DLPC Bilayers Research Articles

A Molecular Dynamics Simulation Study of Outer Membrane Phospholipase a (OMPLA) Structure and Dynamics in an Asymmetric Lipopolysaccharide Membrane

The outer membrane of Gram-negative bacteria is a unique and highly asymmetric lipid bilayer composed of phospholipids in the inner leaflet and mostly lipopolysaccharide (LPS) in the outer leaflet. Outer membrane phospholipase A (OmpLA) is an integral membrane enzyme in Escherichia coli. The structure of monomeric OmpLA consists of a 12-stranded antiparallel β-strands with a convex and a flat side, six loops at the extracellular side and five turns at the periplasmic side of the membrane. Utilizing the latest C36 CHARMM lipid and carbohydrate force field, we have constructed a model of OmpLA embedded in an asymmetric lipid bilayer with rough LPS molecules (without O-antigen) in one leaflet and phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in the other leaflet to model the realistic outer membrane environment. The simulation results will be discussed in terms of the key structural properties of the bacterial outer membrane including hydrophobic thickness, area per lipid, and acyl chain order parameter. We will also show the difference of OmpLA structure and dynamics compared to that in a DLPC bilayer. At the same time a comparison between simulations with different numbers of LPS molecules on the outer leaflet will elucidate the potential technical difficulties in building asymmetric bilayer.

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.

Influence of pH and Histidine Residues on Membrane-Spanning Helical Peptides

Synthetic model peptides such as GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide)provide a useful host framework for investigations of the influence of polar amino acids, for example histidine residues, within the hydrophobic core of a transmembrane helix. Importantly, membrane-spanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra of GWALP23-H12 reveal a marked difference in peptide behavior between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes. To prevent the acid catalyzed degradation of lipids, we employed ether-linked DOPC bilayers to observe the effect of low pH on the L12H mutant. Under acidic conditions GWALP23-H12 is highly dynamic and exhibits multiple states. Indeed, the multi-state behavior of GWALP23-H12, when His is charged between pH 1.5 and pH 3, resembles closely that of GWALP23-R12 at neutral pH (J. Am. Chem. Soc. 132, 5803). The dramatic change in the behavior of GWALP23-H12 indicates a pKa value less than 3 for His near the center of a lipid bilayer. Investigations are in progress to chemically exchange the C2 imidazole hydrogen of His for deuterium in the peptide, toward a goal of enabling direct observation of the His ring by solid-state 2H NMR over a range of pH and buffer conditions.

Exploring Protein-Lipid Interactions using Gramicidin a as a Model System

Gramicidin A (gA) is a 15 amino acid peptide with an alternating L and D sequence that dimerizes to form a membrane-spanning monovalent cation channel. Among gA's key features are four tryptophan (Trp) residues per monomer, which have been implicated as important contributors to the stability and orientation of the channels in bilayers. using molecular dynamics simulations, we have explored the Trp residues' effects on gA channel-lipid interactions. First, a modified gA (Me-gA) was built using 1-methyltryptophan, which disrupts the ability of the indoles to be hydrogen bond donors. These Me-gA dimers were simulated for 100 ns in pure DLPC, DMPC, DOPC, and POPC bilayers, and the results were compared to the previously published results using wild type gA channels. Second, both gA and Me-gA channels were simulated for about 3 μs in bilayer systems with the same average hydrophobic length (equimolar DC16:1PC+DC24:1PC mixture, equimolar DC18:1PC+DC22:1PC mixture, and pure DC20:1PC) to explore the effects of hydrophobic mismatch on lipid distribution adjacent to the protein. In all systems, protein structure and orientation characteristics were calculated. Lipid bilayer properties, such as thickness, surface area per lipid, and lateral compressibility were also quantified as functions of distance from the channel/lipid boundary to elucidate the mutual protein-lipid/ protein-bilayer interactions. Preliminary results show that Me-gA systems exhibit a reduced first shell trough (by 1-3 A) and the bilayers become thicker around Me-gA in all pure bilayer types in comparison to wild type gA. This suggests that the lipids stretch to accommodate the slightly longer hydrophobic thickness of Me-gA. The results from the mixed bilayer simulations indicate lipid redistribution around the channels based on hydrophobic mismatch. These redistribution events appear to begin about 500 ns after beginning the simulations.

