Lipid bilayer ultrastructure. Electron density profiles and chain tilt angles as determined by X-ray diffraction.

High resolution (6A) electron density profiles have been computed on an absolute electron density scale for bilayers composed of both saturated fatty acids and fatty acids associated with the alkaline earth series of divalent cations. Lowangle X-ray diffraction data have been interpreted by an isomorphous replacement technique. The position on the X-ray film of discrete wide-angle reflections has provided direct information on the hydrocarbon chain packing and chain tilt in these bilayers. These results have been correlated to an electron microscopy study of the same bilayers (Waldbilling, R. C., Robertson, J.D. and McIntosh, T. J. (1976) Biochim. Biophys. Acta 448, 1-14) and also to X-ray diffraction studies of fatty acid crystals. A method for forming and structurally analyzing bilayers of well defined chemical asymmetry is also described.     

Images of divalent cations in unstained symmetric and asymmetric lipid bilayers

Divalent cations have been microscopically visualized in association with simple lipid bilayers. Symmetric and asymmetric oriented bilayers were constructed from fatty acid monolayers and were cut in thin transverse sections for examination by bright field electron microscopy in the absence of stains, fixatives or embedding materials. It has been found that bilayers formed of lipid molecules having alkaline earth head groups exhibit natural electron contrast. The intrinsic image has been linked to local variations in the bilayer absolute electron density profile determined by X-ray diffraction analysis of the same specimens (McIntosh, T.J., Waldbillig, R. C. and Robertson J. D. (1976) Biochim. Biophys. Acta 448, 15–33). By combining the microscopic, chemical and X-ray evidence it has been estimated that local increments of about 1 g/cm³ can produce detectable electron contrast in 500 Å transverse sections of bilayers.     

Hydration force and bilayer deformation: a reevaluation

The hydration repulsive force between lipid bilayers and the deformability of both gel and liquid-crystalline bilayers have been quantitated by an X-ray diffraction analysis of osmotically stressed liposomes. Both sampling theorem reconstructions and electron density distributions were calculated from diffraction data obtained from multilayers with applied osmotic pressures of 0-50 atm. The bilayer thickness and area per lipid molecule remain nearly constant (to within about 4%) in this pressure range, as adjacent bilayers move from their equilibrium separation in excess water to within 2-4 A of each other. This analysis indicates that the bilayers are relatively incompressible. This results differs from previously published X-ray diffraction studies of bilayer compressibility but agrees with direct mechanical measurements of the bilayer compressibility modulus. It is also found that the hydration repulsive force decays exponentially with separation between bilayers with a decay constant of 1.4 A for gel-state dipalmitoylphosphatidylcholine and 1.7 A for liquid-crystalline egg phosphatidylcholine bilayers. This implies that the exponential decay constant is not necessarily equal to the diameter of a water molecule, as has been previously suggested on experimental and theoretical grounds.     

Organization of skin stratum corneum extracellular lamellae: diffraction evidence for asymmetric distribution of cholesterol

Lipid suspensions containing 2:1:1 skin ceramides:palmitic acid:cholesterol, similar to the lipid composition found in the extracellular matrix of skin stratum corneum, were analyzed by X-ray diffraction methods. These suspensions gave a sharp wide-angle reflection at 4.1 A, indicating tight hydrocarbon chain packing that would function as a water barrier, and low-angle lamellar diffraction with a repeat period near 130 A, similar to that previously recorded from intact stratum corneum. The lamellar repeat increased from 121 A at pH 6 to 133 A at pH 8.5, allowing phase angles of the lamellar data to be obtained by a sampling theorem "swelling" analysis. Electron density profiles showed that each repeating unit contained two asymmetric bilayers, with a fluid space on one side of the bilayer that increased with increasing pH, due to electrostatic repulsion between bilayers because of ionization of the palmitic acid. Profiles obtained from lamellae with cholesterol sulfate partially substituted for cholesterol showed large density increases on that same side of the bilayer, indicating that cholesterol is asymmetrically distributed in each bilayer. A molecular model was developed postulating that this asymmetry is due to the exclusion of cholesterol from lipid monolayers containing the ester-linked unsaturated (linoleic) hydrocarbon chain of skin ceramide 1. This model can explain the altered organization of extracellular lamellae in epidermal cysts (P. W. Wertz, D. C. Swartzendruber, K. C. Madison, D. T. Downing. 1987. J. Invest. Dermatol. 89:419-425) where the ester-linked chains have a higher percentage of saturated fatty acids than found in normal epidermis.     

