G. Graham Shipley

Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine bilayers. Kinetic, hydration and structural study

Abstract Differential scanning calorimetry and fast-recording X-ray diffraction methods are used to study the kinetics, hydration and structural changes of the L β , gel→L c ‘crystal’ conversion at −2°C for less than maximally hydrated (27.2 wt% H 2 O) and maximally hydrated (48.9 wt% H 2 O) 1,2-dipalmitoyl- l -phosphatidylcholine (DPPC) dispersions. Equilibration of DPPC dispersions at −2°C for increasing time periods results in a progressive increase in the sub-transition temperature to a limiting value of 17–19°C, while the enthalpy of the sub-transition also increases on reaching an enthalpy maximum ΔH =5.6–5.8 kcal/mol DPPC after 2 days equilibration. Corresponding X-ray diffraction experiments demonstrate two time domains involving structural alterations. An initial time domain involves a rapid shift of the two characteristic L β , wide angle reflections, 1 4.18 A −1 and 1 4.08 A −1 , to 1 4.3 A −1 and 1 4.0 A −1 , respectively, while there is no significant change in the lamellar periodicity. A slower structural alteration subsequently occurs involving a progressive decrease in lamellar periodicity to its limiting dimension, d ≅ 59.5 A and further shifts in the two wide angle reflections to final values of 1 4.4 A −1 and 1 3.86 A −1 . These changes are indicative of alterations in both the bilayer organization and the hydrocarbon chain packing. Hydration studies over the range 10.1 to 48.9 wt% H 2 O demonstrate that at 4°C the L c bilayer phase has a reduced hydration limit of 11 mol H 2 O/mol DPPC compared to 19 mol H 2 O/mol DPPC and 25 mol H 2 O/mol DPPC for the L β′ and L α bilayer phases, respectively. It is concluded that the L β′ → L c conversion involves dehydration and hydrocarbon chain ordering. The data suggest a crystallization of DPPC and 11 H 2 O molecules presumably involving tightly bound water molecules in an interbilayer matrix characterized by water-water and water-DPPC hydrogen bonding. A structural interpretation of the changes occurring in the two-dimensional hydrocarbon chain packing modes during the transitions between the hydrated L c , L β′ , P β′ and L α bilayer forms of DPPC is proposed.

Characterization of the sub-transition of hydrated dipalmitoylphosphatidylcholine bilayers: X-ray diffraction study

Abstract The structural changes accompanying the recently described sub-transition of hydrated dipalmitoylphosphatidylcholine (Chen, S.C., Sturtevant, J.M. and Gaffney, B.J. (1980) Proc. Natl. Acad. Sci. USA 77, 5060–5063) have been defined using X-ray diffraction methods. Following prolonged storage at −4°C the usual L β′ gel form of hydrated dipalmitoylphosphatidylcholine (DPPC) is converted into a more ordered stable ‘crystal’ form. The bilayer periodicity is 59.1 A and the most striking feature is the presence of a number of X-ray reflections in the wide angle region. The most prominent of these are a sharp reflection at 1 4.4 A −1 and a broader reflection at 1 3.9 A −1 . This diffraction pattern is indicative of more ordered molecular and hydrocarbon chain packing modes in this low temperature ‘crystal’ bilayer form. At the sub-transition ( T rmsub = 15–20° C ) an increase in the bilayer periodicity occurs ( d=63.6 A ) and a strong reflection at approx. 1 4.2 A −1 with a shoulder at approx. 1 4.1 A −1 is observed. This diffraction pattern is identical to that of the bilayer gel (L β′ ) form of hydrated DPPC. Thus, the sub-transition corresponds to a bilayer ‘crystal’ → bilayer L β′ gel structural rearrangement accompanied by a decrease in the lateral hydrocarbon chain interactions. Differential scanning calorimetry and X-ray diffraction show that on further heating the usual structural changes L β′ → P β′ and P β′ → L α occur at the pre- and main transitions, at approx. 35°C and 41°C, respectively.

Thermal transitions in human plasma low density lipoproteins

Thermal analysis of human plasma low density lipoproteins reveals a broad reversible transition encompassing body temperature. The calorimetric and x-ray scattering data identify this transition as a cooperation, liquid-crystalline to liquid phase change involving the cholesterol esters in the lipoprotein. This behavior requires the presence of a region rich in cholesterol ester within the lipoprotein.

