Membrane insertion of a lipidated ras peptide studied by FTIR,solid-state NMR,and neutron diffraction spectroscopy

Membrane binding of a doubly lipid modified heptapeptide from the C-terminus of the human N-ras protein was studied by Fourier transform infrared, solid-state NMR, and neutron diffraction spectroscopy. The 16:0 peptide chains insert well into the 1,2-dimyristoyl-sn-glycero-3-phosphocholine phospholipid matrix. This is indicated by a common main phase transition temperature of 21.5 degrees C for both the lipid and peptide chains as revealed by FTIR measurements. Further, (2)H NMR reveals that peptide and lipid chains have approximately the same chain length in the liquid crystalline state. This is achieved by a much lower order parameter of the 16:0 peptide chains compared to the 14:0 phospholipid chains. Finally, proton/deuterium contrast variation of neutron diffraction experiments indicates that peptide chains are localized in the membrane interior analogous to the phospholipid chains. In agreement with this model of peptide chain insertion, the peptide part is localized at the lipid-water interface of the membrane. This is revealed by (1)H nuclear Overhauser enhancement spectra recorded under magic angle spinning conditions. Quantitative cross-peak analysis allows the examination of the average location of the peptide backbone and side chains with respect to the membrane. While the backbone shows the strongest cross-relaxation rates with the phospholipid glycerol, the hydrophobic side chains of the peptide insert deeper into the membrane interior. This is supported by neutron diffraction experiments that reveal a peptide distribution in the lipid-water interface of the membrane. Concurring with these experimental findings, the amide protons of the peptide show strong water exchange as seen in NMR and FTIR measurements. No indications for a hydrogen-bonded secondary structure of the peptide backbone are found. Therefore, membrane binding of the C-terminus of the N-ras protein is mainly due to lipid chain insertion but also supported by interactions between hydrophobic side chains and the lipid membrane. The peptide assumes a mobile and disordered conformation in the membrane. Since the C-terminus of the soluble part of the ras protein is also disordered, we hypothesize that our model for membrane binding of the ras peptide realistically describes the membrane binding of the lipidated C-terminus of the active ras protein.     

Membrane localization and flexibility of a lipidated ras peptide studied by molecular dynamics simulations

Lipid-modified membrane-binding proteins are essential in signal transduction events of the cell, a typical example being the GTPase ras. Recently, membrane binding of a doubly lipid-modified heptapeptide from the C-terminus of the human N-ras protein was studied by spectroscopic techniques. It was found that membrane binding is mainly due to lipid chain insertion, but it is also favored by interactions between apolar side chains and the hydrophobic region of the membrane. Here, 10 explicit solvent molecular dynamics simulations for a total time of about 150 ns are used to investigate the atomic details of the peptide-membrane association. The 16:0 peptide lipid chains are more mobile than the 14:0 phospholipid chains, which is in agreement with (2)H NMR experiments. Peptide-lipid and peptide-solvent interactions, backbone and side-chain distributions, as well as the effects of lipidated peptide insertion onto the structure, and dynamics of a 1,2-dimyristoylglycero-3-phosphocholine bilayer are described. The simulation results validate the structural model proposed by the analysis of spectroscopic data and highlight the main aspects of the insertion mechanism. The peptide in the membrane is rather rigid over the simulation time scale of about 10 ns, but different partially extended conformations devoid of backbone hydrogen bonds are observed in different trajectories.     

Impact on lipid membrane organization by free branched-chain fatty acids

Here, we exploit the non-invasive techniques of solid-state NMR (nuclear magnetic resonance) and differential scanning calorimetry (DSC) to study the effect of free iso and ante-iso branched chain fatty acids (BCFAs) on the physicochemical properties of lipid membranes. Free fatty acids are present in biological membranes at low abundance, but can influence the cellular function by modulating the membrane organization. Solid state NMR spectra of dimyristoylphosphatidylcholine (DMPC) lipid membranes containing either free 12-methyltetradecanoic acid (a15:0) or free 13-methyltetradecanoic acid (i15:0), show significant differences in their impact on the lipid bilayer. Chain order profiles obtained by deuterium NMR on fully deuterated DMPC-d(67) bilayers revealed an ordering effect induced by both fatty acids on the hydrophobic membrane core. This behavior was also visible in the corresponding DSC thermograms where the main phase transition of DMPC bilayers-indicative of the hydrophobic membrane region-was shifted to higher temperatures, with the iso isomer triggering more pronounced changes as compared to the ante-iso isomer. This is probably due to a higher packing density in the core of the lipid bilayer, which causes reduced diffusion across membranes. By utilizing the naturally occurring spin reporters nitrogen-14 and phosphorus-31 present in the hydrophilic DMPC headgroup region, even fatty acid induced changes at the membrane interface could be detected, an observation reflecting changes in the lipid headgroup dynamics.     

