Dimyristoylphosphatidylcholine Bilayer Membrane Research Articles

Diffusion and spectroscopy of water and lipids in fully hydrated dimyristoylphosphatidylcholine bilayer membranes

Microscopic structure and dynamics of water and lipids in a fully hydrated dimyristoylphosphatidylcholine phospholipid lipid bilayer membrane in the liquid-crystalline phase have been analyzed with all-atom molecular dynamics simulations based on the recently parameterized CHARMM36 force field. The diffusive dynamics of the membrane lipids and of its hydration water, their reorientational motions as well as their corresponding spectral densities, related to the absorption of radiation, have been considered for the first time using the present force field. In addition, structural properties such as density and pressure profiles, a deuterium-order parameter, surface tension, and the extent of water penetration in the membrane have been analyzed. Molecular self-diffusion, reorientational motions, and spectral densities of atomic species reveal a variety of time scales playing a role in membrane dynamics. The mechanisms of lipid motion strongly depend on the time scale considered, from fast ballistic translation at the scale of picoseconds (effective diffusion coefficients of the order of 10(-5) cm(2)/s) to diffusive flow of a few lipids forming nanodomains at the scale of hundreds of nanoseconds (diffusion coefficients of the order of 10(-8) cm(2)/s). In the intermediate regime of sub-diffusion, collisions with nearest neighbors prevent the lipids to achieve full diffusion. Lipid reorientations along selected directions agree well with reported nuclear magnetic resonance data and indicate two different time scales, one about 1 ns and a second one in the range of 2-8 ns. We associated the two time scales of reorientational motions with angular distributions of selected vectors. Calculated spectral densities corresponding to lipid and water reveal an overall good qualitative agreement with Fourier transform infrared spectroscopy experiments. Our simulations indicate a blue-shift of the low frequency spectral bands of hydration water as a result of its interaction with lipids. We have thoroughly analyzed the physical meaning of all spectral features from lipid atomic sites and correlated them with experimental data. Our findings include a "wagging of the tails" frequency around 30 cm(-1), which essentially corresponds to motions of the tail-group along the instantaneous plane formed by the two lipid tails, i.e., in-plane oscillations are clearly of bigger importance than those along the normal-to-the plane direction.

Application of pressure perturbation calorimetry to the aqueous system of phospholipid vesicle dispersion

Pressure perturbation calorimetry (PPC) is a relatively new technique which provides the thermal expansion coefficient α2 of a solute. We give a detailed description on the theoretical background of this technique for a binary solution and a theoretical explanation for the method to estimate the volume change Δ v app, 2 with a phase transition. In this theory, a reversible isothermal pressure change is assumed, although an irreversible pressure change is applied in the PPC experiment. In order to verify the effect of this inconsistency, we carried out the PPC measurements for the dimyristoylphosphatidylcholine bilayer membrane. The Δ v app, 2 value with the gel-to-liquid-crystalline transition was estimated at 18.3 ml mol−1, which is in agreement with the literature values obtained by other techniques. This indicates that the irreversibility has virtually no effect on the Δ v app, 2 value and also supports the validity of the method for estimating the Δ v app, 2 value that we adopted.

Effects of Fatty Acid Inclusion in a DMPC Bilayer Membrane

Free fatty acids in biomembranes have been proposed to be a central component in several cellular control and regulatory mechanisms. To elucidate some fundamental elements underlying this, we have applied molecular dynamics simulations and experimental density measurements to study the molecular packing and structure of oleic acid (HOA) and stearic acid (HSA) in fluid bilayers of dimyristoylphosphatidylcholine (DMPC). The experimental data show a small but consistent positive excess volume for fatty acid concentrations below 10 mol %. At higher concentrations the fatty acids mix ideally with fluid DMPC. The simulations, which were benchmarked against the densitometric data, revealed interesting differences in the structure and location of the fatty acids depending on their protonation status. Thus, the protonated (uncharged) acid is located rather deeply in the membrane with an average position of the carboxy group near the second carbon segment of the lipid chains with a typical end-to-end distance of 16-18 A. This structure of the fatty acid brings about a rather tight lateral packing in the mixed membrane and a moderate ordering and hence stretching of the lipid chains. Deprotonation of the fatty acids is associated with a pronounced movement of their carboxy group to a more hydrated position at the membrane interface and a lateral expansion driven by the mutual repulsion of the anions. These changes increase both the disorder and the degree of interdigitation of the lipid chains, and they make the membrane thinner by 2-3 A.