Influence of Histidine Residues on Transmembrane Helix Alignment

To investigate His residues in lipid bilayer membranes, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as a favorable host peptide. Importantly, membrane-spanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra reveal a marked difference between the L12H mutant and the L12H-A13H double mutant. GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes, suggesting that the bilayer-incorporated histidine side chain is neutral (deprotonated) at an experimental pH of 5-6. By contrast, GWALP23-H12,13 is highly dynamic and exhibits multiple states which are in slow exchange on the NMR timescale. Indeed, the multi-state behavior of GWALP23-H12,13 between pH 4 and pH 9 resembles closely that of GWALP23-R12 (J. Am. Chem. Soc. 132, 5803). Further aspects of the pH dependence of transmembrane helices having one or two histidines are under investigation.

An Extension and Further Validation of an All-Atomistic Force Field for Biological Membranes.

Biological membranes are versatile in composition and host intriguing molecular processes. In order to be able to study these systems, an accurate model Hamiltonian or force field (FF) is a necessity. Here, we report the results of our extension of earlier developed all-atomistic FF parameters for fully saturated phospholipids that complements an earlier parameter set for saturated phosphatidylcholine lipids (J. Phys. Chem. B, 2012, 116, 3164-3179). The FF, coined Slipids (Stockholm lipids), now also includes parameters for unsaturated phosphatidylcholine and phosphatidylethanolamine lipids, e.g., POPC, DOPC, SOPC, POPE, and DOPE. As the extended set of parameters is derived with the same philosophy as previously applied, the resulting FF has been developed in a fully consistent manner. The capabilities of Slipids are demonstrated by performing long simulations without applying any surface tension and using the correct isothermal-isobaric (NPT) ensemble for a range of temperatures and carefully comparing a number of properties with experimental findings. Results show that several structural properties are very well reproduced, such as scattering form factors, NMR order parameters, thicknesses, and area per lipid. Thermal dependencies of different thicknesses and area per lipid are reproduced as well. Lipid diffusion is systematically slightly underestimated, whereas the normalized lipid diffusion follows the experimental trends. This is believed to be due to the lack of collective movement in the relatively small bilayer patches used. Furthermore, the compatibility with amino acid FFs from the AMBER family is tested in explicit transmembrane complexes of the WALP23 peptide with DLPC and DOPC bilayers, and this shows that Slipids can be used to study more complex and biologically relevant systems.

Molecular Dynamics Investigations of PRODAN in a DLPC Bilayer

Molecular dynamics computer simulations have been performed to identify preferred positions of the fluorescent probe PRODAN in a fully hydrated DLPC bilayer in the fluid phase. In addition to the intramolecular charge-transfer first vertical excited state, we considered different charge distributions for the electronic ground state of the PRODAN molecule by distinct atomic charge models corresponding to the probe molecule in vacuum as well as polarized in a weak and a strong dielectric solvent (cyclohexane and water). Independent on the charge distribution model of PRODAN, we observed a preferential orientation of this molecule in the bilayer with the dimethylamino group pointing toward the membrane's center and the carbonyl oxygen toward the membrane's interface. However, changing the charge distribution model of PRODAN, independent of its initial position in the equilibrated DLPC membrane, we observed different preferential positions. For the ground state representation without polarization and the in-cyclohexane polarization, the probe maintains its position close to the membrane's center. Considering the in-water polarization model, the probe approaches more of the polar headgroup region of the bilayer, with a strong structural correlation with the choline group, exposing its oxygen atom to water molecules. PRODAN's representation of the first vertical excited state with the in-water polarization also approaches the polar region of the membrane with the oxygen atom exposed to the bilayer's hydration shell. However, this model presents a stronger structural correlation with the phosphate groups than the ground state. Therefore, we conclude that the orientation of the PRODAN molecule inside the DLPC membrane is well-defined, but its position is very sensitive to the effect of the medium polarization included here by different models for the atomic charge distribution of the probe.

