Lipid-gramicidin interactions: dynamic structure of the boundary lipid by 2D-ELDOR

The use of 2D-electron-electron double resonance (2D-ELDOR) for the characterization of the boundary lipid in membrane vesicles of DPPC and gramicidin A' (GA) is reported. We show that 2D-ELDOR, with its enhanced spectral resolution to dynamic structure as compared with continuous-wave electron spin resonance, provides a reliable and useful way of studying lipid-protein interactions. The 2D-ELDOR spectra of the end-chain spin label 16-PC in DPPC/GA vesicles is composed of two components, which are assigned to the bulk lipids (with sharp auto peaks and crosspeaks) and to the boundary lipids (with broad auto peaks). Their distinction is clearest for higher temperatures and higher GA concentrations. The quantitative analysis of these spectra shows relatively faster motions and very low ordering for the end chain of the bulk lipids, whereas the boundary lipids show very high "y-ordering" and slower motions. The y-ordering represents a dynamic bending at the end of the boundary lipid acyl chain, which can then coat the GA molecules. These results are consistent with the previous studies by Ge and Freed (1999) using continuous-wave electron spin resonance, thereby supporting their model for GA aggregation and H(II) phase formation for high GA concentrations. Improved instrumental and simulation methods have been employed.     

Studies on lipid membranes by two-dimensional Fourier transform ESR: Enhancement of resolution to ordering and dynamics.

The first two-dimensional Fourier-transform electron spin resonance (2D-FT-ESR) studies of nitroxide-labeled lipids in membrane vesicles are reported. The considerable enhancement this experiment provides for extracting rotational and translational diffusion rates, as well as orientational ordering parameters by means of ESR spectroscopy, is demonstrated. The 2D spectral analysis is achieved using theoretical simulations that are fit to experiments by an efficient and automated nonlinear least squares approach. These methods are applied to dispersions of 1-palmitoyl-2oleoyl-sn-glycerophosphatidylcholine (POPC) model membranes utilizing spin labels 1-palmitoyl-2-(16-doxyl stearoyl) phosphatidylcholine and the 3-doxyl derivative of cholestan-3-one (CSL). Generally favorable agreement is obtained between the results obtained by 2D-FT-ESR on vesicles with the previous results on similar systems studied by continuous wave (cw) ESR on aligned samples. The precision in determining the dynamic and ordering parameters is significantly better for 2D-FT-ESR, even though the cw ESR spectra from membrane vesicles are resolved more poorly than those from well aligned samples. Some small differences in results by the two methods are discussed in terms of limitations of the methods and/or theoretical models, as well as possible differences between dynamic molecular structure in vesicles versus aligned membranes. An interesting observation with CSL/POPC, that the apparent homogeneous linewidths seem to increase in "real time," is tentatively attributed to the effects of slow director fluctuations in the membrane vesicles.     

An electron spin resonance study of interactions between gramicidin A'' and phosphatidylcholine bilayers.

The model of microscopic order and macroscopic disorder was used to stimulate electron spin resonance spectra of spin-labeled lipids, 5-PC, 10-PC, and 16-PC in multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) containing gramicidin A' (GA) at temperatures above the gel-to-liquid crystal transition of DPPC. The simulations show that at a lower concentration of GA (i.e., molar ratios of DPPC/GA greater than 3), GA has only a slight effect on the acyl chain dynamics. The rotational diffusion rate around the axis parallel to the long hydrocarbon chain remains unchanged or increases slightly, while the rate around the perpendicular axes decreases slightly. These spectra from DPPC/GA mixtures could only be fit successfully with two or more components consistent with the well-known concept of "boundary lipids," that is, the peptide induces structural inhomogeneity in lipid bilayers. However, the spectra were significantly better fit with additional components that exhibit increased local ordering, implying decreased amplitude of rotational motion, rather than immobilized components with sharply a reduced rotational rate. The largest relative effects occur at the end of the acyl chains, where the average local order parameter St of 16-PC increases from 0.06 for pure lipid to 0.66 for 1:1 DPPC/GA. The inhomogeneity in ordering in DPPC bilayers due to GA decreases with increasing temperature. The hyperfine tensor component Azz increases for 10-PC and 16-PC when GA is incorporated into DPPC bilayers, indicating that water has deeply penetrated into the DPPC bilayers. Simulations of published electron spin resonance spectra of 14-PC in dimyristoylphosphatidylcholine/cytochrome oxidase complexes were also better fit by additional components that were more ordered, rather than immobilized. The average local order parameter in this case is found to increase from 0.11 for pure dimyristoylphosphatidylcholine to 0.61 for a lipid/protein ratio of 50. These spectra and their simulations are similar to the results obtained with 16-PC in the DPPC/GA mixtures. The relevance to studies of lipid-protein interactions for other proteins is briefly discussed.     

