The effect of N-(1-methyldodecyl)-N,N-dimethylaminoxide on the conformation of hydrocarbon chains in phospholipid bilayers isolated from Escherichia coli

Using the method of ESR spectroscopy of stearic acid spin probes labeled by the doxyl group on the 12th or 16th carbon, it has been found that bactericidal surfactant N-(1-methyldodecyl)-N,N-dimethylamine oxide increases the effective energy difference between trans- and gauche conformers Eg and decreases the probability of gauche conformers formation pg in lipid hydrocarbon chains in multilamellar liposomes prepared from Escherichia coli-isolated phospholipids, at low surfactant concentrations. Above the surfactant: phospholipid molar ratio of 1:14 to 1:17, the value of Eg decreases and that of pg increases. The results are interpreted using the cluster model of lipid bilayer. At low concentrations the surfactant molecules are inserted into the dynamical defects between the clusters, thereby increasing the packing density of chains in the bilayer. At high concentrations the surfactant molecules penetrate into the clusters perturbing the dense packing of chains in clusters.     

A self-consistent chain model for the phase transitions in lipid bilayer membranes

We presented a mechanical model of a lipid bilayer membrane. The internal conformations of a polar head group and double hydrocarbon chains in a lipid molecule were described on the basis of the isomeric bond-rotation scheme. The thermodynamic properties of the lipid membranes were represented by a density matrix that described the rotational isomeric states of the head groups and chains. The parameters that determined the density matrix were obtained in the presence of the intermolecular interactions, which depend on the conformation of the molecules. The interchain interaction was given by the Kihara potential, which depends on the shape of the chains. The Coulomb interaction between the polar head groups and the lateral pressure were considered. The calculation was made for the three lipid molecules corresponding to DMPC, DPPC, and DSPC. The model agreed well with the following experimental results: the temperature, the latent heat of the gel-to-liquid crystalline phase transition, the temperature dependencies of (a) the intermolecular distance, (b) the number of gauche bonds in a hydrocarbon chain, (c) the order parameter for the bond orientation, (d) the volume of the membrane, (e) the thermal expansion coefficients, and (f) the birefringence.     

Behavior of cholesterol and its effect on head group and chain conformations in lipid bilayers: a molecular dynamics study.

Cholesterol molecules were put into a computer-modeled hydrated bilayer of dimyristoyl phosphatidyl choline molecules, and molecular dynamics simulations were run to characterize the effect of this important molecule on membrane structure and dynamics. The effect was judged by observing differences in order parameters, tilt angles, and the fraction of gauche bonds along the hydrocarbon chains between lipids adjacent to cholesterol molecules and comparing them with those further away. It was observed that cholesterol causes an increase in the fraction of trans dihedrals and motional ordering of chains close to the rigid steroid ring system with a decrease in the kink population. The hydrogen-bonding interactions between cholesterol and lipid molecules were determined from radial distribution calculations and showed the cholesterol hydroxyl groups either solvated by water, or forming hydrogen bond contacts with the oxygens of lipid carbonyl and phosphate groups. The dynamics and conformation of the cholesterol molecules were investigated and it was seen that they had a smaller tilt with respect to the bilayer normal than the lipid chains and furthermore that the hydrocarbon tail of the cholesterol was conformationally flexible.     

Interaction of surfactants with model and biological membranes. II. Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine bilayers as studied by spin probe ESR

The interaction of N-alkyl-N,N,N-trimethylammonium (CnTMA, n = 6-18) salts (iodides and/or bromides) with model membranes prepared by hydration of egg yolk phosphatidylcholine (EYPC) over aqueous salt solutions has been studied by m-doxyl stearic acid (m-DSA, m = 12 and 16) spin probe method. In disoriented EYPC bilayers the CnTMA salts decrease the orientational order parameter S33 of m-DSA evaluated from the powder pattern ESR spectra. This effect is maximal for C6TMA. In oriented EYPC bilayers prepared by the parallel-beam sputtering method and hydrated over saturated NaCl solution the order parameter S33 calculated from the angular dependence of the nitrogen hyperfine splitting is decreased in the presence of C6TMA. The order parameter S11 obtained from the angular dependence of line positions indicates deviation of m-DSA motion from axial symmetry. C6TMA increases the probability of gauche conformations of the lipid chains by about 13-14%, and decreases the effective energy difference between the trans and gauche conformations by about 420-480 J/mol, at molar ratio of EYPC/C6TMA = 2:1. The angular dependence of linewidths is analysed by employing a theory of spin relaxation based on the strong collision model for molecular reorientations. The correlation time tau 0 of the reorientation of an axis orthogonal to the doxyl ring of 16-DSA is decreased in the presence of C6TMA, while that of 12-DSA is not influenced by it. The ratio of tau 2/tau 0 is increased in the presence of C6TMA for the both spin probes. The results are explained using the free-volume model of the CnTMA-EYPC membrane interaction.     