The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using ¹⁵N-NMR and quantitative determination of ¹H-¹³C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the Cα sites and lower in the side chains (0.37 for the CH₂ and 0.18 for the CH₃ groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 μs (with one exception, where the trajectory length was 10 μs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins.     

Influence of perfluorinated compounds on the properties of model lipid membranes

The influence of selected perfluorinated compounds (PFCs), perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS), on the structure and organization of lipid membranes was investigated using model membranes-lipid monolayers and bilayers. The simplest model--a lipid monolayer--was studied at the air-water interface using the Langmuir-Blodgett technique with surface pressure and surface potential measurements. Lipid bilayers were characterized by NMR techniques and molecular dynamics simulations. Two phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), characterized by different surface properties have been chosen as components of the model membranes. For a DPPC monolayer, a phase transition from the liquid-expanded state to the liquid-condensed state can be observed upon compression at room temperature, while a DMPC monolayer under the same conditions remains in the liquid-expanded state. For each of the two lipids, the presence of both PFOA and PFOS leads to the formation of a more fluidic layer at the air-water interface. Pulsed field gradient NMR measurements of the lateral diffusion coefficient (DL) of DMPC and PFOA in oriented bilayers reveal that, upon addition of PFOA to DMPC bilayers, DL of DMPC decreases for small amounts of PFOA, while larger additions produce an increased DL. The DL values of PFOA were found to be slightly larger than those for DMPC, probably as a consequence of the water solubility of PFOA. Furthermore, 31P and 2H NMR showed that the gel-liquid crystalline phase transition temperature decreased by the addition of PFOA for concentrations of 5 mol % and above, indicating a destabilizing effect of PFOA on the membranes. Deuterium order parameters of deuterated DMPC were found to increase slightly upon increasing the PFOA concentration. The monolayer experiments reveal that PFOS also penetrates slowly into already preformed lipid layers, leading to a change of their properties with time. These experimental observations are in qualitative agreement with the computational results obtained from the molecular dynamics simulations showing a slow migration of PFCs from the surrounding water phase into DPPC and DMPC bilayers.     

The lipidated membrane anchor of full length N-Ras protein shows an extensive dynamics as revealed by solid-state NMR spectroscopy

Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5-10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized (2)H or (13)C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static (2)H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of (1)H-(13)C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.     

Effects of lysophospholipids on membrane order of phosphatidylcholine

Lysophospholipids are known to play a role in a wide range of cellular processes involving membrane–protein or membrane–membrane interactions; however lysolipids–lamellar lipids interactions remain unclear. The effects of lysolipids on membrane order and dynamics were examined using optical birefringence and fluorescence techniques. We found that lysophosphatidic acid (LPA) induces a considerable disorder in chain orientation for synthetic lipid of dimyristoyl-phosphatidylcholines (DMPC), whereas a slight order for natural lipid of egg yolk phosphatidylcholine (Egg-PC), e.g. the chain order decreases by 10% at 0.1 mole ratio for DMPC in comparison with the membranes without LPA and increases by 3.4% at 0.09 mole ratio for Egg-PC. Also, membrane fluidity corresponds with the change in the chain disorder, namely, the fluidity increases for DMPC membranes, while decreases for Egg-PC membranes by addition of LPA. The difference in the effects of LPA is interpreted by a difference in the chain packing between the synthetic and the natural lipid bilayers. LPA can be incorporated into natural lipid membranes without disturbance, and readjusts itself to a more favorable hydrophobic match with the bilayers. Lysophophatidylcholine (LPC) also induces a disorder in DMPC membranes, but the decrease in chain order is only half compared with that for LPA.     

Structural properties of docosahexaenoyl phospholipid bilayers investigated by solid-state 2H NMR spectroscopy.