ESR studies of spin-labeled membranes aligned by isopotential spin-dry ultracentrifugation: lipid-protein interactions.

Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol% BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol% GA/DPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptide- and protein-containing membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer.     

2H NMR evidence for antibiotic-induced cholesterol immobilization in biological model membranes

The interaction of the polyene antibiotic filipin with membrane sterols has been studied by deuterium nuclear magnetic resonance of the molecular probes [2,2,3,4,4,6-2H6]cholesterol and 1-myristoyl-2-[4',4',14',14',14'-2H5]myristoyl-sn-glycero-3-phospho- choline. At physiological temperatures, there is evidence of filipin-induced cholesterol immobilization in the membrane. The 2H NMR spectra of cholesterol show two domains in which ordering and dynamics are very different. In one of these, cholesterol is static on the 2H NMR time scale, whereas in the other it undergoes rapid axially symmetric motions similar to those it exhibits in the drug-free membrane; this indicates that the jumping frequency of cholesterol between the labile and immobilized domains is less than 10(5) s-1. The distribution of cholesterol between these two sites is temperature dependent; at 0 degrees C all sterol molecules are immobilized, whereas at 60 degrees C they are almost totally in the labile site. In contrast to cholesterol, the phospholipids sense only one type of environment, at both the top and center of the bilayer, indicating that cholesterol acts as a screen, preventing the lipids from direct interaction with the antibiotic. At low temperature, the ordering of the lipid in the presence of cholesterol does not change upon filipin addition, whereas at elevated temperatures the local ordering of both the lipid and the labile cholesterol is significantly lower than that in the absence of the drug. Moreover, there is a very important difference between the degree of local ordering as measured by the lipids and by cholesterol at high temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apocytochrome c binding to negatively charged lipid dispersions studied by spin-label electron spin resonance

The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

alpha-methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: (2)H-NMR quadrupole splittings from individual lipids in mixed bilayers

A Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with alpha-deuterated oleic acid. Phosphatidylglycerol (PG), the glucolipids monoglucosyldiacylglycerol (MGlcDAG), diglucosyldiacylglycerol (DGlcDAG) and monoacyldiglucosyldiacylglycerol, and the phosphoglucolipid glycerophosphoryldiglucosyldiacylglycerol (GPDGlcDAG) were purified, and the phase behaviour and molecular ordering for the individual lipids, as well as for mixtures of the lipids, were studied by (2)H-, (31)P-NMR and X-ray scattering methods. The chemical structure of all the A. laidlawii lipids, except PG, has been determined and verified previously; here also the chemical structure of PG was verified, utilising mass spectrometry and (1)H and (13)C high resolution NMR spectroscopy. For the first time, lipid dimers were found in the mass spectrometry measurements. The major findings in this work are: (1) addition of 50 mol% of PG to the non-lamellar-forming lipid MGlcDAG does not significantly alter the transition temperature between lamellar and non-lamellar phases; (2) the (2)H-NMR quadrupole splitting patterns obtained from the lamellar liquid crystalline phase are markedly different for PG on one hand, and DGlcDAG and GPDGlcDAG on the other hand; and (3) mixtures of PG and DGlcDAG or MGlcDAG give rise to (2)H-NMR spectra consisting of a superposition of splitting patterns of the individual lipids. These remarkable features show that the local ordering of the alpha-carbon of the acyl chains is different for PG than for MGlcDAG and DGlcDAG, and that this difference is preserved when PG is mixed with the glucolipids. The results obtained are interpreted in terms of differences in molecular shape and hydrophilicity of the different polar headgroups.     

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of ?90%. ²H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar ²H spectra. Below 25°C the spectra exhibit a broad gel phase component which at 0°C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30°C) the A. laidlawii B membrane lipids are ～85–90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.     