Study of the topography of cannabinoids in model membranes using X-ray diffraction

Small-angle X-ray diffraction was used to determine the topography of (-)-delta 8-tetrahydrocannabinol in partially hydrated dimyristoylphosphatidylcholine bilayers. Electron density profiles of lipid bilayers in the presence and absence of the cannabinoid were calculated using Fourier transform. Step-function equivalent profiles were then constructed to obtain the absolute electron density scale. We have compared the electron density profiles of the above preparations to determine the location of the drug molecule in the bilayer. By using (-)-5'-iodo-delta 8-tetrahydrocannabinol in parallel experiments, we were also able to locate the iodine atom in the bilayer and deduce the conformation of the cannabinoid side alkyl chain. All comparisons were made between different preparations having the same mesomorphic form and total period repeat distance. To achieve this, we have carried out X-ray diffraction experiments at various temperatures to cover the different mesomorphic phases and combined our data with the corresponding results from differential scanning calorimetry. Based on the results of this work and previous data on the orientation of the cannabinoid in model membranes, we concluded that the phenolic hydroxy group of the drug molecule exists near the carbonyl groups of DMPC and that the average position of the iodine atom is approx. 5.5 A from the center (terminal methyl region) of the DMPC bilayer. This requires the cannabinoid side-chain to assume an orientation parallel to the bilayer chains.     

High-resolution electron density profiles reveal influence of fatty acids on bilayer structure.

Small-angle x-ray diffraction studies were performed on gel phase-oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC containing 40 mol% of either palmitic acid (PA) or palmitic acid brominated at the 2-position (BPA). Oriented samples were prepared using a method developed by us, which is as simple as powder sample preparations while offering all the advantages of oriented samples made by traditional methods. Phases were determined using swelling experiments with structure factors plotted in reciprocal space, creating a relatively smooth curve as the amount of water between the bilayers was changed. Continuous Fourier transforms were also calculated to further test the consistency of the phase assignments. The diffraction data were used to calculate absolute electron density profiles for different bilayers to a resolution of 5-6 A. Analysis indicates the following: (a) The electron density profiles for the three preparations are virtually identical in the hydrocarbon chain region. (b) There is a decrease in the electron density of the glycerol backbone-headgroup region and d-space in DPPC-PA compared to DPPC. (c) The bromine of fatty-acid brominated at the 2-position is in the vicinity of the glycerol backbone. (d) The bilayer thickness of DPPC containing either brominated or unbrominated fatty acid remains relatively constant with increased levels of hydration, unlike DPPC bilayers.     

Structure of polymerizable lipid bilayers. I--1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, a tubule-forming phosphatidylcholine

This report presents the first X-ray diffraction data on diacetylenic phospholipids. The tubule-forming polymerizable lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), was studied by low angle X-ray diffraction from partially dehydrated oriented multibilayers in both polymerized and unpolymerized form. Bilayers of this material were found to be highly ordered, yielding as many as 16 orders of lamellar diffraction, in both the polymerized and unpolymerized states. The unit cell dimension was very small for a lipid of this size. In addition to the features usually observed in the electron density profile structure of phospholipid bilayers, the electron-dense diacetylenic portions of the fatty acyl chain produced electron density maxima at two well-defined levels on each side of the bilayer approximately 15 A and 9 A from the bilayer midplane. A model molecular conformation deduced from the one-dimensional electron density map features all-trans acyl chains tilted at approximately 28 degrees from the bilayer normal that are interdigitated with chains of the opposing monolayer by approximately two carbons at the bilayer center. The linear diacetylene moieties on beta- and gamma-chains appear at different positions along the bilayer normal axis and are roughly parallel to the bilayer surface. This model is discussed in terms of a polymerization mechanism.     

omega-6 and omega-3 fatty acids: monolayer packing and effects on bilayer permeability and cholesterol exchange.