Polyunsaturated lipids in cellular membranes are known to play key roles in such diverse biological processes as vision, neuronal signaling, and apoptosis. One hypothesis is that polyunsaturated lipids are involved in second messenger functions in biological signaling. Another current hypothesis affirms that the functional role of polyunsaturated lipids relies on their ability to modulate physical properties of the lipid bilayer. The present research has employed solid-state 2H NMR spectroscopy to acquire knowledge of the molecular organization and material properties of polyunsaturated lipid bilayers. We report measurements for a homologous series of mixed-chain phosphatidylcholines containing a perdeuterated, saturated acyl chain (n:0) at the sn-1 position, adjacent to docosahexaenoic acid (DHA, 22:6omega3) at the sn-2 position. Measurements have been performed on fluid (L(alpha))-state multilamellar dispersions as a function of temperature for saturated acyl chain lengths of n = 12, 14, 16, and 18 carbons. The saturated sn-1 chains are therefore used as an intrinsic probe with site-specific resolution of the polyunsaturated bilayer structure. The 2H NMR order parameters as a function of acyl position (order profiles) have been analyzed using a mean-torque potential model for the chain segments, and the results are discussed in comparison with the homologous series of disaturated lipid bilayers. At a given absolute temperature, as the sn-1 acyl length adjacent to the sn-2 DHA chain is greater, the order of the initial chain segments increases, whereas that of the end segments decreases, in marked contrast with the corresponding disaturated series. For the latter, the order of the end segments is practically constant with acyl length, thus revealing a universal chain packing profile. We find that the DHA-containing series, while more complex, is still characterized by a universal chain packing profile, which is shifted relative to the homologous saturated series. Moreover, we show how introduction of DHA chains translates the order profile along the saturated chains, making more disordered states accessible within the bilayer central region. As a result, the area per lipid headgroup is increased as compared to disaturated bilayers. The systematic analysis of the 2H NMR data provides a basis for studies of lipid interactions with integral membrane proteins, for instance in relation to characteristic biological functions of highly unsaturated lipid membranes.     

Detection of peptide-phospholipid interaction sites in bilayer membranes by 13C NMR spectroscopy: observation of 2H/31P-selective 1H-depolarization under magic-angle spinning

We have developed a solid-state NMR method for observing the signals due to 13C spins of a peptide in the close vicinity of 31P and 2H spins in deuterated phospholipid bilayers. The signal intensities in 13C high-resolution NMR spectra directly indicate the depolarization of 1H by 1H-31P and 1H-2H dipolar couplings under multiple-contact cross-polarization. This method was applied to a fully 13C-, 15N-labeled 14-residue peptide, mastoparan-X (MP-X), bound to phospholipid bilayers whose fatty acyl chains are deuterated. The 13C NMR spectra for the depolarization were simulated from the chemical shifts and structure of membrane-bound MP-X previously determined and the distribution of 2H and 31P spins in lipid bilayers. The minimization of RMSD between the simulated and the experimental spectra showed that the amphiphilic alpha-helix of MP-X was located in the interface between the water layer and the hydrophobic domain of the bilayer, with nonpolar residues facing the phosphorus atoms and alkyl chains of the lipids.     

Study of adsorption and penetration of E2(279-298) peptide into Langmuir phospholipid monolayers

The peptide corresponding to the sequence (279-298) of the Hepatitis G virus (HGV/GBV-C) E2 protein was synthesized, and surface activity measurements, pi-A compression isotherms, and penetration of E2(279-298) into phospholipid monolayers spread at the air-water interface were carried out on water and phosphate buffer subphases. The results obtained indicated that the pure E2(279-298) Langmuir monolayer exhibited a looser packing on saline-buffered than on pure water subphase and suggest that the increase in subphase ionic strength stabilizes the peptide monolayer. To better understand the topography of the monolayer, Brewster angle microscopy (BAM) images of pure peptide monolayers were obtained. Penetration of the peptide into the pure lipid monolayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) and into mixtures of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) at various initial surface pressures was investigated to determine the ability of these lipid monolayers to host the peptide. The higher penetration of peptide into phospholipids is attained when the monolayers are in the liquid expanded state, and the greater interaction is observed with DMPC. Furthermore, the penetration of the peptide dissolved in the subphase into these various lipid monolayers was investigated to understand the interactions between the peptide and the lipid at the air-water interface. The results obtained showed that the lipid acyl chain length is an important parameter to be taken into consideration in the study of peptide-lipid interactions.     

