Vittorio Luzzati

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.

Structure et polymorphisme des lipides: Étude par diffraction des rayons X du système formé de lipides de mitochondries de coeur de boeuf et d'Eau☆

Abstract A lipid preparation from beef heart mitochondria, carefully extracted and analysed, has been used for the X-ray diffraction study of the structure of the phases present in the lipid-water system. At high temperature, under conditions in which the conformation of the paraffin chains is “liquid”, two phases are observed. One, found at very low water content, is formed by a two-dimensional hexagonal array of narrow water channels, embedded in a paraffin matrix; this type of hexagonal structure is common in lipid-water systems involving phospholipids of biological origin. The other phase is lamellar, formed by equidistant lipid layers of constant thickness, separated by water layers the thickness of which varies from 8 to over 250 A, according to the water content of the system. The distribution of the electron density in the lipid lamellae has been analysed quantitatively, making use of the fact that, for this type of structure, the amplitudes of the reflections are at all concentrations proportional to the Fourier transform of an isolated lamella. As the temperature is lowered gradual organization of the paraffin chains takes place, without change in the thickness of the lipid lamellae. This phenomenon is interpreted by assuming that the centre of the paraffin leaflet is occupied by an ordered layer, involving a fraction of the paraffin chains, which are organized over part of their length: the conformation of the rest of the chains is assumed to remain “liquid”. At still lower temperatures, two other lamellar phases are observed. One is formed of identical lipid leaflets, each containing a double paraffin layer, in which the chains are stiff (but randomly oriented around thin long axes) and organized in a two-dimensional hexagonal lattice. The other phase consists of an alternate sequence of two types of lipid leaflet: one is the low-temperature form just described, the other is the high-temperature lamellar form, with part of the chains disordered. The agreement of the observed and calculated structure factors provides strong confirmation of the two models. The phase transitions observed in this system certainly involve a segregation of the different lipid molecules. This phenomenon is discussed with reference to the known chemical heterogeneity of the lipids of this system.

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.

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.

Shape and size of bovine rhodopsin: A small-angle X-ray scattering study of a rhodopsin-detergent complex☆

Abstract Rhodopsin is extracted from rod outer segments of retinas with dodecyldimethylamine oxide (DDAO), a non-ionie detergent. The rhodopsin-DDAO complex is characterized by binding experiments, gel filtration, sedimentation, densimetry; its homogeneity, chemical composition, weight and partial specific volume are determined. The complex turns out to be a reasonably monodisperse association of one rhodopsin and 156 DDAO molecules. The rhodopsin-DDAO complex and the detergent micelles are studied by small-angle X-ray scattering techniques using a water/sucrose solvent of variable density. The experiments are performed on an absolute scale; mainly the value and curvature of the scattering curves at zero angle are exploited. The structure of the complex and of the micelles is shown to be independent of sucrose. Under these conditions the final result of the X-ray scattering study of each type of particle is the numerical value of a set of five parameters: molecular weight, volume and radius of gyration of the volume occupied by the particles, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of rhodopsin and of the detergent moiety are very near to each other. The analysis of these parameters leads to the determination of the size and shape of the detergent micelles and to an estimate of the size and shape of the volumes occupied by protein and by detergent in the complex. We find rhodopsin to be a very elongated molecule (maximum diameter ~95 A) which spans a flat detergent micelle. These results suggest that in the rod outer segment discs the rhodopsin molecules span the membranes, that the rhodopsin molecules of the two opposite membranes of each disc come near to each other and that a high fraction of the intra-disc space is occupied by rhodopsin.

THE STRUCTURE OF NUCLEOHISTONES AND NUCLEOPROTAMINES.

