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ABSTRACT: Synchrotron radiation X-ray scattering experiments were performed on unmyelinated pike olfactory nerves. The difference between the meridional and the equatorial traces of the 2-D spectra yielded the 1-D equatorial intensity of the macromolecular components oriented with respect to the nerve: axonal membranes, microtubules and other cytoskeletal filaments. These 1-D spectra display a diffuse band typical of bilayer membranes and, at small s, a few sharper bands reminiscent of microtubules. All the spectra merge at large s. The intensity of the axonal membrane was determined via a noise analysis of the nerve-dependent spectra, involving also the notion that the thickness of the membrane is finite. The shape of the intensity function indicated that the electron density profile is not centrosymmetric. The knowledge of intensity and thickness paved the way to the electron density profile via an ab initio solution of the phase problem. An iterative procedure was adopted: (i) choose the lattice D of a 1-D pseudo crystal, interpolate the intensity at the points sh = h/D, adopt an arbitrary set of initial phases and compute the profile; (ii) determine the phases corresponding to this profile truncated by the thickness D/2; (iii) repeat the operation with the updated phases until a stable result is obtained. This iterative procedure was carried out for different D-values, starting in each case from randomly generated phases: stable results were obtained in less than 10,000 iterations. Most importantly, for D in the vicinity of 200 A, the overwhelming majority of the profiles were congruent with each other. These profiles were strongly asymmetric and otherwise typical of biological membranes.
Journal of Molecular Biology 11/2004; 343(1):187-97. · 4.00 Impact Factor
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ABSTRACT: The effects of several agents, sugars, isotonic KCl, and a variety of drugs, on the structure of the axonal membranes of unmyelinated pike olfactory nerve have been studied by synchrotron radiation X-ray scattering experiments. The main effects of the sugars are: (i) to increase the electron density of the extra-axonal space and thereby yield the absolute scale of the electron density profile; (ii) to osmotically stress the membrane and thus yield its elastic modulus of area compressibility, since the related strain, thickness dilation, is directly determined by the X-ray scattering experiments. Exposure to isotonic KCl, a depolarizing agent, induces membrane thickness to increase. The energy liberated in this process is a function of the amplitude of the dilation and of the elastic modulus of the membrane. This energy turns out to be close to the thermal energy liberated by the pike olfactory nerve during the initial phase of action potential that has previously been measured by others. Electrical depolarization thus seems to be accompanied by a thickness dilation of the axonal membrane. Another effect of isotonic KCl is to induce a large fraction of the membranes to pair by tight apposition of their extra-axonal faces. Local anaesthetics and some drugs have the effect of altering membrane thickness. All these observations are interpreted in terms of a modulation of the conformational disorder of the hydrocarbon chains of the lipid molecules.
Journal of Molecular Biology 11/2004; 343(1):199-212. · 4.00 Impact Factor
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ABSTRACT: The observed intensities of the reflections from the body-centered cubic phase of lipid systems are shown to be incompatible with a recently reported model consisting of straight, indefinitely long rods.
Proceedings of the National Academy of Sciences 09/1981; 78(8):4683. · 9.68 Impact Factor
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Nature 10/1969; 223(5211):1116-21. · 36.28 Impact Factor
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ABSTRACT: The general aim of our current work is to calibrate spectroscopic techniques, useful for the structural analysis of biological membranes, against model systems of well-defined structure. In this study i-anilino-8-naphthalene sulfonate (ANS) and tryptophan fluorescence and X-ray diffraction analyses were carried out on a variety of protein-lipid-water phases. The fluorescence parameters-emission spectrum, quantum yield and efficiency of energy transfer—were found to display a remarkable correlation with the structure of the phases, and with the nature of the two main types of protein-lipid interactions, namely electrostatic and hydrophobic. The sites of fixation of ANS in the different phases are discussed.
Biochimica et Biophysica Acta (BBA) - Biomembranes.