Solid-state 2H NMR studies of the effects of cholesterol on the acyl chain dynamics of magnetically aligned phospholipid bilayers

We report the utilization of magnetically aligned phospholipid bilayers (bicelles) to study the effects of cholesterol in phospholipid bilayers for both chain perdeuterated DMPC and partially deuterated alpha-[2,2,3,4,4,6-d(6)]-cholesterol using (2)H solid-state NMR spectroscopy. The quadrupolar splittings at 40 degrees C were 25.5 and 37.7 kHz, respectively, for the 2,4-(2)H(eq) and 2,4-(2)H(ax) deuterons when the bilayer normal of the discs was aligned perpendicular to the static magnetic field. The quadrupolar splittings were doubled when Yb(3+) ions were added to flip the bicelles 90 degrees such that the bilayer normal was colinear with the magnetic field. The results suggest that cholesterol is incorporated into the bicelle discs. For chain perdeuterated DMPC-d(54), incorporated into DMPC-DHPC bicelle discs, the individual quadrupolar splittings of the methylene and methyl groups doubled on going from the perpendicular to the parallel alignment. Also, the presence of cholesterol increased the overall ordering of the acyl chains of the phospholipids. S(CD) (i) calculations were extracted directly from the (2)H quadrupolar splittings of the chain perdeuterated DMPC. The order parameter, S(CD) (i), calculations clearly indicated an overall degree of ordering of the acyl chains in the presence of cholesterol. We also noted a disordering effect at higher temperatures. This study demonstrates the ease with which (2)H order parameters can be calculated utilizing magnetically aligned phospholipid bilayers when compared with randomly dispersed membrane samples.     

Computational analysis of local membrane properties

In the field of biomolecular simulations, dynamics of phospholipid membranes is of special interest. A number of proteins, including channels, transporters, receptors and short peptides are embedded in lipid bilayers and tightly interact with phospholipids. While the experimental measurements report on the spatial and/or temporal average membrane properties, simulation results are not restricted to the average properties. In the current study, we present a collection of methods for an efficient local membrane property calculation, comprising bilayer thickness, area per lipid, deuterium order parameters, Gaussian and mean curvature. The local membrane property calculation allows for a direct mapping of the membrane features, which subsequently can be used for further analysis and visualization of the processes of interest. The main features of the described methods are highlighted in a number of membrane systems, namely: a pure dimyristoyl-phosphatidyl-choline (DMPC) bilayer, a fusion peptide interacting with a membrane, voltage-dependent anion channel protein embedded in a DMPC bilayer, cholesterol enriched bilayer and a coarse grained simulation of a curved palmitoyl-oleoyl-phosphatidyl-choline lipid membrane. The local membrane property analysis proves to provide an intuitive and detailed view on the observables that are otherwise interpreted as averaged bilayer properties.     

Orientation and conformational preference of leucine-enkephalin at the surface of a hydrated dimyristoylphosphatidylcholine bilayer: NMR and MD simulation

The morphogenic opiate pentapeptide leucine-enkephalin (lenk) in a hydrated dimyristoylphosphatidylcholine (DMPC) bilayer is studied using NMR spectroscopy and molecular dynamics simulation. Contrary to the frequent assumption that the peptide attains a single fixed conformation in the presence of membranes, we find that the lenk molecule is flexible, switching between specific bent conformations. The constraints to the orientation of the aromatic rings that are identified by the NMR experiment are found by the MD simulation to be related to the depth of the peptide in the bilayer. The motion of the N-H vectors of the peptide bonds with respect to the magnetic field direction as observed by MD largely explain the magnitude of the observed residual dipolar coupling (RDC), which are much reduced over the static (15)N-(1)H coupling. The measured RDCs are nevertheless significantly larger than the predicted ones, possibly due the absence of long-time motions in the simulations. The conformational behavior of lenk at the DMPC surface is compared to that in the aqueous solution, both in the neutral and in the zwitterionic forms.     

Structure and dynamics of phospholipid bilayers using recently developed general all-atom force fields

Two fully hydrated pure-species phospholipids bilayers, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), in the fluid phase and explicit solvent have been studied using molecular dynamics simulation. Atom interactions were modeled using recently developed force fields based on AMBER with full atomistic details. Several representative liquid phase properties for the structure and dynamics of lipids with different length of hydrocarbon chains and different level of saturation have been reproduced without artificially biasing the system in order to match experimental data. In particular, as the new GAFF (General Amber Force Field) has not been explicitly developed to reproduce lipid characteristics and is naturally compatible with standard AMBER nucleic acids and proteins parameters, it is here proven a promising tool to study mixed lipid-protein processes as protein activity dependence on membrane composition, permeation of solute across membranes, and other cellular processes.     