Molecular dynamics investigation of the structure of a fully hydrated gel-phase dipalmitoylphosphatidylcholine bilayer.

We report the results of a constant pressure and temperature molecular dynamics simulation of a gel-phase dipalmitoylphosphatidylcholine bilayer with nw = 11.8 water molecules/lipid at 19 degrees C. The results of the simulation were compared in detail with a variety of x-ray and neutron diffraction data. The average positions of specific carbon atoms along the bilayer normal and the interlamellar spacing and electron density profile were in very good agreement with neutron and x-ray diffraction results. The area per lipid and the details of the in-plane hydrocarbon chain structure were in excellent agreement with wide-angle x-ray diffraction results. The only significant deviation is that the chains met in a pleated arrangement at the bilayer center, although they should be parallel. Novel discoveries made in the present work include the observation of a bimodal headgroup orientational distribution. Furthermore, we found that there are a significant number of gauche conformations near the ends of the hydrocarbon chains and, in addition to verifying a previous suggestion that there is partial rotational ordering in the hydrocarbon chains, that the two chains in a given molecule are inequivalent with respect to rotations. Finally, we have investigated the lipid/water interface and found that the water penetrates beneath the headgroups, but not as far as the carbonyl groups, that the phosphates are strongly hydrated almost exclusively at the nonesterified oxygen atoms, and that the hydration of the ammonium groups is more diffuse, with some water molecules concentrated in the grooves between the methyl groups.     

A difference infrared spectroscopic study of gramicidin A, alamethicin and bacteriorhodopsin in perdeuterated dimyristoylphosphatidylcholine

Difference infrared spectroscopy has been used to study the way in which the intrinsic molecules gramicidin A, alamethicin and bacteriorhodopsin perturb their environment when present within a lipid bilayer structure. Dimyristoylphosphatidylcholine containing perdeuterated chains has been used to enable the lipid chain C-2H stretching absorption band to be separated from the C-H bands arising from the intrinsic polypeptide or protein. The C-2H stretching bands of the phospholipid are sensitive to two different types of chain conformation. The C-2H stretching frequency provides information about the static order of the lipid chains, whilst the half-maximum bandwidth provides a measure of chain librational and torsional motion. From the measurements it is concluded that: (1) Above the lipid phase transition temperature tc, low concentrations of either gramicidin A or alamethicin cause a small decrease in lipid chain gauche isomers whilst bacteriorhodopsin in the lipid bilayer has no effect. At higher concentrations each intrinsic molecule causes an increase to occur in lipid chain gauche isomers. (2) The lipid acyl chain motion, as deduced from the bandwidths is increased by the presence of a low concentration of gramicidin A within the lipid bilayer. The presence of the other intrinsic molecules studied have little effect. A higher concentration of alamethicin causes a decrease in chain motion whilst gramicidin A and bacteriorhodopsin have no effect. (3) Below tc each of the intrinsic molecules when present in the lipid bilayer causes an increase in gauche isomers to occur as well as an increase in the lipid chain motion. A broadening of the lipid phase transition occurs as the concentration of the polypeptide increases.     

Transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations.