Multinuclear solid-state-NMR and FT-IR-absorption investigations on lipid/trichogin bilayers

Solid-state NMR and FT-IR absorption spectroscopy are employed to study the molecular properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids as a function of trichogin-OMe content, a membrane-active analogue of the peptaibol trichogin GA IV. Variable-temperature NMR studies are performed, comprising (13)C-, (31)P-, and (14)N-NMR line-shape and relaxation experiments, to provide information about the mobility and ordering of the phospholipid head group and the acyl-chain region in the absence and presence of trichogin-OMe. Likewise, variable-temperature FT-IR-absorption studies are performed, and the conformation-sensitive CH(2) stretching bands are analyzed to examine the conformational state of the acyl chain. At lower trichogin-OMe concentrations, the peptide exhibits no remarkable influence on the dynamics and ordering features of the phospholipid molecules. It is concluded that, in this case, trichogin-OMe is embedded in the lipid bilayer, with its helix axis laying parallel to the bilayer plane, the more hydrophobic part pointing towards the inner part of the bilayer ('carpet-like' superstructure). The lipid dynamics are probed by rotating-frame spin-lattice-relaxation (T(1rho)) experiments for the (13)C and (31)P nuclei, which are assumed to be dominated by collective-order fluctuations. Variation of T(1rho) with sample composition is attributed to changes of the membrane stiffness. For the sample with the highest lipid/peptide (L/P) molar ratio, i.e., L/P 5 : 1, phase separation as a result of membrane disruption occurs. In this case, a second spectroscopic component can be separated in the (31)P-NMR spectra. In addition, the (motionally averaged) magnetic interactions are greatly reduced, the actual values differing for both components. The second spectroscopic component refers to membrane components with high trichogin-OMe concentration and to strong lipid-trichogin-OMe interactions, as reflected by significant changes of the head-group orientation (toroidal model). At the same time, DMPC molecules exist with minor lipid-trichogin-OMe interactions, most probably in smaller liposomes, trichogin-OMe being embedded in a 'carpet-like' manner. Moreover, lipid ordering is generally reduced for the highly concentrated sample, which may result from fast lateral lipid motion along the curved bilayer surface.     

Spectroscopic signatures of bilayer ordering in native biological membranes

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (L_o) and liquid-disordered (L_d) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, L_o microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In ¹³C-¹³C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.     

Domain-formation in DOPC/SM bilayers studied by pfg-NMR: effect of sterol structure

Pulsed field gradient (pfg)-NMR spectroscopy was utilized to determine lipid lateral diffusion coefficients in oriented bilayers composed of 25 mol % sterol and equimolar amounts of dioleoylphosphatidylcholine and sphingomyelin. The occurrence of two lipid diffusion coefficients in a bilayer was used as evidence of lateral phase separation into liquid ordered and liquid disordered domains. It was found that cholesterol, ergosterol, sitosterol, and lathosterol induced domains, whereas lanosterol, stigmasterol, and stigmastanol resided in homogeneous membranes in the temperature interval of 24-70 degrees C. Among the domain-forming sterols, differences in the upper miscibility temperature indicated that the stability of the liquid ordered phase could be modified by small changes in the sterol structure. The domain-forming capacity for the different sterols is discussed in terms of the ordering effect of the sterols on the lipids, and it is proposed that the driving force for the lateral phase separation is the reduced solubility of the unsaturated lipid in the highly ordered phase.     

Progressive ordering with decreasing temperature of the phospholipids of influenza virus

Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 degrees C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 degrees C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.     

Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study

Four chain spin labels and a spin-labeled cholestane were used to study the dynamic structure of plasma membrane vesicles (PMV) prepared from RBL-2H3 mast cells at temperatures ranging from 22 degrees C to 45 degrees C. Analysis shows that the spectra from most labels consist of two components. The abundant spectral components exhibit substantial ordering that is intermediate between that of a liquid-ordered (Lo) phase, and that of a liquid-crystalline (Lc) phase as represented by model membranes. Also, rotational diffusion rates of the spin labels are comparable to those in the Lo phase. In contrast, the ordering for the less abundant components is much lower. These results indicate that a Lo-like region or phase (the abundant component) and an Lc-like region or phase (the less abundant component) coexist in the PMV. In contrast, membranes reconstituted from extracted lipids exhibit the more ordered phase only. This suggests that membrane-associated proteins are important for the coexistence of Lo-like and Lc-like regions in the plasma membrane. In addition, binding of the myristoylated protein, ARF6 to PMV, leads to a new spectral component for a headgroup lipid spin label that indicates the formation of plasma membrane defects by this low molecular weight GTPase.     