Structure of gramicidin a in a lipid bilayer environment determined using molecular dynamics simulations and solid-state NMR data

Two different high-resolution structures recently have been proposed for the membrane-spanning gramicidin A channel: one based on solid-state NMR experiments in oriented phospholipid bilayers (Ketchem, R. R.; Roux, B.; Cross, T. A. Structure 1997, 5, 1655-1669; Protein Data Bank, PDB:1MAG); and one based on two-dimensional NMR in detergent micelles (Townsley, L. E.; Tucker, W. A.; Sham, S.; Hinton, J. F. Biochemistry 2001, 40, 11676-11686; PDB:1JNO). Despite overall agreement, the two structures differ in peptide backbone pitch and the orientation of several side chains; in particular that of the Trp at position 9. Given the importance of the peptide backbone and Trp side chains for ion permeation, we undertook an investigation of the two structures using molecular dynamics simulation with an explicit lipid bilayer membrane, similar to the system used for the solid-state NMR experiments. Based on 0.1 micros of simulation, both backbone structures converge to a structure with 6.25 residues per turn, in agreement with X-ray scattering, and broad agreement with SS backbone NMR observables. The side chain of Trp 9 is mobile, more so than Trp 11, 13, and 15, and undergoes spontaneous transitions between the orientations in 1JNO and 1MAG. Based on empirical fitting to the NMR results, and umbrella sampling calculations, we conclude that Trp 9 spends 80% of the time in the 1JNO orientation and 20% in the 1MAG orientation. These results underscore the utility of molecular dynamics simulations in the analysis and interpretation of structural information from solid-state NMR.     

Elastic deformation of membrane bilayers probed by deuterium NMR relaxation

In deuterium ((2)H) NMR spectroscopy of fluid lipid bilayers, the average structure is manifested in the segmental order parameters (S(CD)) of the flexible molecules. The corresponding spin-lattice relaxation rates (R(1Z) depend on both the amplitudes and the rates of the segmental fluctuations, and indicate the types of lipid motions. By combining (2)H NMR order parameter measurements with relaxation studies, we have obtained a more comprehensive picture of lipids in the liquid-crystalline (L(alpha)) state than formerly possible. Our data suggest that a lipid bilayer constitutes an ordered fluid, in which the phospholipids are grafted to the aqueous interface via their polar headgroups, whereas the fatty acyl chains are in effect liquid hydrocarbon. Studies of (2)H-labeled saturated lipids indicate their R(1Z) rates and S(CD) order parameters are correlated by a model-free, square-law functional dependence, signifying the presence of relatively slow bilayer fluctuations. A new composite membrane deformation model explains simultaneously the frequency (magnetic field) dependence and the angular anisotropy of the relaxation. The results imply the R(1Z) rates are due to a broad spectrum of 3-D collective bilayer excitations, together with effective axial rotations of the lipids. For the first time, NMR relaxation studies show that the viscoelastic properties of membrane lipids at megahertz frequencies are modulated by the lipid acyl length (bilayer thickness), polar headgroups (bilayer interfacial area), inclusion of a nonionic detergent (C(12)E(8)), and the presence of cholesterol, leading to a range of bilayer softness. Our findings imply the concept of elastic deformation is relevant on lengths approaching the bilayer thickness and less (the mesoscopic scale), and suggest that application of combined R(1Z) and S(CD) studies of phospholipids can be used as a simple membrane elastometer. Heuristic estimates of the bilayer bending rigidity kappa and the area elastic modulus K(a) enable comparison to other biophysical studies, involving macroscopic deformation of thin membrane lipid films. Finally, the bilayer softness may be correlated with the lipid diversity of biomembranes, for example, with regard to membrane curvature, repulsive interactions between bilayers, and lipid-protein interactions.     

Lipid dynamics and protein-lipid interactions in 2D crystals formed with the β-barrel integral membrane protein VDAC1

We employ a combination of (13)C/(15)N magic angle spinning (MAS) NMR and (2)H NMR to study the structural and functional consequences of different membrane environments on VDAC1 and, conversely, the effect of VDAC1 on the structure of the lipid bilayer. MAS spectra reveal a well-structured VDAC1 in 2D crystals of dimyristoylphosphatidylcholine (DMPC) and diphytanoylphosphatidylcholine (DPhPC), and their temperature dependence suggests that the VDAC structure does not change conformation above and below the lipid phase transition temperature. The same data show that the N-terminus remains structured at both low and high temperatures. Importantly, functional studies based on electrophysiological measurements on these same samples show fully functional channels, even without the presence of Triton X-100 that has been found necessary for in vitro-refolded channels. (2)H solid-state NMR and differential scanning calorimetry were used to investigate the dynamics and phase behavior of the lipids within the VDAC1 2D crystals. (2)H NMR spectra indicate that the presence of protein in DMPC results in a broad lipid phase transition that is shifted from 19 to ~27 °C and show the existence of different lipid populations, consistent with the presence of both annular and bulk lipids in the functionally and structurally homogeneous samples.     

