Annette Tardieu

Structure and polymorphism of the hydrocarbon chains of lipids: A study of lecithin-water phases☆

This work describes the structure of a variety of lecithin-water phases observed below the “melting” temperature of the hydrocarbon chains, with special emphasis on the conformation of the chains. The lecithins studied in this work are the homologous series dioctanoyl to distearoyl, 2-decanoyl-1-stearoyl, and a preparation from hen eggs. The hydrocarbon chains are found to adopt a variety of conformations in addition to type α, the liquid-like organization observed above the melting temperature. Type β: the chains are stiff and parallel, oriented at right angles to the plane of the lamellae and packed with rotational disorder in a two-dimensional hexagonal lattice (a ~ 4.85 A). Type β′: similar to β, but with the chains tilted with respect to the normal to the lamellae. Type δ: the chains are probably coiled into helices, whose axes are perpendicular to the plane of the polar groups and are packed with rotational disorder in a two-dimensional square lattice (a ~ 4.80 A), α is the predominant conformation, common to most lipids in the presence of water and at sufficiently high temperature, and the one more relevant to membranes; β is observed at lower temperatures in lipids whose chains are heterogeneous and in the presence of very small amounts of water; β′ is found in synthetic lecithins with identical chains, in the presence of variable amounts of water; δ is observed in dry lecithins. A highly ordered crystalline phase, yet displaying rotational disorder of the chains, is observed in almost dry lecithins. Most of the phases are lamellar, and contain one lipid bilayer per repeat unit. Two phases display two-dimensional lattices: Pδ, formed by ribbon-like elements with the chains in the δ conformation; Pβ′, formed by lamellae of type β′ distorted by periodic ripples. The results emphasize the clear-cut difference between the liquid-like and the other types of partly ordered conformations, as well as the correlations which exist between the chemical composition and the structure of the lipids below the melting temperature of the chains.

Lamellar and hexagonal lipid phases visualized by freeze-etching

Abstract 1. 1. Rapid freezing of lipid-water preparations preserves the structure of the high temperature phases. Both lamellar and hexagonal phases can be readily demonstrated by electron microscopy and X-ray observations. 2. 2. Without careful controls, contaminants are readily deposited on fresh fracture faces. The contamination can take the form of particles resembling those found on natural membranes. 3. 3. Neither degree of saturation, degree of hydration, nor cholesterol admixture significantly affects the appearance of lamellar fracture faces which appear uniformly smooth. On uncontaminated specimens, no structures were found which resembled the particulate material of natural membranes.

Structure of the cubic phases of lipid-water systems.

IN the course of the structure analysis of the mesomorphic phases occurring in lipid–water systems1–4 we have observed an optically isotropic phase, the X-ray reflexions of which can be indexed on a face-centred cubic lattice2,3. We assumed that its structure consists of spherical particles, packed in a face-centred cubic lattice. We further postulated that the spheres consist of micelles containing lipid2,3; but we pointed out that the alternative model, formed by water spheres embedded in a hydrocarbon matrix, could not be discarded solely on the basis of the X-ray data5. We intend to show in this communication that the latter structure is in fact more satisfactory, when other lines of evidence are taken into account.

Second virial coefficient: variations with lysozyme crystallization conditions

Interparticle lysozyme interactions in solution have been studied by small angle X-ray scattering (SAXS) as a function of salt type (NaCl, NaNO 3 , NaSCN and NaOAc). salt concentration, and as a function of temperature between 30 C and 10°C. The choice of conditions was made to cover variations from (undersaturated) solutions to (supersaturated) crystallization conditions. The second virial coefficients (A 2 ) were determined from the X-ray structure factors extrapolated to the origin, as a function of protein concentration. The A 2 values which correspond to lysozyme crystallization conditions were found to be in a range from about zero to - 8.0 × 10 -4 mol ml g -2 , in agreement with previous determinations by other groups. The variations of the second virial coefficient from positive (repulsive interactions) to negative (attractive interactions) were found to follow the efficiency of salts to induce crystallization. The choice of the second virial coefficient as a tool to predict crystallization conditions is discussed.

Order-disorder conformational transitions of the hydrocarbon chains of lipids☆

Abstract In many lipid-containing systems (intact membranes, lipid-water and proteinlipid-water phases) the hydrocarbon chains are known to undergo a reversible temperature-dependent transition between a highly disordered (type α) and a partly ordered (type β) conformation; in the β conformation the chains, stiff and all parallel, are packed with rotational disorder according to a two-dimensional hexagonal lattice. This work describes an X-ray diffraction and freeze-fracturing electron microscope study of the phases involved in this conformational transition. Several lipid-water systems were studied: mitochondrial lipids; phosphatidic acid, synthetic lecithin; hen egg lecithin. The conformational transition is found to be a complex phenomenon dependent upon the chemical composition of the lipids, the amount of water and temperature. When the lipid is a pure chemical species the transition involves two phases; one with all the chains in the α conformation the other with all the chains in the β conformation. If the chains are heterogeneous, then from the onset of the transition from type α, they segregate into regions with different conformation, presumably according to their length and degree of saturation. One of the phases (Lαβ) consists of regularly stacked lipid lamellae, each of which is a disordered mosaic of two types of domains; one with the chains in the α, the other in the β conformation. In another phase (Lγ) each lipid lamella is formed by one monolayer of type α and one of type β, joined by their apolar faces. Two other phases (Pγ and Pαβ) display two-dimensional lattices, and consist of lipid lamellae distorted by wave-like ripples, with an ordered segregation of domains in the α and in the β conformation. The number and the structure of the phases involved in the conformational transition are strongly dependent upon the heterogeneity of the hydrocarbon chains and upon the charge and hydration of the polar groups. The results of this study have a bearing on the conformation of the chains in membranes, and on the possible biological significance of conformational transitions.