To probe the fundamentals of membrane/protein interactions, all-atom multi-nanosecond molecular dynamics simulations were conducted on a single transmembrane poly(32)alanine helix in a fully solvated dimyristoyphosphatidylcholine (DMPC) bilayer. The central 12 residues, which interact only with the lipid hydrocarbon chains, maintained a very stable helical structure. Helical regions extended beyond these central 12 residues, but interactions with the lipid fatty-acyl ester linkages, the lipid headgroups, and water molecules made the helix less stable in this region. The C and N termini, exposed largely to water, existed as random coils. As a whole, the helix tilted substantially, from perpendicular to the bilayer plane (0 degree) to a 30 degrees tilt. The helix experienced a bend at its middle, and the two halves of the helix at times assumed substantially different tilts. Frequent hydrogen bonding, of up to 0.7 ns in duration, occurred between peptide and lipid molecules. This resulted in correlated translational diffusion between the helix and a few lipid molecules. Because of the large variation in lipid conformation, the lipid environment of the peptide was not well defined in terms of "annular" lipids and on average consisted of 18 lipid molecules. When compared with a "neat" bilayer without peptide, no significant difference was seen in the bilayer thickness, lipid conformations or diffusion, or headgroup orientation. However, the lipid hydrocarbon chain order parameters showed a significant decrease in order, especially in those methylene groups closest to the headgroup.     

Lipid-binding proteins: structure of the phospholipid ligands

Dihedral angles are evaluated for the phospholipid ligands of the lipid-binding proteins found in the Protein Data Base (PDB). Phospholipid structures occur with a trans C1-C2 configuration of the glycerol backbone and oppositely extended chains, in addition to the gauche C1-C2 rotamers found in membranes. Headgroup conformations are not restricted to the single bent-down configuration and gauche-gauche configuration of the phosphodiester that is found in phospholipid crystals. Additionally, fully extended headgroups and orientations directed away from the lipid chains are found for phospholipids in the protein binding pockets. On average, the hydrocarbon chains of the protein-bound lipids are conformationally more disordered than in fluid bilayer membranes. However, much of this configurational disorder arises from energetically disallowed skew conformations. This suggests a configurational heterogeneity in the lipids at a single binding site: Eclipsed conformations occur also in some lipid headgroups and glycerol backbones. Stereochemical violations appear for some of the ester carboxyl groups of the protein-bound phospholipids in the PDB, and two glycerol backbones have the incorrect enantiomeric configuration.     

Alterations in rat alveolar surfactant system induced by treatment with carbicron (O-[(2-butenoic acid)-N,N-dimethylamide-3-yl]-O,O-dimethylphosphate)

1. Intoxication of rats with carbicron (O-([2-butenoic acid)-N,N-dimethylamide-3-yl]-O,O-dimethylphosphate) induced a reduction of the total phospholipids and phosphatidylcholine in lung alveolar surfactant.2. The lipid transfer protein activity was inhibited due to carbicron treatment.3. No alterations were observed in phospholipase A₂ activity in the alveolar surfactant of intoxicated animals. The structural order parameter (S_DPH) of bilayer liposomes, prepared from surfactant phospholipids of carbicron-treated rats also remained unchanged.     

Lipid conformation in crystalline bilayers and in crystals of transmembrane proteins

Dihedral torsion angles evaluated for the phospholipid molecules resolved in the X-ray structures of transmembrane proteins in crystals are compared with those of phospholipids in bilayer crystals, and with the phospholipid conformations in fluid membranes. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1-C2 rotamers found invariably in phospholipid bilayer crystals. Lipid headgroup conformations in protein crystals also do not conform solely to the bent-down conformation, with gauche-gauche configuration of the phospho-diester, that is characteristic of phospholipid bilayer membranes. This suggests that the lipids that are resolved in crystals of membrane proteins are not representative of the entire lipid-protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from energetically disallowed skew conformations. This indicates a configurational heterogeneity in the lipids at a single binding site: eclipsed conformations occur also in some glycerol backbone torsion angles and C-C torsion angles in the lipid headgroups. Stereochemical violations in the protein-bound lipids are evidenced by one-third of the ester carboxyl groups in non-planar configurations, and certain of the carboxyls in the cis configuration. Some of the lipid structures in protein crystals have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in structures of the phytanyl chains associated with bacteriorhodopsin crystals are in the incorrect S-configuration.     

Constant-pressure molecular dynamics investigation of cholesterol effects in a dipalmitoylphosphatidylcholine bilayer.