Characterization of Antimicrobial Peptides Relating to Shortened RWALP Model Peptides

In the face of increasing bacterial drug resistance, small membrane-active peptides offer potentially attractive avenues to alternative antimicrobial agents. The goals of this project are to characterize the lipid interactions and analyze the antimicrobial efficacy of model peptides that are longer than the lactoferrin-derived, surface-acting LfB6 (RRWQWR-NH2), yet shorter than the transmembrane RWALP23 (acetyl-GRALW(LA)6LWLARA-NH2). New-generation RWALP peptides, of general sequence (ac-GRnWm(LA)jLWmRnA-NH2) were designed with varying total lengths (13-15) and numbers of Arg and Trp residues. 2H-alanines were incorporated at several positions to serve as probes for recording solid-state NMR spectra from mechanically aligned samples of the peptides in bilayers of DLPC, DMPC or DOPC. Labeled RWALP13 (j=3, n=1, m=1) exhibits partial water solubility, no antimicrobial activity, and 2H-NMR spectra characteristic of isotropic motion, even in the presence of lipid bilayers. Circular dichroism spectra of RWALP13 suggest partial α-helical character in water that is enhanced when lipids are present. The 2H-NMR spectra indicate that the longer 14- or 15-residue peptides are aligned to varying degrees in the different lipid bilayer membranes. Antimicrobial assays reveal that peptides with four arginines have higher activity than those with only two arginines. Interestingly, within the 4-Arg category, RRWALP15 (j=3, n=2, m=1) shows higher activity (MIC of 6.25 μ1/4g/ml) against E. coli than does RRWWALP15 (j=2, n=2, m=2; MIC of 25 μ1/4g/ml). The 2H-NMR spectra of RRWALP15 suggest significant alignment of the peptide with respect to lipid bilayer membranes. The combined antimicrobial and spectral features make RRWALP15 an especially good candidate for further analysis.

Exploring Hydrophobic Mismatch's Impacts on Dissociation of Gramicidin a Channels using Molecular Dynamics Free Energy Simulations

Gramicidin A (gA) channels, formed by trans-bilayer association of two non-conducting subunits, have been used as probes to explore how changes in lipid bilayer composition, including the adsorption of small molecules, alter lipid bilayer properties through measurements of gA channels’ appearance frequency, lifetime, conductance, and gating. However, the molecular mechanism of the channel association/dissociation remains an enigma. Because the gA channel's association/dissociation can be a multi-dimensional reaction coordinate problem, it also is challenging to computationally explore these mechanisms in a fully atomistic system. As a first step, based on our previous molecular dynamics (MD) simulation systems of gA in DLPC, DMPC, DOPC, and POPC bilayers, we have performed umbrella sampling MD free energy simulations (MD/FES) of gA dissociation along the membrane normal (i.e., the Z axis). In this presentation, the MD/FES results of gA dissociation in the four different bilayers will be discussed in terms of (1) the resulting free energy profiles, (2) gA subunits’ structure, orientation, and XY displacement, (3) the subunit-subunit hydrogen bonding (number and pattern), (4) the pore water molecules’ distribution and (5) the bilayer properties (thickness, per-lipid surface area, lateral compressibility, and order parameters) along the reaction coordinate.

Outer membrane phospholipase A in phospholipid bilayers: A model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers

Understanding the forces that stabilize membrane proteins in their native states is one of the contemporary challenges of biophysics. To date, estimates of side chain partitioning free energies from water to the lipid environment show disparate values between experimental and computational measures. Resolving the disparities is particularly important for understanding the energetic contributions of polar and charged side chains to membrane protein function because of the roles these residue types play in many cellular functions. In general, computational free energy estimates of charged side chain partitioning into bilayers are much larger than experimental measurements. However, the lack of a protein-based experimental system that uses bilayers against which to vet these computational predictions has traditionally been a significant drawback. Moon & Fleming recently published a novel hydrophobicity scale that was derived experimentally by using a host–guest strategy to measure the side chain energetic perturbation due to mutation in the context of a native membrane protein inserted into a phospholipid bilayer. These values are still approximately an order of magnitude smaller than computational estimates derived from molecular dynamics calculations from several independent groups. Here we address this discrepancy by showing that the free energy differences between experiment and computation become much smaller if the appropriate comparisons are drawn, which suggests that the two fields may in fact be converging. In addition, we present an initial computational characterization of the Moon & Fleming experimental system used for the hydrophobicity scale: OmpLA in DLPC bilayers. The hydrophobicity scale used OmpLA position 210 as the guest site, and our preliminary results demonstrate that this position is buried in the center of the DLPC membrane, validating its usage in the experimental studies. We further showed that the introduction of charged Arg at position 210 is well tolerated in OmpLA and that the DLPC bilayers accommodate this perturbation by creating a water dimple that allows the Arg side chain to remain hydrated. Lipid head groups visit the dimple and can hydrogen bond with Arg, but these interactions are transient. Overall, our study demonstrates the unique advantages of this molecular system because it can be interrogated by both computational and experimental practitioners, and it sets the stage for free energy calculations in a system for which there is unambiguous experimental data. This article is part of a Special Issue entitled: Membrane protein structure and function.