It has been suggested that the polyunsaturated omega-3 fatty acid, docosahexaenoic acid (DHA), can adopt unique closely packed arrays in lipid bilayers (Glomset and Applegate. (1986) J. Lipid Res. 27, 658-680). These conformations are predicted on the basis of molecular dynamics calculations and are in contrast to the expanded conformations characteristic of omega-6 unsaturated fatty acids. It has also been suggested that close packing of omega-3 acyl chains could have a substantial affect on the physical properties of lipid bilayers (e.g. permeability). We report here some experimental tests of these predictions. Surface pressure-area experiments have been carried out on DHA and its mixtures with stearic and oleic acids. At low surface pressures DHA is more expanded than oleic acid. Extrapolation to the high surface pressures characteristic of lipid bilayers indicates that the area per molecule of DHA is only marginally less than that for oleic acid. Thus there is no compelling evidence to suggest that the average area per molecule of the omega-3 fatty acid is substantially different from the omega-6 fatty acid at high surface pressures. Experiments also show that the permeability of bilayers to glucose and the rates of dissociation of pyrenyl cholesterol from bilayers were similar for bilayers containing DHA compared to bilayers containing oleic acid or linoleic acid.     

Electrostatic interaction of poly(L-lysine) with dipalmitoylphosphatidic acid studied by X-ray diffraction

Structure of dipalmitoylphosphatidic acid (DPPA) bilayers in the presence of poly(L-lysine) is proposed from the results of X-ray diffraction obtained by a storage phosphor detector with a high resolution called an imaging plate. The small-angle X-ray diffraction pattern exhibits that DPPA/poly(L-lysine) complex forms a highly ordered multilamellar structure. The electron density profile of the DPPA/poly(L-lysine) complex draws that only one poly(L-lysine) layer is intercalated between the neighboring DPPA bilayers. The wide-angle X-ray diffraction pattern suggests that the presence of poly(L-lysine) hardly affects the nature of hydrocarbon chain packing in the DPPA bilayers. The X-ray reflection from the DPPA/poly(L-lysine) complex indicates that the poly(L-lysine) molecules adopt a beta-sheet conformation on the surface of the DPPA bilayers. The both surface areas occupied by a headgroup of the DPPA and by a lysine residue in poly(L-lysine) are estimated from the observed spacings. The number ratio of lysine residues to DPPA headgroups per unit area is greater than unity. Therefore, one DPPA headgroup interacts with more than one lysine residue electrostatically, i.e., the electric charge distributions in both the surface of a DPPA bilayer and the poly(L-lysine) beta-sheet are incommensurate.     

Reconstitution of the lipid-depleted pyruvate oxidase system of Escherichia coli: the palmitic acid effect

Previous work has shown that the coupling of the soluble Escherichia coli pyruvate oxidase to a lipid-depleted membrane terminal electron transport system requires the addition of ubiquinone and a neutral lipid fraction (C. Cunningham and L. P. Hager (1975) J. Biol. Chem. 250, 7139-7146). The active factor present in the neutral lipid fraction has now been isolated and characterized. NMR, uv, and mass spectroscopic analysis identifies palmitic acid as the active component. A comparison of palmitic acid with other fatty acids of varying chain lengths indicates that most fatty acids having chain lengths in the range C12 to C20 have comparable activity to palmitic acid. Exceptions are stearic and arachidic acid which have greatly reduced activity. Fatty acids of C6 to C10 chain length showed about one third the activity of palmitic acid. Fatty acids having chain lengths of 2 to 5 carbon atoms are essentially inactive. The carboxyl function of the fatty acid is required for activity. Derivatives of fatty acids in which the carboxyl group had been modified to an alcohol, aldehyde, or methyl ester function show greatly diminished activity. Both the cis and trans forms of unsaturated long-chain fatty acids are active. The stimulation of the electron transfer reaction by fatty acids occurs at the ubiquinone level of the electron transport chain. Ubiquinone-30 is rapidly reduced by pyruvate oxidase only in the presence of palmitic acid.     

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.     