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ABSTRACT: Of the 7 cubic phases clearly identified in lipid-containing systems, 2 are bicontinuous, 4 micellar. 3 of these are of type I: one (Q$^{223}$) consists of two types of micelles, the two others of identical quasi-spherical micelles close-packed in the face-centred (Q$^{225}$) or the body-centred mode (Q$^{229}$). These structures, mush like foams, can be described as systems of space-filling polyhedra: distorted 12- and 14-hedra in Q$^{223}$, rhombic dodecahedra in Q$^{225}$, truncated octahedra in Q$^{229}$. In foams the geometry of the septa and of their junctions are generally assumed to obey Plateau's conditions, at least at vanishing water content: these conditions are satisfied in Q$^{223}$, can be satisfied in Q$^{229}$ by introducing subtle distortions in the hexagonal faces, but cannot be satisfied in Q$^{225}$. Alternatively, these structures can be represented in terms of infinite periodic minimal surfaces (IPMS) since it is found that two types of IPMS, F-RD in Q$^{225}$ and I-WP in Q$^{229}$, almost coincide with one particular equi-electron-density surface of the 3D electron density maps. These IPMS partition 3D space into two non-congruent labyrinths: in the case of the lipid phases one of the labyrinths contains the hydrated micelles, the other is filled by water. If interfacial interactions are associated with these surfaces, then the surfaces being minimal, the interactions may also be expected to be minimal. Another characteristic of the micellar phases is that the dimensions of their hydrophobic core, computed assuming that headgroups and water are totally immiscible with the chains, often are incompatible with the fully extended length of the chains. This paradox is evaded if headgroups and chains are allowed to be partiallly miscible with each other. Des 7 phases cubiques clairement identifiées dans les systèmes lipidiques, 2 sont bicontinues, 4 micellaires. Parmi ces dernières, 3 sont du type I : une (Q$^{223}$) comporte deux types de micelles, deux autres sont formées de micelles identiques quasi-sphériques, empaquetées selon le mode face-centré (Q$^{225}$) ou centré (Q$^{229}$). Tout comme les mousses, ces structures peuvent être décrites en termes d'assemblages denses de polyhèdres : 12-hèdres et 14-hèdres déformés dans Q$^{223}$, dodecahèdres rhombiques dans Q$^{225}$, octahèdres tronqués dans Q$^{229}$. Dans les mousses la géométrie des faces et de leurs jonctions est censée obéir aux conditions de Plateau, du moins aux faibles teneurs en eau : ces conditions sont satisfaites dans Q$^{223}$, peuvent être satisfaites dans Q$^{229}$ au prix d'une petite distortion des faces hexagonales, mais il est impossible de les satisfaire dans Q$^{225}$. On peut également représenter ces structures en termes de surfaces minimales périodiques et infinies (IPMS) : on trouve, en effet, que deux types de IPMS, F-RD pour Q$^{225}$ et I-WP pour Q$^{229}$, coincident avec l'une des surfaces d'égale densité électronique des cartes 3D. Ces IPMS partagent l'espace en deux labyrinthes non congruents: dans les phases micellaires un de ces labyrinthes contient les micelles hydratées, l'autre l'eau. Si on associe à ces surfaces des interactions interfaciales, on peut alors penser que ces interactions sont minimales puisque les surfaces sont minimales. Une autre caractéristique des phases micellaires est que le rayon de leur noyau apolaire, déterminé en faisant l'hypothèse que les chaînes paraffiniques sont totalement immiscibles avec les têtes polaires et l'eau, dépasse souvent la longueur des chaînes étirées. On peut éviter ce paradoxe en admettant que chaînes et têtes polaires sont partiellement miscibles entre elles.
http://dx.doi.org/10.1051/jp2:1996100.
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Vittorio Luzzati
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ABSTRACT: The purpose of this paper is to use (X-ray or neutron) scattering spectra to assess the degree of order — more precisely, translational entropy — in a fluid of monodisperse isotropic particles, avoiding to rely on microscopic models and on computer simulations. The mathematical approach, borowed from information theory, is based upon an ideal stochastic process : a particle is cast in a box containing a known number of particles, with a probability density corresponding to the distribution of interparticle distances defined by the scattering experiment. If the a priori probability density (i.e. before the X-ray scattering experiment) is uniform, then the information associated with the pair of probability densities can be determined : its expression is a straightforward function of the radial distribution function of the interparticle distances, $g(r)$. The information, moreover, is proportional to the derivative, with respect to concentration, of the (translational) entropy in excess over the perfect gas. The correlation with the thermodynamic properties of the system is discussed. By way of illustration, the treatment is applied to neutron scattering experiments performed on Ar and Kr : the agreement of the entropy determined by the thermodynamic and the scattering procedures is quite satisfactory. The validity of the treatment, and more generally the very possibility of determining the function $g(r)$ from the scattering data is shown to require that the function [ $g(r)-1$] have a finite support.
http://dx.doi.org/10.1051/jp2:1993204.