Solid‐state NMR spectroscopic studies on the interaction of sorbic acid with phospholipid membranes at different pH levels

²H, ³¹P, and ¹H‐magic‐angle‐spinning (MAS) solid‐state NMR spectroscopic methods were used to elucidate the interaction between sorbic acid, a widely used weak acid food preservative, and 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) bilayers under both acidic and neutral pH conditions. The linewidth broadening observed in the ³¹P NMR powder pattern spectra and the changes in the ³¹P longitudinal relaxation time (T₁) indicate interaction with the phospholipid headgroup upon titration of sorbic acid or decanoic acid into DMPC bilayers over the pH range from 3.0 to 7.4. The peak intensities of sorbic acid decrease upon addition of paramagnetic Mn²⁺ ions in DMPC bilayers as recorded in the ¹H MAS NMR spectra, suggesting that sorbic acid molecules are in close proximity with the membrane/aqueous surface. No significant ²H quadrupolar splitting (Δν_Q) changes are observed in the ²H NMR spectra of DMPC‐d₅₄ upon titration of sorbic acid, and the change of pH has a slight effect on Δν_Q, indicating that sorbic acid has weak influence on the orientation order of the DMPC acyl chains in the fluid phase over the pH range from 3.0 to 7.4. This finding is in contrast to the results of the decanoic acid/DMPC‐d₅₄ systems, where Δν_Q increases as the concentration of decanoic acid increases. Thus, in the membrane association process, sorbic acids are most likely interacting with the headgroups and shallowly embedded near the top of the phospholipid headgroups, rather than inserting deep into the acyl chains. Thus, antimicrobial mode of action for sorbic acid may be different from that of long‐chain fatty acids. Copyright © 2009 John Wiley & Sons, Ltd.     

Interaction of Polyunsaturated Fatty Acids with Cholesterol: A Role in Lipid Raft Phase Separation

Unequal affinity between lipids has been hypothesized to be a mechanism for the formation of microdomains/rafts in membranes. Our studies focus upon the interaction of cholesterol with polyunsaturated fatty acid (PUFA)-containing phospholipids. They support the proposal that steric incompatibility of the rigid steroid moiety for highly disordered PUFA chains, in particular docosahexaenoic acid (DHA), provides a sensitive trigger for lateral segregation of lipids into PUFA-rich/sterol-poor and PUFA-poor/sterol-rich regions. Solid state ²H NMR and x-ray diffraction (XRD) demonstrate that the solubility of cholesterol is reduced in 1-palmitoyl-2-docosahexaenoyl-phosphatidylethanolamine (16-0:22:6PE) bilayers. In mixed membranes of phosphatidylethanolamine (PE) with the lipid raft forming molecules egg sphingomyelin (SM) and cholesterol, diminished affinity of the sterol for 16:0-22:6PE relative to 1-palmitoyl-2-oleoylphosphatidylethanolamine (16:0-18:1PE) is identified by ²H NMR order parameters and detergent extraction. Phase separation of the PUFA-containing phospholipid from SM/cholesterol rafts is the implication, which may be associated with the myriad of health benefits of dietary DHA.     

The cell-penetrating peptide TAT(48-60) induces a non-lamellar phase in DMPC membranes.

Cell-penetrating peptides (CPPs) are short polycationic sequences that can translocate into cells without disintegrating the plasma membrane. CPPs are useful tools for delivering cargo, but their molecular mechanism of crossing the lipid bilayer remains unclear. Here we study the interaction of the HIV-derived CPP TAT (48-60) with model membranes by solid-state NMR spectroscopy and electron microscopy. The peptide induces a pronounced isotropic (31)P NMR signal in zwitterionic DMPC, but not in anionic DMPG bilayers. Octaarginine and to a lesser extent octalysine have the same effect, in contrast to other cationic amphiphilic membrane-active peptides. The observed non-lamellar lipid morphology is attributed to specific interactions of polycationic peptides with phosphocholine head groups, rather than to electrostatic interactions. Freeze-fracture electron microscopy indicates that TAT(48-60) induces the formation of rodlike, presumably inverted micelles in DMPC, which may represent intermediates during the translocation across eukaryotic membranes.