A model of attractive interactions to account for fluid–fluid phase separation of protein solutions

Concentrated γ‐crystallin and lysozyme solutions have been reported to undergo a fluid–fluid phase separation when cooled below a critical temperature. This behavior is under control of the weak forces acting in solution between macromolecules. We have used small angle x‐ray scattering at the synchrotron radiation facility LURE (Orsay, France) to analyze the interaction potentials. A model of attractive interactions which depends upon three parameters, protein diameter, potential depth, and range, is able to account for both the x‐ray structure factors measured at high temperature and for the low temperature phase separation. Although van der Waals forces could be at the origin of the attractive interaction potentials, other more specific effects also contribute to the protein phase diagrams.

On the structure of human serum low density lipoprotein

Abstract Human serum low density lipoprotein was studied in solution by small-angle X-ray scattering techniques, in the presence of variable amounts of NaBr (used with the purpose of raising the electron density of the solvent). The observation of a few diffraction fringes separated by low minima indicates that the low density lipoprotein preparations are fairly homogeneous and that the particles display a spherical symmetry, at least at low resolution; under these conditions the spherical average of the electron density distribution can be determined directly. The analysis of the structure is based upon these electron density distributions and upon the application of Guiniers law to the intensity scattered at very small angles. Quite unexpectedly, the particles are found to contain a spherical lipid bilayer, whose average radius is 65 A. The outer surface appears to be covered by a loose two-dimensional network of protein subunits, probably 60 in number, with icosahedral symmetry; the molecular weight of these subunits is approximately 8000 daltons. The distribution of the two major lipid components, phospholipids and cholesterol esters, is uniform on the two sides of the bilayer; besides, it appears that the protein subunits interact specifically with the cholesterol moiety of the cholesterol esters and that on the outer face of the bilayer the polar groups of the phospholipids are exposed to the solvent. Indirect arguments suggest that the centre of the particle is occupied by a protein core.

Protein interactions in the calf eye lens: interactions between β-crystallins are repulsive whereas in γ-crystallins they are attractive

Non-specific interactions in β- and γ-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7°C and 31°C. Two types of interactions were observed. With β-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with α-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with γ-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.

Structure of human serum lipoproteins in solution: II. Small-angle X-ray scattering study of HDL3 and LDL☆☆☆

Abstract Two human serum lipoprotein particles, HDL 3 and LDL, were studied in solution in solvents of variable density (NaBr in water) by small-angle X-ray scattering using a position-sensitive proportional counter. The data were analysed using the theoretical approach outlined in the accompanying paper (Luzzati et al. , 1976). The structures of the particles were found to be independent of the salt concentration of the solution (i.e. the particles are impenetrable to NaBr). Density heterogeneities are negligible and size and shape heterogeneities appear to be small. The particle structures could be quantitatively described in terms of a set of parameters and of a few one-dimensional functions. The parameters are the volume, radius of gyration and surface area of the shape functions; the second moment and square average of the electron density contrast at buoyancy; the electron density level, volume, radius of gyration and surface area of the hydrocarbon and polar regions. The one-dimensional functions are: the distribution of chords, the spherical average of the shape function and of the electron density at buoyancy, and the fraction of each spherical shell occupied by the hydrocarbon and polar regions. These parameters and functions are internally consistent and agree with the chemical data confirming the assumptions made in their derivation. The results are compatible with the shape of the particle being compact and quasi-spherical although with deeply convoluted surfaces. They also indicate that the outer layers of the particles are occupied by the proteins and the polar groups of phospholipids and free cholesterol, and the cores by neutral lipids. The maximum diameters of the particles are 130A and 280A for HDL 3 and LDL, respectively, while the hydrocarbon cores have diameters of 80A and 230A, respectively. The solvent is considered to penetrate to 25A from the center of the HDL 3 particle with a minimum solvation at a radius of 45A. In the case of LDL, the solvent penetrates to 55A from the center of the particle. The lipids in the cores of the particles, particularly the cholesterol esters, appear to display a micelle-like organization with the steroid nuclei segregated in regions distinct from those occupied by the hydrocarbon chains. Although the data are consistent with several aspects of previously proposed models, they indicate that the structures of the HDL 3 and LDL particles are more complex than previously believed.

A novel cubic phase—A cage-like network of rods with enclosed spherical micelles

Abstract Among the numerous lipid-water phases a few crystallize in the cubic system and offer interesting crystallographic problems. One of these (space group Ia3d) consists of rods of finite length, joined three by three and forming two three-dimensional networks, mutually interwoven and unconnected. We describe the structure of another widespread cubic phase, space group Pm3n. The structure consists of a three-dimensional networks of rods of finite length, all crystallographically equivalent, which meet three of three at one end and four by four at the other and form the bars of a system of cages, each of which encloses a spherical micelle (chlathrate-type structure). The “liquid” hydrocarbons occupy the interior of the rods and spheres, the water fills the interstices. The hydrocarbon-water interface is covered by the polar groups of the lipid molecules.