Incorporation of Outer Membrane Protein OmpG in Lipid Membranes: Protein−lipid Interactions and β-Barrel Orientation

OmpG is an intermediate size, monomeric, outer membrane protein from Escherichia coli, with n beta = 14 beta-strands. It has a large pore that is amenable to modification by protein engineering. The stoichiometry ( N b = 20) and selectivity ( K r = 0.7-1.2) of lipid-protein interaction with OmpG incorporated in dimyristoyl phosphatidylcholine bilayer membranes was determined with various 14-position spin-labeled lipids by using EPR spectroscopy. The limited selectivity for different lipid species is consistent with the disposition of charged residues in the protein. The conformation and orientation (beta-strand tilt and beta-barrel order parameters) of OmpG in disaturated phosphatidylcholines of odd and even chain lengths from C(12:0) to C(17:0) was determined from polarized infrared spectroscopy of the amide I and amide II bands. A discontinuity in the protein orientation (deduced from the beta-barrel order parameters) is observed at the point of hydrophobic matching of the protein with lipid chain length. Compared with smaller (OmpA; n beta = 8) and larger (FhuA; n beta = 22) monomeric E. coli outer membrane proteins, the stoichiometry of motionally restricted lipids increases linearly with the number of beta-strands, the tilt (beta approximately 44 degrees ) of the beta-strands is comparable for the three proteins, and the order parameter of the beta-barrel increases regularly with n beta. These systematic features of the integration of monomeric beta-barrel proteins in lipid membranes could be useful for characterizing outer membrane proteins of unknown structure.

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the 9- cis and 13- cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all- trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13- cis and 9- cis zeaxanthin, respectively; whereas, incorporation of all- trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all- trans < 9- cis ≤ 13- cis. Obtained results suggest that the investigated cis-isomers of zeaxanthin, similar to the all- trans isomer, are located in the membrane interior, adopting transmembrane orientation with the polar terminal hydroxyl groups located in the opposite leaflets of the bilayer. However, the existence of the second pool of cis-zeaxanthin molecules located in the one leaflet and anchored by the terminal hydroxyl groups in the same polar headgroup region cannot be completely ruled out.

TOAC Spin Labels in the Backbone of Alamethicin: EPR Studies in Lipid Membranes

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe) 7,18,19 variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC n alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic 14N-hyperfine couplings indicate that TOAC 8 and TOAC 16 are situated in the hydrophobic core of the membrane, whereas the TOAC 1 label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.

Effects of pH-induced variations of the charge of the transmembrane α-helical peptide Ac-K 2(LA) 12K 2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the transmembrane α-helical peptide Ac-K 2(LA) 12K 2-amide ((LA) 12) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 °C when measured at pH 7.0, by 1.6 °C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA) 12. Apparent p K as of the lysine side chain amino groups of (LA) 12 in DMPC bilayer are 8.6 and ∼10.9, as compared with the p K a value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA) 12 are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA) 12-rich regions are faster than 0.5 μs, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA) 12 do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA) 12 on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA) 12 membrane interior.

High-field spin-label EPR of lipid membranes

High-field EPR of spin-labelled lipid chains has proved to be an extremely productive means for biophysical investigations of phospholipid bilayer membranes. Results on the following three topics are reviewed: 1. Non-axial ordering of lipid chains in cholesterol-containing membranes; 2. Transmembrane polarity profiles and water penetration in lipid membranes; 3. Role of HF-EPR in multi-frequency spectral simulations to investigate lipid-chain dynamics in membranes. These results are obtained from phosphatidylcholine spin probes with the nitroxide systematically stepped down the sn-2 chain, in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine (DMPC)-containing cholesterol.

Membrane disordering induced by chloroform and carbon tetrachloride

We studied effects of chloroform and carbon tetrachloride on bilayer membranes of dimyristoyl-phosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (Egg-PC) by birefringence, dynamic light scattering and fluorescence methods. It is shown that interference light due to the membrane birefringence considerably decreases by addition of the organohalogen compounds for both lipid membranes, indicating a significant decrease in membrane order. In addition, results of dynamic light scattering and turbidity measurements show a rupture of multilamellar DMPC vesicles induced by addition of chloroform at concentrations above 0.2 v/v%. No rupture of the vesicles is observed within the limit of solubility of carbon tetrachloride in water, but excessive addition of carbon tetrachloride (above 0.2 v/v%) induces the vesicle rupture. Chain orientational order was estimated from the interference light intensity at low concentrations of the organohalogen compounds without the occurrence of the vesicle rupture. The estimation shows a monotonic decrease in the chain order with increasing the concentration. The decreases in DMPC chain order by chloroform and by carbon tetrachloride are about 17% at 0.2 v/v% and 23% at 0.05 v/v%, respectively. The reduction in the chain order is correlated with an increase in the membrane fluidity observed by excimer fluorescence of pyrene incorporated to the membrane. Behavior of membrane disordering of Egg-PC is approximately similar with that of DMPC. This implies the strong interaction between the organohalogen compounds and the lipid chains, whether or not the bilayer has the vacancy resulted from unsaturated double bonds and different chains in length. The results of this work suggest that damages of biological membranes by chloroform and tetrachloride are not only induced by a direct attack on proteins but also by a significant membrane disorder.