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Vittorio Luzzati
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ABSTRACT: The original purpose of this work was to seek an explanation of the empirical observation that pairs of phases in thermodynamic equilibrium often display an epitaxial relationship. Considering that the polar/apolar interfacial interactions appear to play a predominant role among all the forces that stabilize the phases, there is ground for the proposition that the two phases involved in any particular phase transition consist of structure elements whose area/volume ratio is invariant. Volume and area of the structure elements can be expressed as functions of the water content and the cell parameters of the two coexisting phases: their values can thus be determined experimentally. The volume ratio (structure elements)/(lipid molecules) is equivalent to a partition coefficient. These ideas were applied to a large variety of data available in the literature. The partition coefficient was found to display wide variations, remarkably correlated with the chemical and the physical parameters of the system, suggesting that the segregation of the hydrocarbon chains away from the polar headgroups is not as sharp as it is commonly assumed. The notion of a variable polar/apolar partition is a novelty in the field; moreover, this partition coefficient may well turn into an interesting thermodynamic parameter. As to the significance of the epitaxial relationships, a search through the literature shows that its very existence has many exceptions. In order to explain these observations the conjecture is put forward that the epitaxial coincidences have a kinetic effect on the phase transitions. In particular, it is suggested that any transition involving epitaxially related phases is unlikely to display metastable states. The possibility is also evoked that a selective advantage (be it technological, biological or experimental) may be associated with the existence of epitaxial relationships. This conjecture is illustrated by several examples drawn from the literature. De nombreuses observations expérimentales ont montré que les deux phases qui coexistent lors d'une transition respectent souvent des relations épitaxiales : le but initial de ce travail était d'expliquer ce phénomène étrange. Puisque les intéractions interfaciales semblent jouer un rôle prédominant dans l'équilibre énergétique il y a lieu de supposer que les deux phases à l'équilibre consistent d'éléments de structure dont le volume et l'aire superficielle sont invariants. Ces deux paramètres peuvent s'exprimer en fonction de la teneur en eau et des dimensions des mailles élémentaires des deux phases : leurs valeurs peuvent donc être déterminées à partir de données expérimentales. On note, en outre, que le rapport entre le volume des éléments de structure et celui de la composante lipidique du système équivaut à un coefficient de partage. Lorsqu'on applique ces idées aux données de la littérature on observe que le coefficient de partage varie fortement et que ses valeurs manifestent des corrélations remarquables avec d'autres paramètres chimiques et physiques du système. Ceci suggère que la séparation des chaînes paraffiniques et des têtes polaires des lipides n'est pas aussi nette qu'on a l'habitude de le penser. Cette notion d'un partage polaire/apolaire variable est une nouveauté dans ce domaine; le coefficient de partage, par ailleurs, pourrait jouer un rôle thermodynamique intéressant. En ce qui concerne les relations épitaxiales une analyse des données publiées montre que de telles relations sont fréquentes, mais avec beacoup d'exceptions. Pour expliquer ces observations on émet l'hypothèse que le rôle des relations épitaxiales est surtout cinétique. Cette hypothèse conduit à associer aux relations épitaxiales un avantage sélectif (dans un sens large). Plusieurs exemples sont cités à l'appui de cette hypothèse.
http://dx.doi.org/10.1051/jp2:1995205.
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ABSTRACT: 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 ϑ-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 . 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 centred 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.
Journal of Molecular Biology.