Structure and dynamics of the lipid modifications of a transmembrane α-helical peptide determined by 2H solid-state NMR spectroscopy

The fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments. Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusiogenically active transmembrane segments in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776–14781). From this series, we have investigated the peptide LV16 (KKKW LVLV LVLV LVLV LVLV KKK), which was synthesized featuring either a free N-terminus or a saturated N-acylation of 2, 8, 12, or 16 carbons. We used 2H and 31P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C2 chain, all peptide acyl chains were found to insert well into the membrane. This can be explained by the high local lipid concentrations the N-terminal lipid chains experience. Further, the insertion of these peptides did not influence the membrane structure and dynamics as seen from the 2H and 31P NMR data. In spite of the fact that the longer acyl chains insert into the membrane, they do not adapt their lengths to the thickness of the bilayer. Even the C16 lipid chain on the peptide, which could match the length of the POPC palmitoyl chain, exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2H NMR square law plots reveal motions of slightly larger amplitudes for the peptide lipid chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acylations, the fraction of gauche defects in the inserted chains is constant.

Solid State NMR Studies on Acylated Transmembrane Peptides Driving Membrane Fusion

The fusion of biological membranes is mediated by integral membrane proteins with α-helical transmembrane segments (TMSs). Additionally, those proteins are often modified by the covalent attachment of hydrocarbon chains. Previously, a series of de novo designed α-helical peptides with mixed Leu/Val sequences was presented, mimicking fusogenic TMSs in model membranes (Hofmann et al., Proc. Natl. Acad. Sci. USA 101 (2004) 14776-14781). From this series, we have investigated the peptide LV16 (KKKWLVLVLVLVLVLVLVLVKKK), which was synthesized presenting either a free N-terminus or an N-acylation of 2, 8, 12, or 16 carbons. We used 2H and 31P NMR spectroscopy to investigate the structure and dynamics of those peptide lipid modifications in POPC and DLPC bilayers and compared them to the hydrocarbon chains of the surrounding membrane. Except for the C-2 chain, all peptide acyl chains were found to insert well into the membrane. This can be understood from the high local lipid concentration, which the N-terminal lipid chains experience. The insertion of these peptides did not influence the membrane structure and dynamics as seen from 2H and 31P NMR. In spite of the fact that the longer acyl chains insert into the membrane, there is no perfect length adaptation. Even the C-16 chain on the peptide, which could match the length of the POPC palmitoyl chain exhibited lower order parameters in the upper chain, which get closer and finally reach similar values in the lower chain region. 2H NMR square law plots reveal a slightly more dynamic characteristic of the peptide acyl chains compared to the surrounding phospholipids. In spite of the significantly different chain lengths of the acyl chains, the fraction of gauche defects in the inserted chains is constant, suggesting similar entropies of the inserted chains.

Charged and Aromatic Anchoring Amino Acids Affect the Orientation of Transmembrane Peptides: A Deuterium NMR Study

In integral proteins, the membrane-spanning segments are often flanked by aromatic and/or charged amino acids, which serve as anchoring residues, promoting protein-lipid interactions. These residues have specific polarity preferences, which are reflected in their typical immersion depths. Namely, tryptophan (W) is localized primarily to the carbonyl region of lipid acyl chains, while charged lysine (K) and arginine (R) prefer more polar regions. Although such patterns are widely recognized, little is known regarding the contributions of these residues for the orientations of transmembrane segments. Solid-state NMR spectroscopy offers a way to approach this problem. Peptides of the “WALP” family, GWW(LA)nLWWA, have served as useful models. Membrane-spanning proteins, nevertheless, typically contain more diverse sets of anchoring amino acids, which may act together to influence molecular geometry and orientation. To address these issues, we have designed the X2,22W5,19ALP23 peptides (acetyl-GXALW5(LA)6LW19LAXA-[ethanol]amide) to encompass two different anchor residues. In these peptides the inner anchor is kept constant (W), while the outer “anchor” (separated by a short Leu-Ala spacer) is varied to either W, K, R or the control residue G. Solid-state 2H NMR spectra were recorded for peptides having Ala CD3 groups in aligned bilayers of DLPC, DMPC and DOPC. The “Geometric Analysis of Labeled Alanines” provided a means to deduce the apparent average peptide orientations. Introduction of charged anchors (X = K or R) resulted in a 2-5° increase in the apparent tilt angle compared to X = G. Conversely, for X = W, the apparent tilt angle decreases 2-9°, accompanied by a significant change in the tilt direction.