We report a 1.4-ns constant-pressure molecular dynamics simulation of cholesterol at 12.5 mol% in a dipalmitoylphosphatidylcholine (DPPC) bilayer at 50 degrees C and compare the results to our previous simulation of a pure DPPC bilayer. The interlamellar spacing was increased by 2.5 A in the cholesterol-containing bilayer, consistent with x-ray diffraction results, whereas the bilayer thickness was increased by only 1 A. The bilayer/water interface was more abrupt because the lipid headgroups lie flatter to fill spaces left by the cholesterol molecules. This leads to less compensation by the lipid headgroups of the oriented water contribution to the membrane dipole potential and could explain the experimentally observed increase in the magnitude of the dipole potential by cholesterol. Our calculations suggested that 12.5 mol% cholesterol does not significantly affect the conformations and packing of the hydrocarbon chains and produces only a slight reduction in the empty free volume. However, cholesterol has a significant influence on the subnanosecond time scale lipid dynamics: the diffusion constant for the center-of-mass "rattling" motion was reduced by a factor of 3, and the reorientational motion of the methylene groups was slowed along the entire length of the hydrocarbon chains.     

Chain ordering in liquid crystals: II. Structure of bilayer membranes

The ordering of the hydrocarbon chain interior of bilayer membranes has been calculated using the molecular field approximation developed in previous work on liquid crystals. Different statistical averages are evaluated by exact summation over all conformations of a single chain in the field due to neighboring molecules. The internal energy of each conformation, as well as contributions arising from interaction with the molecular field and from a lateral pressure on the chain have been included.The results describe properties of both lipid monolayers and bilayers. For monolayers, the calculated pressure-area relationships are in good agreement with experimental observations. The order parameter for hydrocarbon chains in bilayers (or monolayers) as a function of temperature, lateral pressure and position along the chain, is shown and compared with the available NMR data. Combining the results of calculation and NMR measurements we obtain the value for intrinsic lateral pressure within bilayer membranes, in excellent agreement with direct measurements on surface monolayers.The calculation also gives average length of hydrocarbon chains, thermal expansion coefficient and fraction of bonds in gauche conformations. The effect of cholesterol and proteins within the bilayer is qualitatively described, and the contribution of the bilayer interior to membrane elasticity is determined.     

Structural and dynamical studies of mixed chlorophyll/phosphatidylcholine bilayers via x-ray diffraction, absorption polarization spectroscopy and nuclear magnetic resonance.

The structure and dynamics of phosphatidylcholine bilayers containing chlorophyll were studied by X-ray diffraction and absorption polarization spectroscopy in the form of hydrated orientated multilayers below the thermal phase transition of the lipid chains and by nuclear magnetic resonance in the form of single-wall vesicles above the thermal transition. Our results show that (a) chlorophyll is incorporated into the phosphatidylcholine bilayers with its porphyrin ring located anisotropically in the polar headgroup layer of the membrane and with its phytol chain penetrating in a relatively extended form between the phosphatidylcholine fatty acid chains in the hydrocarbon core of the mixed bilayer membrane and (b) the intramolecular anisotropic rotational dynamics of the host phosphatidylcholine molecules are significantly perturbed upon chlorophyll incorporation into the bilayer at all levels of the phosphatidylcholine structure. These dynamics for the host phosphatidylcholine fatty acids chains are qualitatively different from that of the incorporated chlorophyll phytol chains on a 10(-9)-10(-10)s time scale in the ideally mixed two-component bilayer.     

The physiological and morphological phenotype of a yeast mutant resistant to the quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride

We investigated the action of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride) on Saccharomyces cerevisiae yeast cells. Changes in the yeast cell ultrastructure were confirmed by electron microscopy. We treated resistant mutant cells with QAS, and confirmed destruction of the mutant cytoplasm, an increase in the thickness of the cell wall, separation of the cell wall from the cytoplasm, and the accumulation of numerous lipid droplets. We also observed a relatively high production of lipids in the cells of the parental wild-type strain Σ1278b and in its IM-resistant (IM^R) mutant in the presence of the QAS. The IM^R mutant showed increased sensitivity to CaCl₂ and SDS, and resistance to ethidium bromide, chloramphenicol, erythromycin and osmotic shock. It also tolerated growth at low pH. We suggest that the resistance to IM could be connected with the level of permeability of the cell membrane because the IM^R mutant was sensitive to this compound in vivo in the presence of SDS and guanidine hydrochloride, which cause increased permeability of the cell plasma membrane.     