Phase behavior and bilayer properties of fatty acids: hydrated 1:1 acid-soaps

The physical properties in water of a series of 1:1 acid-soap compounds formed from fatty acids and potassium soaps with saturated (10-18 carbons) and omega-9 monounsaturated (18 carbons) hydrocarbon chains have been studied by using differential scanning calorimetry (DSC), X-ray diffraction, and direct and polarized light microscopy. DSC showed three phase transitions corresponding to the melting of crystalline water, the melting of crystalline lipid hydrocarbon chains, and the decomposition of the 1:1 acid-soap compound into its parent fatty acid and soap. Low- and wide-angle X-ray diffraction patterns revealed spacings that corresponded (with increasing hydration) to acid-soap crystals, hexagonal type II liquid crystals, and lamellar liquid crystals. The lamellar phase swelled from bilayer repeat distances of 68 (at 45% H2O) to 303 A (at 90% H2O). Direct and polarized light micrographs demonstrated the formation of myelin figures as well as birefringent optical textures corresponding to hexagonal and lamellar mesophases. Assuming that 1:1 potassium hydrogen dioleate and water were two components, we constructed a temperature-composition phase diagram. Interpretation of the data using the Gibbs phase rule showed that, at greater than 30% water, hydrocarbon chain melting was accompanied by decomposition of the 1:1 acid-soap compound and the system changed from a two-component to a three-component system. Comparison of hydrated 1:1 fatty acid/soap systems with hydrated soap systems suggests that the reduced degree of charge repulsion between polar groups causes half-ionized fatty acids in excess water to form bilayers rather than micelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipopolysaccharide bilayer structure: effect of chemotype, core mutations, divalent cations, and temperature.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a protective and permeability barrier. X-ray diffraction analysis of osmotically stressed LPS multilayers was used to determine the structure and interactive properties of LPSs from strains containing the minimum number of sugars necessary for bacterial survival (Re chemotype) to the maximum number of sugars found in rough bacteria (Ra chemotype). At 20 degrees C in the absence of divalent cations, LPS suspensions gave a sharp wide-angle reflection at 4.23 A and a broad low-angle band centered at 50-68 A depending on the chemotype, indicating the presence of gel phase bilayers separated by large fluid spaces. As osmotic pressure was applied, the apposing bilayers were squeezed together and lamellar diffraction at 6 A resolution was obtained. At low applied pressures (<10(6) dyn/cm2), the total repulsive pressure between bilayers could be explained by electrostatic double layer theory. At higher applied pressures, there was a sharp upward break in each pressure-distance relation, indicating the presence of a hydrophilic steric barrier whose range depended strongly on the LPS chemotype. The positions of these upward breaks, along with electron density profiles, showed that the sugar core width systematically increased from 10 A for the Re chemotype to 27 A for the Ra chemotype. In excess buffer, the addition of divalent cations brought the bilayers into steric contact. Electron density profiles were used to determine the locations of cation binding sites and polar substituents on the LPS oligosaccharide core. The area per hydrocarbon chain was approximately 26 A2 in liquid-crystalline LPS bilayers, an indication of an acyl chain packing that is much tighter than that found in bilayers composed of typical membrane lipids. This unusually tight packing could be a critical factor in the permeability barrier provided by LPS.     

Effects of cyclosporin A on model lipid membranes

Cyclosporin A (CSA) is a widely used immunosuppressant drug for transplant therapy, however its limitation is its toxicity. The effect of CSA on model membranes such as dimyristoyl phosphatidylcholine (DMPC) bilayers was studied using small-angle X-ray diffraction and differential scanning calorimetry (DSC). CSA abolishes the pretransition and affects the transition of DMPC model membranes in a concentration-related manner as is shown by DSC. CSA induces a second peak at the high temperature side of the main transition, which is interpreted as a phase separation between areas rich and poor in CSA concentration. Small angle X-ray diffraction shows that the repeat distance of the DMPC bilayers in the lamellar Lalpha state increases as a function of concentration up to 10 mol% and remains constant thereafter. Furthermore, CSA affects the fatty acyl chains of the bilayer, especially the part of the chain proximal to the head group. In conclusion, CSA, as both small-angle X-ray diffraction and DSC show, affects in a concentration-wise manner the DMPC model membranes and perturbs the bilayer, in particular the acyl chain region.     