Comparative Molecular Dynamics Simulation Studies of Protegrin-1 Monomer and Dimer in Two Different Lipid Bilayers

Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated 15N and 13CO chemical shifts of Val16 in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions.

Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation

The molecular motions of membrane proteins in liquid-crystalline lipid bilayers lie at the interface between motions in isotropic liquids and in solids. Specifically, membrane proteins can undergo whole-body uniaxial diffusion on the microsecond time scale. In this work, we investigate the (1)H rotating-frame spin-lattice relaxation (T (1rho)) caused by the uniaxial diffusion of the influenza A M2 transmembrane peptide (M2TMP), which forms a tetrameric proton channel in lipid bilayers. This uniaxial diffusion was proved before by (2)H, (15)N and (13)C NMR lineshapes of M2TMP in DLPC bilayers. When bound to an inhibitor, amantadine, the protein exhibits significantly narrower linewidths at physiological temperature. We now investigate the origin of this line narrowing through temperature-dependent (1)H T (1rho) relaxation times in the absence and presence of amantadine. Analysis of the temperature dependence indicates that amantadine decreases the correlation time of motion from 2.8 +/- 0.9 mus for the apo peptide to 0.89 +/- 0.41 micros for the bound peptide at 313 K. Thus the line narrowing of the bound peptide is due to better avoidance of the NMR time scale and suppression of intermediate time scale broadening. The faster diffusion of the bound peptide is due to the higher attempt rate of motion, suggesting that amantadine creates better-packed and more cohesive helical bundles. Analysis of the temperature dependence of ln(T_1rho(-1)) indicates that the activation energy of motion increased from 14.0 +/- 4.0 kJ/mol for the apo peptide to 23.3 +/- 6.2 kJ/mol for the bound peptide. This higher activation energy indicates that excess amantadine outside the protein channel in the lipid bilayer increases the membrane viscosity. Thus, the protein-bound amantadine speeds up the diffusion of the helical bundles while the excess amantadine in the bilayer increases the membrane viscosity.

Immobilization of the influenza A M2 transmembrane peptide in virus envelope-mimetic lipid membranes: a solid-state NMR investigation.

The dynamic and structural properties of membrane proteins are intimately affected by the lipid bilayer. One property of membrane proteins is uniaxial rotational diffusion, which depends on the membrane viscosity and thickness. This rotational diffusion is readily manifested in solid-state NMR spectra as characteristic line shapes and temperature-dependent line narrowing or broadening. We show here that this whole-body uniaxial diffusion is suppressed in lipid bilayers mimicking the composition of eukaryotic cell membranes, which are rich in cholesterol and sphingomyelin. We demonstrate this membrane-induced immobilization on the transmembrane peptide of the influenza A M2 (AM2-TM) proton channel protein. At physiological temperature, AM2-TM undergoes uniaxial diffusion faster than approximately 10(5) s(-1) in DLPC, DMPC, and POPC bilayers, but the motion is slowed by 2 orders of magnitude, to <10(3) s(-1), in a cholesterol-rich virus envelope-mimetic membrane ("viral membrane"). The immobilization is manifested as near rigid-limit (2)H quadrupolar couplings and (13)C-(1)H, (15)N-(1)H, and (13)C-(15)N dipolar couplings for all labeled residues. The immobilization suppresses intermediate time scale broadening of the NMR spectra, thus allowing high-sensitivity and high-resolution spectra to be measured at physiological temperature. The conformation of the protein in the viral membrane is more homogeneous than in model PC membranes, as evidenced by the narrow (15)N lines. The immobilization of the M2 helical bundle by the membrane composition change indicates the importance of studying membrane proteins in environments as native as possible. It also suggests that eukaryote-mimetic lipid membranes may greatly facilitate structure determination of membrane proteins by solid-state NMR.