Unoriented gels and solutions of DNA, nucleohistone (DNH) and nuoleoprotamine (DNP) have been studied systematically as a function of water content and ionic strength, by small-angle X-ray diffraction techniques. This experimental approach, which is formally equivalent to a phase diagram analysis, permits the investigation of the structure of biological macromolecules under conditions similar to those that exist in the living cell; furthermore, intact biological samples can sometimes be studied by the same technique. Dilute isotropic solutions, in which the solute is randomly dispersed, give a diffuse halo around the incident beam. When the solute particles are rod-like in shape the scattered intensity has a typical angular distribution from which the radius of gyration of the rods around their axis can be determined; if the experiments are carried out on an absolute scale, the mass per unit length of the rods can be determined as well. At higher concentration organized liquid-crystalline structures are encountered, the X-ray diffraction patterns of which are formed by bands or sharp lines. A systematic study, as a function of concentration, of systems containing DNA, DNH and DNP reveals the existence of various types of structure, each characterizing one phase; some of the parameters of each structure can be determined. The results obtained with DNA-water systems, containing variable amounts of electrolyte, show that at all concentrations from 1 to 50% the DNA molecules are rod-like in shape and that the mass per unit length of the rods is in excellent agreement with the Watson-Crick model. Furthermore, at high concentrations the DNA rods are aligned with their axes parallel and organized in hexagonal arrays, each molecule being separated from the others by the solvent. The molecules are effectively independent, the distance between them depending only on the concentration. The structures observed in DNH are more complex. In the dilute solutions rod-like particles are found, the mass per unit length and the axial radius of gyration of which are consistent with a bundle of four DNA molecules surrounded by histone. At higher concentrations, several liquid crystalline phases are observed, some of which give fairly sharp reflections at high spacings (50 A). One phase, which exists at concentrations close to 38%, is formed by parallel associations of pairs of DNA molecules accompanied by histone. Another phase, present in the concentration range 55 to 65%, is a hexagonal array of parallel DNA rods with water and histones filling the gap between the DNA molecules. At still higher concentration (circa >70%) another phase is found, the structure of which is not discussed here. The various phases are observed in a variety of DNH samples; the salient features of the different structures are shown to be typical of the nucleohistones. In the DNP-water system two phases are found: the isotropic dilute solution, which exists at very low DNP concentration and cannot be studied by the X-ray techniques, and a hexagonal organization of DNA molecules, with water and protamine in the interstices. The amount of water that can be taken up by this structure is much smaller than in the case of DNA and DNH. The structure of DNP is more ionic-strength dependent than either DNA or DNH. These structures are highly specific for DNH and DNP; no intermediate structure is found in systems containing a mixture of DNH and DNP. Furthermore, it is shown that the X-ray pictures obtained with intact fowl erythrocyte nuclei and with trout sperm heads are very similar to those either of DNH-water or of DNP-water systems, taken at appropriate concentrations. The results of this small-angle X-ray scattering study are compared with the previous X-ray scattering investigations of oriented nucleoprotein fibres and with the information provided by hydrodynamic and electron microscope techniques.

X-ray diffraction and electron microscope study of the interactions of myelin components. The structure of a lamellar phase with a 150 to 180 Å repeat distance containing basic proteins and acidic lipids☆

Abstract A variety of phases has been studied: those formed by lipids extracted from myelin, the basic myelin proteins A1 (from the central nervous system) and P1 (from the peripheral nervous system) or other basic proteins. A particularly interesting type of phase was observed which consists of one of the basic proteins of myelin, acidic phospholipids and sulphatides; this phase is lamellar and contains two lipid bilayers in its unit cell. The structure of this phase was determined by the pattern recognition technique and by electron microscope observations of OsO 4 -flxed and freeze-etched preparations. It is formed by two different lipid bilayers, one containing mainly the phospholipids with the hydrocarbon chains in a liquid-like conformation and the other containing mainly the sulphatides with at least one fraction of the chains stiff and hexagonally packed. Under the effect of high temperature, or if cholesterol is added, this phase is replaced by other phases which lack the large repeat. The segregation of the lipids and their specific associations with the basic proteins are discussed in relation to the structure of myelin.

Polymorphisme des lipides polaires et des galacto-lipides de chloroplastes de maïs, en présence d'eau

Abstract The lipid-water phase diagrams of polar- and galacto-lipids extracted from maize chloroplasts are described and the structures of the various phases are analysed. The general conformation of the paraffin chains is found to be liquid-like. One phase is lamellar, formed by identical planar lipid leaflets, filled by the paraffin chains and separated by layers of water. In the case of the polar lipids the thickness of the water layer increases more than 150 A with increasing water concentration, whereas in the case of the galacto-lipids only a small amount of water can be incorporated between the lipid leaflets (the maximum thickness of the water layer is 20 A) and the water in excess remains as a separate phase. This property, previously observed with other lipids, is related to the presence of net electrical charges in polar lipids. In the “dry” region of the phase diagram other phases are observed: one is a hexagonal array of infinite stiff rods, filled by the polar elements of the system, the other is formed by rods of similar structure but of finite length, joined three by three to form two interwoven three-dimensional networks, organized in a body-centred cubic lattice. The hexagonal phase is observed for both the polar- and galacto-lipids and the cubic phase only in galacto-lipids. The structures of the lamellar and of the cubic phases are consistent with the analysis of the intensity of the X-ray reflections. The observations are compared to those previously made with other lipid systems, and some conclusions of general interest are drawn.

