molecules between aqueous and lipid phases, and water self-diffusion coefficient in the hydrophobic region of the bilayer are estimated.     

Translational diffusion of lipid monomers determined by spin label electron spin resonance

The collision rates between spin-labelled valeric acid in water, and between the corresponding mixed-chain, spin-labelled phosphatidylcholine in water-methanol mixtures, and also between spin-labelled phosphatidylcholine monomers and micelles in water have been determined from the spin-spin broadening of the electron spin resonance spectrum. In each case the second order rate constants are consistent with a diffusion-controlled process. For spin-labelled valeric acid in water the translational diffusion coefficient at 20°C is 3.4 · 10⁻⁶ cm² · s⁻¹, and for spin-labelled phosphatidylcholine varies between 2.3 · 10⁻⁶ and 3.8 · 10⁻⁶ cm² · s⁻¹ within the range 44 to 88 wt% methanol. The spin-labelled phosphatidylcholine monomer diffusion coefficient in water at 20°C is 2.4 · 10⁻⁶ cm² · s⁻¹, deduced from the monomer-micelle association rate, with an activation energy of 4.0 kcal · mol⁻¹. The much slower on-rates for association of lipid monomers with phospholipid bilayer vesicles reported in the literature, therefore indicate that incorporation into bilayers is not a diffusion-controlled process.     

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.     

Distance Measurements and Conformational Analysis of sn-2-Arachidonoylglycerol-Membrane Sample by 2H–31P REDOR NMR

The purpose of these studies is to determine the intermolecular distances that define the location, orientation, and conformation of 2-AG in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayers using rotational-echo double-resonance (REDOR) NMR. All five protons on the glycerol backbone of 2-AG were replaced with ²H and the distance between the deuterons and naturally occurring ³¹P on the POPC lipid headgroup determined with REDOR. To determine the distance from each deuteron to the phosphorus, the POPC headgroup was arranged in a hexagonal array. The 2-AG intercalates between the lipid molecules and the ²H labels, resulting in an average distance of z directly above or below the center of the parallelogram of the four phosphorus atoms P₁, P₂, P₃, and P₄. For different z values, the ²H–³¹P inter-nuclear distances were 7.6–9.1 Å (²H–³¹P₁ and ²H–³¹P₃₁) and 4.4–6.7 Å (²H–³¹P₂ and ²H–³¹P₄). Each result involved the calculations and summation of 893,101 terms. Based on the curve-fitting parameters, the calculations with z = 0 fits the data the best, which means these methylene ²H atoms are at the same level as the phosphate group of the POPC lipid bilayer. Molecular dynamic simulation data suggested that the ²H atoms at the glycerol backbone of 2-AG are involved in an extended H-bonding network with the phosphorus atoms after 10-ns simulation.     

The effect of H<Subscript>3</Subscript>O<Superscript>+</Superscript> on the membrane morphology and hydrogen bonding of a phospholipid bilayer

At the 2017 meeting of the Australian Society for Biophysics, we presented the combined results from two recent studies showing how hydronium ions (H₃O⁺) modulate the structure and ion permeability of phospholipid bilayers. In the first study, the impact of H₃O⁺ on lipid packing had been identified using tethered bilayer lipid membranes in conjunction with electrical impedance spectroscopy and neutron reflectometry. The increased presence of H₃O⁺ (i.e. lower pH) led to a significant reduction in membrane conductivity and increased membrane thickness. A first-order explanation for the effect was assigned to alterations in the steric packing of the membrane lipids. Changes in packing were described by a critical packing parameter (CPP) related to the interfacial area and volume and shape of the membrane lipids. We proposed that increasing the concentraton of H₃O⁺ resulted in stronger hydrogen bonding between the phosphate oxygens at the water–lipid interface leading to a reduced area per lipid and slightly increased membrane thickness. At the meeting, a molecular model for these pH effects based on the result of our second study was presented. Multiple μs-long, unrestrained molecular dynamic (MD) simulations of a phosphatidylcholine lipid bilayer were carried out and showed a concentration dependent reduction in the area per lipid and an increase in bilayer thickness, in agreement with experimental data. Further, H₃O⁺ preferentially accumulated at the water–lipid interface, suggesting the localised pH at the membrane surface is much lower than the bulk bathing solution. Another significant finding was that the hydrogen bonds formed by H₃O⁺ ions with lipid headgroup oxygens are, on average, shorter in length and longer-lived than the ones formed in bulk water. In addition, the H₃O⁺ ions resided for longer periods in association with the carbonyl oxygens than with either phosphate oxygen in lipids. In summary, the MD simulations support a model where the hydrogen bonding capacity of H₃O⁺ for carbonyl and phosphate oxygens is the origin of the pH-induced changes in lipid packing in phospholipid membranes. These molecular-level studies are an important step towards a better understanding of the effect of pH on biological membranes.     

Intramembrane Water Associated with TOAC Spin-Labeled Alamethicin: Electron Spin-Echo Envelope Modulation by D2O

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D₂O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D₂O-ESEEM intensities decrease from TOAC¹ to TOAC⁸ and TOAC¹⁶ but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC¹⁶ is located in the bilayer leaflet opposite to that of TOAC¹ and TOAC⁸. Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D₂O-ESEEM intensity is greatest for TOAC⁸, indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.