Order-disorder transition in bilayers of diphytanoyl phosphatidylcholine

A comparative study on bilayers of diphytanoyl phosphatidylcholine (DPhPC) and bilayers of dimyristoyl phosphatidylcholine (DMPC) was made by X-ray lamellar diffraction as a function of temperature and the degree of hydration. An order-disorder phase transition of DPhPC reveals an interesting contrast to the standard model of DMPC. Electron density profiles allow us to deduce the conformational changes which occur in the headgroup-glycerol region and in the chain region. The important conclusion is that the lipid headgroup may have different conformational energetics in lipids of different chains. We explain why this is important to protein-membrane interactions.     

Structure and cohesive properties of sphingomyelin/cholesterol bilayers.

Thermal, structural, and cohesive measurements have been obtained for both bovine brain sphingomyelin (BSM) and N-tetracosanoylsphingomyelin (C24-SM) in the presence and absence of cholesterol. A goal of these experiments has been to clarify the mechanisms responsible for the strong interaction between sphingomyelin and cholesterol. Differential scanning calorimetry shows that fully hydrated bilayers of BSM and C24-SM have main endothermic phase transitions at 39 and 46 degrees C, respectively, that reflect the melting of the acyl chains from a gel to a liquid-crystalline phase. For each lipid, the addition of cholesterol monotonically reduces the enthalpy of this transition, so that at equimolar cholesterol the transition enthalpy is zero. The addition of equimolar cholesterol to either BSM or C24-SM coverts the wide-angle X-ray diffraction reflection at 4.15 A to a broad band centered at 4.5 A. Electron density profiles of gel-phase C24-SM bilayers contain two terminal methyl dips in the center of the bilayer, indicating that the lipid hydrocarbon chains partially interdigitate so that the long saturated 24-carbon acyl chains in one monolayer cross the bilayer center and appose the shorter sphingosine chains from the other monolayer. The incorporation of cholesterol adds electron density to the hydrocarbon chain region near the head group and removes the double terminal methyl dip. These wide- and low-angle X-ray data indicate that cholesterol packs into the hydrocarbon chain region near the sphingomyelin head group, fluidizes the methylene chains near the center of the bilayer compared to the gel phase, and reduces the extent of methylene chain interdigitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of porcine heart cytoplasmic malate dehydrogenase: combining X-ray diffraction and chemical sequence data in structural studies

The amino acid sequence of cytoplasmic malate dehydrogenase (sMDH) has been determined by a combination of X-ray crystallographic and chemical sequencing methods. The initial molecular model incorporated an "X-ray amino acid sequence" that was derived primarily from an evaluation of a multiple isomorphous replacement phased electron density map calculated at 2.5-A resolution. Following restrained least-squares crystallographic refinement, difference electron density maps were calculated from model phases, and attempts were made to upgrade the X-ray amino acid sequence. The method used to find the positions of peptides in the X-ray structure was similar to those used for studying protein homology and was shown to be successful for large fragments. For sMDH, X-ray methods by themselves were insufficient to derive a complete amino acid sequence, even with partial chemical sequence data. However, for this relatively large molecule at medium resolution, the electron density maps were of considerable help in determining the linear position of peptide fragments. The N-acetylated polypeptide chain of sMDH has 331 amino acids and has been crystallographically refined to an R factor of 19% for 2.5-A resolution diffraction data.     

Phase transitions and fatty acid spin label behavior in interdigitated lipid phases induced by glycerol and polymyxin

Glycerol and polymyxin have been shown by X-ray diffraction to induce interdigitated bilayers in phosphatidylcholine (PC) and phosphatidylglycerol (PG), respectively (McDaniel, R.V., et al. (1983) Biochim. Biophys. Acta 731, 97-108; Ranck, J.-L. and Tocanne, J.-F. (1982) FEBS Lett. 143, 175-178). In the present study we have investigated the phase behavior of PC and PG in the presence of glycerol and polymyxin by differential scanning calorimetry and the use of fatty acid spin labels. Interdigitation causes a large increase in the order parameter of a fatty acid spin labeled near the terminal methyl, 16-doxylstearate, so that it was similar to that of a fatty acid labeled much closer to the polar head group region, 5-doxylstearate. Thus interdigitation abolishes the fluidity gradient found in a non-interdigitated bilayer. 16-Doxylstearate may be useful in detecting interdigitation of lipid bilayers caused by other substances. The different samples all went through two transitions on heating or cooling, or both. However, use of the fatty acid spin label showed that the molecular events during these transitions varies for different samples. The results suggested that PC-glycerol freezes from the liquid-crystalline phase into a non-interdigitated gel phase. This subsequently becomes interdigitated upon lowering the temperature a few degrees, in a low enthalpy transition. PG-polymyxin shows a similar behavior except that the enthalpy of the non-interdigitated gel to interdigitated phase transition is greater and the transition is reversible on heating. Thus on heating PG-polymyxin first goes through a transition from the interdigitated phase to a non-interdigitated gel phase and then, in a separate transition, to the liquid-crystalline phase. This occurs because the fatty acid chains in the presence of polymyxin become too disordered with increase in temperature to maintain the interdigitated state. PG-glycerol goes into the interdigitated state less readily than the other mixtures. If cooled rapidly, PG-glycerol freezes into a metastable phase which is more disordered than the interdigitated phase. It goes into the interdigitated phase in an exothermic transition on heating. An increase in fatty acid chain length causes greater steric hindrance to interdigitation but also increases the stabilizing energy gained by interdigitation.     