Structure de l'acide désoxyribonucléique en solution: Étude par diffusion des rayons X aux petits angles

The structure of the sodium salt of DNA (DN. Na) in aqueous solution has been investigated by X-ray scattering techniques. The gels that are formed at high DNA concentrations (15 to 60%) have been examined with a small-angle scattering camera ( Luzzati & Nicolaieff, 1959 ). The structure is liquid-crystalline and consists of parallel equidistant rods organized in a hexagonal two-dimensional lattice. Given the size of the lattice and the DNA concentration, it is possible to calculate the linear mass (weight per unit length) of DN. Na. The experimental results show that the linear mass is independent of concentration and is the same in pure water and in saline (1 M - and 3 M -NaCl): its value is in excellent agreement with the Crick & Watson model. The isotropic solutions existing at low DNA concentrations (1 to 6%) have been studied with a new technique based upon the measurements of small-angle scattered intensity on an absolute scale ( Luzzati, 1960 ). When the solute is rod-like in shape this technique gives the linear mass of the rods in addition to the radius of gyration around the axis. In pure water the linear mass of DN. Na is the same as in the liquid-crystalline gels, and agrees very closely with the Crick & Watson model. By analysing the effect of two salts, NaCl and NaBr, added to water in different concentrations, it has been established that the structure of DN. Na is insensitive to the amount of electrolyto and that the distribution of the ions in water is strongly disturbed in the vicinity of DNA molecules. The experimental data have been quantitatively interpreted by assuming that every DNA molecule is surrounded by a definite amount of “bound” water (about 70% of the mass of DNA) which is impenetrable to the ions. Several conclusions have been drawn: (a) DNA is rod-like in shape; the molecules are well dispersed in water, at all concentrations, and there are no associations similar to those we have discovered in nucleoproteins ( Luzzati & Nicolaieff, 1959 ). (b) With our present knowledge of the structure of DNA, the most reasonable interpretation of our experimental observations leads to the conclusion that the whole of the DNA extracted from cells by the usual mild procedures has the same structure and that this structure is essentially that of the Crick & Watson model. (c) The structure of DNA is independent of salt concentration. This feature seems to reflect an intrinsic stability of DNA: it is likely that the same structure is preserved inside the nuclei, where DNA is often associated with basic proteins. (d) The perturbation of the distribution of the ions in the vicinity of the DNA molecules probably has some bearing on the chemical reactivity of DNA. Furthermore it is likely that the amount of “bound” water is a function of the base composition of DNA, and this may explain the correlation of the buoyancy density, measured in concentrated CsCl solution, with base composition.

Cubic phases of lipid-containing systems. The structure of phase Q223 (space group Pm3n). An X-ray scattering study.

The hexagonal (H) and the cubic (Q223) phases of the systems dodecyltrimethylammonium chloride-water and palmitoyllysophosphatidy choline-water have been studied by X-ray scattering techniques. The signs of the reflections of phase H were determined by a systematic study as a function of the water content, those of phase Q223 were assessed using a pattern recognition approach based upon the axiom that the histograms of the electron density maps of phases Q223 and H, extrapolated to the same concentration and properly normalized in scale and shape, are very similar to each other. In the case of phase Q223, all the sign combinations (the phi-sets) compatible with the observed reflections were generated, and each of the corresponding histograms was compared with the histogram of the map of phase H. One novelty of this work is the use of a highly sensitive criterion to estimate the similarity of the histograms, namely the distance in the six-dimensional space of the moments [mean value of (delta rho)n]1/n, for 3 greater than or equal to n greater than or equal to 8. In the two systems, the use of this criterion has led to the unambiguous choice of one electron density map. The maps show that the structure of phase Q223 consists of disjointed micelles (of type I), belonging to two different classes: those of one class are quasi-spherical in shape and are centered at the points a, those of the other class are disc-shaped and are centred at the points c. The results of this work rule out a structure formed by a cage-like distribution of rods enclosing a set of quasi-spherical micelles and is consistent with previous proposals. This is the second example, after that of phase Q227, of a micellar cubic phases in lipid-containing systems; all the known examples of phase Q223 are of type I, those of phase Q227 of type II.