Comparative dynamics and location of chain spin-labelled sphingomyelin and phosphatidylcholine in dimyristoyl phosphatidylcholine membranes studied by EPR spectroscopy

The dynamics and environment of sphingomyelin spin-labelled at different positions in the N-acyl chain have been studied in dimyristoyl phosphatidylcholine bilayer membranes by using electron spin resonance spectroscopy. Comparison was made with phosphatidylcholine spin-labelled on the sn-2 acyl chain in the same host membrane. Spin-labelled sphingomyelin was found to mix well with the host phosphatidylcholine lipids in both gel and fluid phase membranes. At 1 mol%, mutual spin–spin interactions are no greater than for spin-labelled phosphatidylcholine. In the fluid membrane phase, the effective chain order parameters and polarity-sensitive isotropic hyperfine coupling constants of spin-labelled sphingomyelin display a similar dependence on the position of labelling to those of spin-labelled phosphatidylcholine. The values of both parameters are, however, generally larger for sphingomyelin than for phosphatidylcholine at equivalent positions of acyl chain labelling. This difference is attributed to the different chain linkage of sphingo- and glycero-lipids, combined with an offset of approximately one C-atom in transbilayer register between the respective N-acyl and O-acyl chains. In the gel phase, differences in chain configuration between sphingomyelin and phosphatidylcholine are indicated by differences in spin label spectral anisotropy between the two lipids, which appears to reverse towards the terminal methyl chain end.

Molecular Dynamics of a Vasopressin V2 Receptor in a Phospholipid Bilayer Membrane

Molecular dynamics simulations were carried out for a V2 receptor (V2R) model embedded in a dimyristoylphosphatidylcholine (DMPC) bilayer. Both free and ligand-bound states of V2R were modeled. Our initial V2R model was obtained using a rule-based automated method for GPCR modeling and refined using constrained simulated annealing in vacuo. The docking site of the native vasopressin ligand was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. The primary purpose of this work was to investigate the usefulness of MD simulation of an integral membrane protein like a GPCR. receptor, upon inclusion of a carefully parameterized surrounding lipid membrane and water. Physical properties of the system were evaluated and compared with the fully hydrated pure DMPC bilayer membrane. The solvation interactions, individual lipid-protein interaction and fluctuations of the protein, the lipid, and water were analyzed in detail. As expected, the membrane-spanning helices of the protein fluctuate less than the peripheral loops do. The protein appears to disturb the local lipid structure. Simulations were carried out using AMBER 4.1 package upon constant number-pressure-temperature (NPT) conditions on massively parallel computers Cray T3E and IBM SP2.

Protein-Induced Vertical Lipid Dislocation in a Model Membrane System: Spin-Label Relaxation Studies on Avidin-Biotinylphosphatidylethanolamine Interactions

The change in vertical location of spin-labeled N-biotinyl phosphatidylethanolamine in fluid-phase dimyristoyl phosphatidylcholine bilayer membranes, on binding avidin to the biotinyl headgroup, has been investigated by progressive saturation electron spin resonance measurements. Spin-labeled phospholipids were present at a concentration of 1 mol%, relative to total membrane lipids. For avidin-bound N-biotinyl phosphatidylethanolamine spin-labeled on the 8 C atom of the sn-2 chain, the relaxation enhancement induced by 30 mM Ni 2+ ions confined to the aqueous phase was 2.5 times that induced by saturating molecular oxygen, which is preferentially concentrated in the hydrophobic core of the membrane. For phosphatidylcholine also spin-labeled at the 8 position of the sn-2 chain, this ratio was reversed: the relaxation enhancement by Ni 2+ ions was half that induced by molecular oxygen. In the absence of avidin, the enhancement by either relaxant was the same for both spin-labeled phospholipids. For a double-labeled system, in which both N-biotinyl phosphatidylethanolamine and phosphatidylcholine were spin-labeled on the 12 C atom of the sn-2 chain, the relaxation rate in the absence of avidin was greater than that predicted from linear additivity of the corresponding singly labeled systems, because of mutual spin-spin interactions between the two labeled lipid species. On binding of avidin to the N-biotinyl phosphatidylethanolamine, this relaxation enhancement by mutual spin-spin interaction was very much decreased. These results indicate that, on binding of avidin to the lipid headgroup, N-biotinyl phosphatidylethanolamine is lifted vertically within the membrane, relative to the phosphatidylcholine host lipids. The specific binding of avidin to N-biotinyl phosphatidylethanolamine parallels the liftase activity proposed for activator proteins associated with the action of certain gangliosidases.