A refinement analysis of the crystallography of the phospholipid, 1,2-dilauroyl-DL-phosphatidylethanolamine, and some remarks on lipid-lipid and lipid-protein interactions

A least-squares refinement analysis of atomic positional and thermal parameters in a single crystal of 1,2-dilauroyl-DL-phosphatidylethanolamine: acetic acid has been based on the X-ray diffraction intensities of 1132 independent reflexions, assessed by automatic microdensitometry. The final unweighted discrepancy index is 0.16 with e.s.ds of the bond lengths ranging from 0.02 to 0.12 A. The general features of our earlier, lessprecise analysis are confirmed. The close-packed arrangement of the phospholipid molecules is discussed in relation to electron microscopy and diffraction studies of the structures of membranes from Acholeplasma laidlawii and Halobacterium halobium.

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.

Fatty acid composition and thermal behavior of natural sphingomyelins.

We found significant differences in the fatty acid composition of several bovine brain, egg yolk and sheep erythrocyte sphingomyelins. These differences in fatty acid composition influence the thermal behavior of hydrated sphingomyelin as recorded by differentail scanning calorimetry. Significant differences were also found in the temperature and complexity of the order-disorder phase transitions of bovine brain sphingomyelin obtained from different sources which, in general, correlate with the relative content of the saturated fatty acids (palmitic (C16:0) and stearic acid (C18:0) acids) and the long unsaturated nervonic acid (C24:1).

The ternary phase diagram of lecithin, cholesteryl linolenate and water: Phase behavior and structure

Abstract The ternary phase diagram of cholesteryl linolenate-egg lecithin-water has been determined by polarizing light microscopy, calorimetry and X-ray diffraction at 23 °C. Hydrated lecithin forms a lamellar liquid-crystalline structure into which small amounts of cholesteryl linolenate are incorporated. The maximum incorporation of cholesterol ester into this lamellar structure varies with the degree of hydration. Increasing the water concentration from 10 to 15% (w/w) increased the limiting molar ratio of cholesteryl linolenate to lecithin in the lamellar phase from 1:50 to 1:22. At intermediate concentrations (15 to 30% water) the cholesteryl linolenate:lecithin ratio remains constant at 1:22. When water is increased to 42.5%, the maximum water content in the lamellar phase, the molar ratio decreased to 1:32. At low water concentrations the cholesterol ester appears to be entirely in the apolar region of the lecithin bilayer, while at higher water concentrations the ester groups of cholesteryl linolenate may be located at the lipid-water interface. At high water concentrations the ester appears to disorder the alkyl chains of the lecithin, giving rise to a thinner lipid layer and an increased surface area per lipid molecule when compared to the lecithin-water system in the absence of cholesteryl linolenate. The lamellar phase is the only phase (except at water concentrations less than 5%) in which all three components mutually interact. All mixtures of the three components having compositions outside the one-phase (lamellar) zone produce additional phases of cholesteryl linolenate or water, or both. Between 23 °C and 60 °C only minor changes in the phase diagram are observed.

Phase behavior and structural characteristics of hydrated bovine brain gangliosides.

Hydrated bovine brain gangliosides have been studied by differential scanning calorimetry, X-ray diffraction, and polarized light microscopy. Over the hydration range 18-50 wt.% H2O, mixed brain gangliosides exhibit a hexagonal mesophase structure, in which the ganglioside molecules form hexagonally packed rod-like structures. The apolar lipid chains radiate from the center of the rods, with the sugar groups on the cylinder surface in contact with water. At higher water contents, an isotropic micellar solution is formed. Over the hydration range 20-30 wt.% H2O, two small thermal transitions with peak maxima at 30 degrees C and 46 degrees C are observed by differential scanning calorimetry. These transitions broaden and move apart in temperature as the hydration is increased to 50 wt.% H2O. X-ray diffraction data indicate that this double transition is associated with a hydrocarbon chain rearrangement from a disordered state to another, possibly more disordered, state. Thus, the gangliosides, although membrane lipid components, have physical characteristics which are very different from those of the membrane phospholipids.

X-ray scattering of vesicles of N-acyl sphingomyelins. Determination of bilayer thickness

A series of N-acyl sphingomyelins (C16:0, C18:0, C20:0, C22:0, and C24:0) have been synthesized and single bilayer vesicles formed by sonication and ultracentrifugation. X-ray scattering data have been collected from the sphingomyelin vesicles at 50 degrees C in the melted-chain state. The x-ray scattering data have been transformed to the corresponding Patterson functions and Fourier electron density profiles; analysis of these functions has provided the intrabilayer phosphate-phosphate separation dp-p, a measure of the lipid bilayer thickness. The bilayer thickness increases linearly with increasing chain length (increment 1.3-1.4 A) and the intercept, 14.3-15.0 A, suggests a contribution of 7.0-7.5 A for each phosphorylcholine group to the bilayer thickness. The electron-density profiles have features suggestive of chain interdigitation when the length of the N-acyl chain (C20:0, C22:0, and C24:0) exceeds significantly the length of the invariant sphingosine chain.

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).