Cholesterol modifies the short-range repulsive interactions between phosphatidylcholine membranes

Pressure versus distance relationships have been obtained for egg phosphatidylcholine bilayers containing a range of cholesterol concentrations. Water was removed from between adjacent bilayers by the application of osmotic pressures in the range of 0.4-2600 atm (4 x 10(5)-2.6 x 10(9) dyn/cm2), and the distance between adjacent bilayers was obtained by Fourier analysis of X-ray diffraction data. For applied pressures up to about 50 atm and bilayer surface separations of 15-5 A, the incorporation of up to equimolar cholesterol has little influence on plots of pressure versus bilayer separation. However, for the higher applied pressures, cholesterol reduces the interbilayer separation distance by an amount that depends on the cholesterol concentration in the bilayer. For example, the incorporation of equimolar cholesterol reduces the distance between bilayers by as much as 6 A at an applied pressure of 2600 atm. At this applied pressure, electron density profiles show that the high-density head-group peaks from apposing bilayers have merged. This indicates that equimolar concentrations of cholesterol spread the lipid molecules apart in the plane of the bilayer enough to allow the phosphatidylcholine head groups from apposing bilayers to interpenetrate as the bilayers are squeezed together. All of these X-ray and pressure-distance data indicate that, by reducing the volume fraction of phospholipid head groups, cholesterol markedly reduces the steric repulsion between apposing bilayers but has a much smaller effect on the sum of the longer ranged repulsive hydration and fluctuation pressures. Increasing concentrations of cholesterol monotonically increase the dipole potential of egg phosphatidylcholine monolayers, from 415 mV with no cholesterol to 493 mV with equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Area per molecule and distribution of water in fully hydrated dilauroylphosphatidylethanolamine bilayers

The area per lipid molecule for fully hydrated dilauroylphosphatidylethanolamine (DLPE) has been obtained in both the gel and liquid-crystalline states by combining wide-angle X-ray diffraction, electron density profiles, and previously published dilatometry results [Wilkinson, D. A., & Nagle, J. F. (1981) Biochemistry 20, 187-192]. The molecular area increases from 41.0 +/- 0.2 to 49.1 +/- 1.2 A2 upon melting from the gel to liquid-crystalline phase. The thickness of the bilayer, as measured from the electron density profiles, decreases about 4 A upon melting, from 45.2 +/- 0.3 to 41.0 +/- 0.6 A. A somewhat unexpected result is that the fluid layer between fully hydrated bilayers is the same in both gel and liquid-crystalline phases and is only about 5 A thick. From these data, plus the volume of the anhydrous DLPE molecule, it is possible to determine the number of water molecules per lipid and their approximate distribution relative to the lipid molecule. Our analysis shows that there are about 7 and 9 waters per DLPE molecule in the gel and liquid-crystalline phases, respectively. About half of the water is located in the fluid space between adjacent bilayers, and the remaining waters are intercalated into the bilayer, presumably in the head group region. There are significantly fewer water molecules in the fluid spaces between DLPE bilayers than in the fluid spaces in gel- or liquid-crystalline-phase phosphatidylcholine bilayers. This small fluid space in PE bilayers could arise from interbilayer hydrogen bond formation through the water molecules or electrostatic interactions between the amine and phosphate groups on apposing bilayers.