High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes.

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the sn-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic 14N-hyperfine splittings are resolved in the gzz and gyy regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged <Azz> element of the 14N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the 14N-hyperfine manifolds in the gzz region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.

Barotropic Phase Transitions and Pressure-induced Interdigitation on Bilayer Membrane of Dimyristoylphosphatidylcholine

Abstract The thermotropic and barotropic phase transitions of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicle have been studied. Both temperatures of the main transition and pretransition were elevated by pressure. A pressure-induced interdigitation of DMPC bilayer membrane was observed at high pressures above 300 MPa.

Partitioning of local anesthetic dibucaine into bilayer membranes of dimyristoylphosphatidylcholine

Binding of a local anesthetic dibucaine to dimyristoylphosphatidylcholine (DMPC) bilayer membranes was studied by using an ion-selective electrode sensitive to dibucaine cation. DMPC bilayer membrane-buffer partition coefficient was directly determined as a function of anesthetic concentration, temperature and pH. The limiting partition coefficient extrapolated to infinite dilution was employed because the values of partition coefficients were dependent upon the dibucaine concentration. DMPC multilamellar vesicles undergo the thermotropic pretransition from the lamellar gel ( L β′ ) to the ripple gel ( P β′ ) phase as well as the main transition from the P β′ phase to the liquid crystal ( L α ) phase. The limiting partition coefficients at pH 5.4 were 2000, 5500 and 32 000 for the L β′ , P β′ and L α phases, respectively. Each of the three states of membranes exhibited a different receptivity to dibucaine partitioning. The limiting partition coefficients were determined as a function of pH at the temperatures corresponding to three states of membranes. The partition coefficients of charged and uncharged anesthetic dibucaine into the DMPC bilayer membranes were estimated from the pH-dependence of the limiting partition coefficients. The three states of DMPC membranes were more receptive to the uncharged dibucaine than the charged species.

Rotational Motion of Square Planar Copper Complexes in Solution and Phospholipid Bilayer Membranes

Electron spin resonance (ESR) spectra of 3-ethoxy-2-oxobutyraldehydebis(N,N-dimethylthiosemicarbazonato)copper(II) (CuKTSM2) and copper(II) tetraphenylporphyrin (CuTPP), in paraffin oil at various temperatures, as well as in oriented dimyristoylphosphatidylcholine bilayer membranes, at 25 °C, were simulated using the stochastic Liouville equation formalism and a postconvolution method. It has been recognized that because nitrogen hyperfine couplings are nearly isotropic, they can be easily introduced after a simulation that omits ligand nuclear parameters, by convoluting a stick diagram for the nitrogen splitting with the copper spectrum. For the whole range of temperatures studied (from −10 to +120 °C for CuTPP and from −60 to +120 °C for CuKTSM2), the line shapes of ESR spectra for these square planar copper complexes can be accounted for by motion. The ESR spectra of Cu(II) in oriented bilayers are well reproduced for the magnetic field both perpendicular and parallel to the membrane plane. Spectra, fo...

Spin-label studies on the anchoring and lipid-protein interactions of avidin with N-biotinylphosphatidylethanolamines in lipid bilayer membranes.

The specific binding of hen egg white avidin to phosphatidylcholine lipid membranes containing spin-labeled N-biotinylphosphatidylethanolamines (biotin-PESLs) was investigated by using ESR spectroscopy. Spin-labeled biotin-PEs were prepared with the nitroxide group at position C-5, C-8, C-10, C-12, or C-14 of the sn-2 chain and were incorporated at 1 mol % in lipid bilayer membranes of dimyristoylphosphatidylcholine. Binding of avidin produced a strong and selective restriction of the biotin-PE lipid mobility at all positions of chain labeling, as shown by the ESR spectra recorded in the fluid lipid phase. The spectral components of the fraction of the biotin-PESLs that were not complexed by avidin indicated that the mobility of the bulk membrane lipids was unperturbed by binding avidin, as demonstrated by difference spectroscopy. Comparison of the positional profiles and temperature dependences of the outer hyperfine splittings from the biotin-PESLs suggests that the C-12 and C-14 positions of the avidin-bound biotin-PEs are in register with the C-5 and C-7/C-6 positions, respectively, of the chains of the bulk membrane lipids. This indicates that the biotin-PEs are partially withdrawn from the membrane, with a vertical displacement of ca. 7-8 A, on complexation with avidin. In addition, the specific lipid-protein interaction with avidin results in a selective reduction in the rates of lipid chain motion, as shown by the increased ESR line widths. These data define the way in which avidin is anchored to lipid membranes containing biotin-PEs.