Organization of lipids in sarcoplasmic reticulum membrane and Ca2+-dependent ATPase activity.

The organization of lipids in sarcoplasmic reticulum membrane was studied with a variety of stearic spin labels and a phosphatidylcholine spin label. The ESR spectra of the spin-labeled membranes consisted of two components, one due to labels in lipid bilayer structure and the other due to more immobilized labels. The relative intensity of the immobilized component increased when the lipid content of the membrane was decreased by treatment with phospholipase A [EC 3.1.1.4] and subsequent washing with bovine serum albumin. Membrane containing 30% of the intact phospholipid, i.e.0.15 mg of phospholipid per mg of protein, showed a spectrum consisting only of the immobilized component (the overall splitting ranged from 58.5 G to 60.5 G). The immobilized component was ascribed to lipids complexed with protein. The fraction of lipids in the two different organizations was determined from the ESR spectrum. The activity of the Ca2+-Mg2+ dependent ATPase [ATP phosphohydrolase, EC 3.6.1.3] was found to increase almost linearly with the lipid bilayer content in the membrane, whereas phosphoenzyme formation was almost independent of the bilayer content. This indicated that the bilayer structure is necessary for the ATPase to attain its full transport activity.     

Estimation of lipid regions in a cytochrome oxidase-lipid complex using spin labeling electron spin resonance: Distribution effects on the spin label

The distribution of lipid in the cytochrome oxidase-lipid complex from beef heart mitochondria has been studied by the spin labeling electron spin resonance technique. The spectra of a phospholipid spin label incorporated in the complex reveals an immobilized (on the ESR time scale) component in addition to the fluid component which is found in aqueous dispersions of the extracted lipids. The first component corresponds to the domain of lipid influenced by the protein, and the second component to the remaining lipid. A theory taking into account not only the sizes of the lipid regions in which the spin label molecule distributes itself, but also the different affinities of the label for the two domains, has been developed. Taking advantage of the variation in spectra obtained with increasing amounts of spin label, computer calculations have been performed to estimate the distribution of lipid in the different regions of the cytochrome oxidase-lipid complex. An extrapolation of the amount of immobilized spin-labeled phospholipid to zero concentration of label allows a calculation of the number of fatty acid residues interacting with the protein to be made. It has been found that the number of aliphatic chains influenced by the protein is higher than that calculated for a single boundary layer around the protein. The approach used in this paper can be useful for studies of protein-lipid interactions in other systems.     

Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy.

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.     

Alterations in lateral lipid mobility in the plasma membrane of urodelean ectodermal cells during gastrulation

The mobility characteristics of lipids were studied in the plasmalemma of dissociated presumptive ectodermal cells from embryos of Pleurodeles Waltl at different stages of development, from early blastula to early neurula, using a Fluorescence Recovery After Photobleaching technique (FRAP), after incorporation of the lipophilic fluorescent probe 5N-(hexadecanoyl)-aminofluoresceine (HEDAF) into the cell plasma membrane. At all stages of development, fluorescence recovery was found to extrapolate to 100%, which suggested that the lipid phase in these plasma membranes can be regarded as dynamically homogeneous (no immobilized fraction). It appears as a continuum over a wide cell surface area, in which lipids are free to move laterally. The lateral diffusion coefficient of the probe, obtained from statistical analysis of the fluorescence recovery data, was found to decrease significantly from blastula to gastrula, slightly increasing at the neurula stage. These changes in the dynamic properties of the lipid probe HEDAF during gastrulation suggest that the lipid phase of the plasma membrane of these ectodermal cells undergo structural changes. The results lend support to the idea that the plasma membrane of these cells is actively involved in the morphogenetic movements which characterize the development of the embryo.     

Membrane structure of the hepatitis B virus surface antigen particle

Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A(2) from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.