Mechanism of short-range interfacial repulsion between hydrated phosphatidylcholine bilayers: Comparison with phosphatidylethanolamine

The grand canonical Monte Carlo technique is used to reveal the origin of the repulsive pressure operating between supported DLPC bilayers at short separations. By partitioning the interbilayer pressure into physically distinct components, it is shown that the short-range repulsion comes mainly from the direct electrostatic lipid–lipid interaction of the head groups in the opposing leaflets. By contrast, the electrostatic lipid–lipid interaction between DLPE bilayers is strongly attractive, and the short-range repulsion is associated with the hydration (water–lipid) interactions [A. Pertsin, D. Platonov, M. Grunze, Langmuir 23 (2007) 1388]. These findings explain why DLPC bilayers have a much larger interbilayer (fluid) separation at a given pressure, as compared to that for DLPE.

Lipid diffusion compared in outer and inner leaflets of planar supported bilayers

The translational diffusion coefficient (D) of lipids located in the outer and inner leaflets of planar supported DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) bilayers in the fluid phase was measured using fluorescence correlation spectroscopy of dye-labeled lipids at the low concentration of 0.001% and using iodide quenching of dyes in the outer leaflet to distinguish diffusion in the inner leaflet from that in the outer leaflet. To confirm the generality of these findings, the bilayers were prepared not only by vesicle fusion but also by Langmuir-Blodgett deposition. We conclude that regardless of whether the bilayers were supported on quartz or on a polymer cushion, D in the inner and outer leaflets was the same within an experimental uncertainty of +/-10% but with a small systematic tendency to be slower (by <5%) within the inner leaflet.

Morphological Behavior of Lipid Bilayers Induced by Melittin near the Phase Transition Temperature

Morphological changes of DMPC, DLPC, and DPPC bilayers containing melittin (lecithin/melittin molar ratio of 10:1) around the gel-to-liquid crystalline phase transition temperatures (Tc) were examined by a variety of biophysical methods. First, giant vesicles with the diameters of ∼20μm were observed by optical microscopy for melittin-DMPC bilayers at 27.9°C. When the temperature was lowered to 24.9°C (Tc=23°C for the neat DMPC bilayers), the surface of vesicles became blurred and dynamic pore formation was visible in the microscopic picture taken at different exposure times. Phase separation and association of melittin molecules in the bilayers were further detected by fluorescent microscopy and mass spectrometry, respectively. These vesicles disappeared completely at 22.9°C. It was thus found that the melittin-lecithin bilayers reversibly undergo their fusion and disruption near the respective Tcs. The fluctuation of lipids is, therefore, responsible for the membrane fusion above the Tc, and the association of melittin molecules causes membrane fragmentation below the Tc. Subsequent magnetic alignments were observed by solid-state 31P NMR spectra for the melittin-lecithin vesicles at a temperature above the respective Tcs. On the other hand, additional large amplitude motion induced by melittin at a temperature near the Tc breaks down the magnetic alignment.

膜環境感受性色素Ｌａｕｒｄａｎは何を感じているか

Laurdan, one of the environment-sensitive fluorescent probes, is useful for monitoring membrane fluidity and is widely used for spectroscopic and imaging applications. In the imaging applications, the generalized polarization parameter (GP), defined by two fluorescent intensities at characteristic wavelengths, is used as a measure of membrane fluidity and polarity. However, time-resolved fluorescent spectra of laurdan revealed that the three excited states concern with the emission spectra. We have measured the steady-state fluorescence spectra and the fluorescence anisotropy of laurdan, prodan and NBD-PE in the DPPC and DLPC bilayers at various temperatures. The spectral dependence on the excitation wavelength is interpreted as the red edge effects that are related to the existence of excited-state distribution of fluorophores on their interaction energy with the environment and the slow dielectric relaxation of this environment. The emission peak of laurdan in the liquid-crystalline phase is independent of the red edge excitation, while prodan and NBD-PE show significant red edge effects. This results strongly suggest that laurdan changes its electronic excited state by the specific interaction with a few water molecules in the bilayers and that in addition the non-specific solvent relaxation causes further spectral shifts in the case of prodan in the bilayers and laurdan in the micella. A new three state model for the absorption and emission of laurdan is proposed. The availability and limitation of the GP method are disscussed.