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ABSTRACT: The ($T, c$)-dependent phase diagrams of two gangliosides, GM1 and GM1(acetyl), have been explored, in spite of the frequent occurrence of metastable states. In GM1 two lamellar, one hexagonal and two cubic (aspects # 5 and 13) phases were identified, in addition to the isotropic micellar solution. In GM1(acetyl) two of the phases are cubic (aspects # 8 and 13), one is the fluid isotropic solution. The structure of the lamellar and the hexagonal phases are trivial. The structure of the cubic phases was determined using a combination of freeze-fracture electron microscope and X-ray scattering experiments. The three cubic phases consist of lipid micelles of type I (oil-in-water); in two of the phases (Q$^{225}$ and Q$^{229})$ the micelles are all identical and of quasi-spherical shape. Phase Q$^{223}$ was previously known to contain two types of micelles, one quasi-spherical, the other slightly flattened. The radii of the micelles determined from the dimensions of the electron density troughs were consistent with the chemical data. In keeping with what is known of the micellar solutions, the size of the micelles of the cubic phases of GM1(acetyl) is compatible with a spherical shape, whereas the micelles of GM1 seem to be somewhat too large to be compatible with the length of the molecules and with a spherical shape. Such a wealth of micellar cubic phases is unusual in lipid-water systems. Le diagramme de phases de deux gangliosides, GM1 et GM1(acétyl), a été exploré malgré la présence d'états métastables. On a identifié les phases suivantes : dans GM1 deux phases lamellaires, une hexagonale, deux cubiques (aspects # 5 et 13), une solution micellaire ; dans GM1(acetyl) deux phases cubiques (aspects # 8 et 13) et une solution micellaire. La structure des phases lamellaires et hexagonale est triviale. La structure des phases cubiques a été déterminée par l'usage combiné de microscopie électronique et de diffraction des rayons X. Les trois phases cubiques sont formées de micelles de type I (huile dans l'eau) ; dans deux de ces phases (Q$^{225}$ et Q$^{229})$ les micelles sont toutes identiques et de forme presque sphérique. La phase Q$^{223}$ est connue ; elle est formée par deux types de micelles, les unes presque sphériques, les autres légèrement aplaties. Les rayons des micelles déterminés sur les cartes de densité électronique sont en excellent accord avec les données chimiques. Dans les deux phases cubiques la taille des micelles de GM1(acétyl) est compatible avec une forme sphérique, tandis que les micelles de GM1 semblent être un peu trop grandes par rapport à la longueur des molécules : ces observations sont en excellent accord avec ce que l'on sait sur les solutions micellaires de ces deux lipides. Cette richesse de phases cubiques micellaires est inhabituelle dans les systèmes lipide-eau.
http://dx.doi.org/10.1051/jp2:1995143.
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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 Guinier's 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 Å. 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.
Journal of Molecular Biology.
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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 Å, 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.
Journal of Molecular Biology. 27(2):303-322.
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ABSTRACT: It has recently been shown that the structure of two of the six cubic phases so far identified in lipid-containing systems is micellar, one (Q223) of type I, the other (Q227) of type II. The micelles of both phases belong to two distinct classes, those of each class being centred at one of the special positions of the space group. From the chemical viewpoint, phase Q227 seems to require a heterogenous mixture of water-miscible and water-immiscible lipids, whereas phase Q223 has been observed with chemically pure lipids. Also, the area/volume ratio measured at the polar/apolar interface takes the same value in the two types of micelles of phase Q223, different values in those phase Q227, in keeping with the notion that the area/molecule ratio is closely related to the chemical activity of the lipid components. The topological properties of the micellar phases are profoundly different from those of the bicontinuous phases. The bicontinuous cubic phases (Q230, Q224, Q229) are often presented as paradigms of the infinite periodic minimal surfaces (IPMS). Some authors have generalized that notion and sought in the IPMS a unified theory underlying the entire field of lipid polymorphism. These analogies entertain some confusion between the mathematical concept of surface and the physical notion of interface. A few electron density maps are presented to document the distance that separates the polar/apolar interfaces from the IPMS. The maps also show that some of the geometric singularities (points, lines, surfaces) of the structures coincide with the locus of the CH3, ends of the chains and with the very centre of the water matrix, i.e. with the regions where the short-range disorder is highest. We introduce the expression chaotic zones to designate these regions. In all the lipid phases the chaotic zones are found to occupy special geometric positions, either related to the symmetry elements or to the IPMS. It thus appears that it is energetically more advantageous to adopt an orderly disposal of the disposal of the short-range disorder than to minimize the area of the polar/apolar interfaces. Finally, regarding the possible biological significance of lipid polymorphism, the point is stressed that among the phases that are observed in equilibrium with excess water (these phases are also the most likely candidates for a biological role) those with a cubic symmetry deserve special attention. We have previously involved one of the bicontinuous phases (Q224) in speculations regarding the digestion of fats and the structure of the outer membrane of thermoacidophilic archaebacteria. One of the micellar cubic phases (Q227) is also interesting: on the one hand, its chemical composition is related to the enzymatic degradations that lipids may undergo in biological membranes; its physical structure, on the other hand, makes that phase impervious to water. Since the structure of phase Q224 is closely related to that of phase Q227, a "patch-the-puncture" process can be imagined whereby an enzymatic attack of the lipid, leading potentially to a leak in the membrane, might also induce a local transition to a water-tight structure, eventually stopping the leak. That process could play a role in a variety of circumstances of biological interest.
Journal of Molecular Biology.