Interaction of local anesthetics with lipid bilayers investigated by (1)H MAS NMR spectroscopy

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by (31)P and (1)H magic-angle spinning (MAS) NMR spectroscopy. The (31)P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.     

Membrane hydration and structure on a subnanometer scale as seen by high resolution solid state nuclear magnetic resonance: POPC and POPC/C12EO4 model membranes.

The position on a subnanometer scale and the dynamics of structurally important water in model membranes was determined using a combination of proton magic-angle spinning NMR (MAS) with two-dimensional NOESY NMR techniques. Here, we report studies on phosphocholine lipid bilayers that were then modified by the addition of a nonionic surfactant that is shown to dehydrate the lipid. These studies are supplemented by 13C magic-angle spinning NMR investigations to get information on the dynamics of segmental motions of the membrane molecules. It can be shown that the hydrophilic chain of the surfactant is positioned at least partially within the hydrophobic core of the lipid bilayer. With the above NMR approach, we are able to establish molecular contacts between water and the lipid headgroup as well as with certain groups of the hydrocarbon chains and the glycerol backbone. This is possible because high resolution proton and 13C-NMR spectra of multilamellar bilayer membranes are obtained using MAS. A phase-sensitive NOESY must also be applied to distinguish positive and negative cross-peaks in the two-dimensional plot. These studies have high potential to investigate membrane proteins hydration and structural organization in a natural lipid bilayer surrounding.     

Lipid chains and cholesterol in model membranes: a Monte Carlo Study

The Monte Carlo method has been employed to study the equilibrium properties of a planar array of hydrocarbon chains interacting with a cholesterol molecule. The chains are arranged to model one monolayer of a lipid bilayer and within this monolayer are allowed to move laterally and change conformations by gauche rotations. In the simulation cell there are 90 lipid chains and a single cholesterol molecule. Periodic boundary conditions are imposed upon the cell. The primary results of the calculations are order parameter profiles for the C-C bonds. These are calculated for (i) all chains, (ii) the 6 chains which are nearest neighbors to the cholesterol, and (iii) the 12 chains which are next-nearest neighbors to the cholesterol. Calculations are carried out for C-14, C-16, and C-18 chains. The results show that cholesterol strongly affects the upper portions of the chains, leaving them less able to change conformations. For C-16 and C-18 chains, the chain termini of the cholesterol neighbors are more disordered than the bulk chain termini. The magnitude of the effect depends strongly on the chain length. The results suggest that the changes in the lipid phase transition caused by cholesterol are a consequence of each cholesterol hindering the rotameric freedom of five to seven lipid chains.     

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.     

Clusters in a liquid-crystalline lipid bilayer

From analysis of the position and width of the diffuse maximum from X-ray scattering on hydrocarbon chains of phospholipid molecules, the average distance between neighbour chains in a bilayer and the values of interaction (correlation) radia were estimated. Comparison with the results of other methods applied shows that the cluster model of molecule packing in lipid bilayers explains the experimental data in the best way. The minimal dimensions of the clusters, average areas per molecule and approximate fraction of molecules in the clusters were estimated.     

Saturation with cholesterol increases vertical order and smoothes the surface of the phosphatidylcholine bilayer: a molecular simulation study

Molecular dynamics (MD) simulations of a mono-cis-unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer and a POPC bilayer containing 50mol% cholesterol (POPC-Chol50) were carried out for 200ns to compare the spatial organizations of the pure POPC bilayer and the POPC bilayer saturated with Chol. The results presented here indicate that saturation with Chol significantly narrows the distribution of vertical positions of the center-of-mass of POPC molecules and POPC atoms in the bilayer. In the POPC-Chol50 bilayer, the same moieties of the lipid molecules are better aligned at a given bilayer depth, forming the following clearly separated membrane regions: the polar headgroup, the rigid core consisting of steroid rings and upper fragments of the acyl chains, and the fluid hydrocarbon core consisting of Chol chains and the lower fragments of POPC chains. The membrane surface of the POPC-Chol50 bilayer is smooth. The results have biological significance because the POPC-Chol50 bilayer models the bulk phospholipid portion of the fiber-cell membrane in the eye lens. It is hypothesized that in the eye lens cholesterol-induced smoothing of the membrane surface decreases light-scattering and helps to maintain lens transparency.