Evidence for domains in deposited lipid bilayers

We have modeled dimyristoylphosphatidylcholine bilayer membranes deposited on argon-sputtered glass or polyacrylamide substrates in order to understand the dependence of the lipid self-diffusion coefficient upon temperature. We used a lattice model that has been successful in understanding some of the thermodynamics of lipid bilayers. We considered two modifications of this model : model I in which the bilayer was described by a distribution of noninteracting bilayer domains within and between which lipids could move and model II in which lipid-lipid interactions were weakened in a random way thereby permitting large diffusion coefficients at low temperatures. We modeled the lateral exchange oflipids as being permitted only when either an adjacent pair of lipids or a mutually adjacent trio of lipids were in their excited states. We carried out a direct computer simulation of lipid movement or related the self-diffusion coefficient to pair or triplet correlation functions. We found that the results predicted by the unperturbed model are in general agreement with measurements of the self-diffusion coefficient on oxidized silica wafers as a prototype of a polished surface. This permitted us to apply the perturbed models to study the other cases for which we found that only model I could account for measurements on argon-sputtered glass and on polyacrylamide films. Argon-sputtered glass yielded domain distributions with a most probable size of ∼250 to 600 lipids per half-bilayer, while polyacrylamide films yielded a most probable domain size of ∼600 lipids per half-bilayer. On the basis of these results, we have made predictions about the average number of gauche bonds per molecule as a function of temperature.

Accessibility of spin-labeled phospholipids in anionic and zwitterionic bilayer membranes to paramagnetic relaxation agents. Continuous wave power saturation EPR studies

The location of phospholipids, spin-labeled in the headgroup or at various positions of the sn-2 chain, incorporated in bilayer membranes of dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol or dioleoylphosphatidylglycerol, has been calibrated in terms of their accessibility to paramagnetic relaxation agents. A power saturation approach has been used to determine the spin-label relaxation times, which in turn is influenced by spin—spin interactions with the different paramagnetic species. The effect of different paramagnetic relaxation agents on the power saturation behaviour of the spin-labeled lipids has been used to determine the relaxation enhancement which is quantified in terms of an accessibility parameter. Molecular oxygen, which dissolves preferentially in the lipid phase and the water-soluble, membrane-impermeant chromium oxalate anion are shown to report reliably on the accessibility of spin-labels located in one of the two phases. On the other hand, an uncharged, polar nickel-iminodiacetic acid complex is shown to enhance relaxation of spin-labels in both phases. These calibrations are essential for the study of the interaction of basic proteins with anionic lipid membranes.

Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram

Small-angle neutron scattering has been used to study structural features of lamellar bilayer membranes of dimyristoylphosphatidylcholine (DMPC) and DMPC mixed with various amount of cholesterol. The studies were recorded at a fixed hydration level of 17% 2H 2O, i.e. just below saturation. Bragg reflections gives information on the ripple structure and on the bilayer periodicity. The crystalline L c phase, which was stabilized after long time storage at low temperature, exhibits major small angle scattering when cholesterol is mixed into the membrane. The intermediate P β′ gel-phase, which is characteristic by the rippled structure, is dramatically stabilized by the introduction of cholesterol. The ripple structure depends significantly both on the cholesterol content and on the temperature. At high temperatures, T>15°C, the inverse ripple periodicity varies basically linearly with cholesterol content, and approach zero (i.e. periodicity goes to infinite) at 20 mol% cholesterol, approximately. At lower temperatures the correlation is more complex. The data indicate additional phase boundaries below 2 mol% and at approx. 8 mol%. Secondary rippled structures are observed in the low temperature L β′-phase for cholesterol content below approx. 8 mol%. The data gives detailed insight into the phosphatidylcholine cholesterol phase diagram, which is discussed on the basis of a simple model in which the cholesterol complexes are fixed to the defect stripes of the rippled structure.