Kinetics of the lamellar-inverse hexagonal phase transition determined by time-resolved X-ray diffraction.

The kinetics of the lamellar (L alpha)-inverse hexagonal (HII) phase transition in diacylphosphatidylethanolamine (PE)--water systems were probed with time-resolved X-ray diffraction. Transition kinetics in the fast time regime (approximately 100 ms) were studied by initiating large temperature jumps (up to 30 degrees C) with a 50-ms electrical current pulse passed through a lipid-salt water dispersion, resulting in ohmic heating of the sample. Diffraction with a time resolution to 10 ms was acquired at the National Synchrotron Light Source. The time constant for the phase transition for 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was on the order of 100 ms for the largest temperature jumps recorded. Faster transition behavior was found for a 1,2-dielaidoyl-sn-glycero-3-PE mixture. The HII lattice parameters for both systems were seen to swell from an initial value commensurate with the lamellar lattice to the final equilibrium value. The rate of swelling was seen to be independent of the magnitude of the temperature jump. For small temperature jumps (less than 10 degrees C), the phase transition kinetics slow dramatically, and transition studies can readily be performed on a conventional rotating anode X-ray source. At 4 degrees C, a DOPE sample was observed to slowly convert to the hexagonal phase over the course of a week, with the decay in the lamellar intensity fitting a power law behavior over four decades of time. This power law behavior is shown to have interesting consequences to the determination of the phase transition temperature of lipid-water dispersions by conventional methods such as calorimetry.     

Effect of heat on frog sciatic nerve determined by X-ray diffraction

Low-angle X-ray diffraction patterns have been recorded from frog sciatic nerves in Ringer's solution after heat treatment from 20 to 80°C. The X-ray patterns were obtained from the heat treated specimens after cooling to room temperature. The normal X-ray pattern of frog sciatic nerve in Ringer's solution with $\text{d=171}\text{A}\text{?}$ was maintained from 20 to 58°C. Above 58°C, a new high temperature pattern based on a repeat period of $\text{d?435}\text{A}\text{?}$ was recorded from the nerve in Ringer's solution. The physical state of nerve myelin after heat teratment at a temperature ?58°C has been identified as the anomalous swollen state. Anomalous swelling takes place in units of four membranes.     

Difficult macromolecular structures determined using X-ray diffraction techniques

Macromolecular crystallography has been, for the last few decades, the main source of structural information of biological macromolecular systems and it is one of the most powerful techniques for the analysis of enzyme mechanisms and macromolecular interactions at the atomic level. In addition, it is also an extremely powerful tool for drug design. Recent technological and methodological developments in macromolecular X-ray crystallography have allowed solving structures that until recently were considered difficult or even impossible, such as structures at atomic or subatomic resolution or large macromolecular complexes and assemblies at low resolution. These developments have also helped to solve the 3D-structure of macromolecules from twin crystals. Recently, this technique complemented with cryo-electron microscopy and neutron crystallography has provided the structure of large macromolecular machines with great precision allowing understanding of the mechanisms of their function.     

The intermediate filament architecture as determined by X-ray diffraction modeling of hard alpha-keratin

Despite investigation since the 1950s, the molecular architecture of intermediate filaments has not yet been fully elucidated. Reliable information about the longitudinal organization of the molecules within the filaments and about the lateral interfilament packing is now available, which is not the case for the transverse architecture. Interesting results were recently obtained from in vitro microscopy observations and cross-linking of keratin, desmin, and vimentin analyses. The structural features that emerge from these analyses could not be fully representative of the in vivo architecture because intermediate filaments are subject to polymorphism. To bring new light to the transverse intermediate filament architecture, we have analyzed the x-ray scattering equatorial profile of human hair. Its comparison with simulated profiles from atomic models of a real sequence has allowed results to be obtained that are representative of hard alpha-keratin intermediate filaments under in vivo conditions. In short, the alpha-helical coiled coils, which are characteristic of the central rod of intermediate filament dimers, are straight and not supercoiled into oligomers; the radial density across the intermediate filament section is fairly uniform; the coiled coils are probably assembled into tetrameric oligomers, and finally the oligomer positions and orientations are not regularly ordered. These features are discussed in terms of filament self-assembling and structural variability.     

Position and orientation of phalloidin in F-actin determined by X-ray fiber diffraction analysis

Knowledge of the phalloidin binding position in F-actin and the relevant understanding of the mechanism of F-actin stabilization would help to define the structural characteristics of the F-actin filament. To determine the position of bound phalloidin experimentally, x-ray fiber diffraction data were obtained from well-oriented sols of F-actin and the phalloidin-F-actin complex. The differences in the layer-line intensity distributions, which were clearly observed even at low resolution (8 A), produced well-resolved peaks corresponding to interphalloidin vectors in the cylindrically averaged difference-Patterson map, from which the radial binding position was determined to be approximately 10 A from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be approximately 84 degrees and 0.5 A relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH)(7) residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11.     

Solvent structure in crystals of trypsin determined by X-ray and neutron diffraction.

The solvent structure in orthorhombic crystals of bovine trypsin has been independently determined by X-ray diffraction to 1.35 A resolution and by neutron diffraction to 2.1 A resolution. A consensus model of the water molecule positions was obtained using oxygen positions identified in the electron density map determined by X-ray diffraction, which were verified by comparison to D2O-H2O difference neutron scattering density. Six of 184 water molecules in the X-ray structure, all with B-factors greater than 50 A2, were found to be spurious after comparison with neutron results. Roughly two-thirds of the water of hydration expected from thermodynamic data for proteins was localized by neutron diffraction; approximately one-half of the water of hydration was located by X-ray diffraction. Polar regions of the protein are well hydrated, and significant D2O-H2O difference density is seen for a small number of water molecules in a second shell of hydration. Hydrogen bond lengths and angles calculated from unconstrained refinement of water positions are distributed about values typically seen in small molecule structures. Solvent models found in seven other bovine trypsin and trypsinogen and rat trypsin structures determined by X-ray diffraction were compared. Internal water molecules are well conserved in all trypsin structures including anionic rat trypsin, which is 65% homologous to bovine trypsin. Of the 22 conserved waters in trypsin, 19 were also found in trypsinogen, suggesting that they are located in regions of the apoprotein that are structurally conserved in the transition to the mature protein. Seven waters were displaced upon activation of trypsinogen. Water structure at crystal contacts is not generally conserved in different crystal forms. Three groups of integral structural water molecules are highly conserved in all solvent structures, including a spline of water molecules inserted between two beta-strands, which may resemble an intermediate in the formation of beta sheets during the folding of a protein.     

A high-pressure form of sulfuric acid monohydrate as determined by X-ray and neutron diffraction

Two high-pressure polymorphs of sulfuric acid monohydrate (oxonium hydrogensulfate) have been obtained at ambient temperature by crystallisation at high pressure from the liquid at 1.3 GPa (form III) and by direct compression of the ambient-pressure form I first to 1.26 GPa (form II) and then to 1.72 GPa (form III). The structure of form III was solved by single crystal X-ray diffraction and this structure was used as the basis for the refinement of hydrogen positions using high-pressure neutron powder diffraction data. Form III crystallises in the orthorhombic crystal system at 1.97 GPa, and features parallel chains of hydrogensulfate ions linked by oxonium ions to form a three-dimensional hydrogen-bonded network. On further compression to 3.05 GPa, the direction of maximum compressibility is found to be along the a-axis and is associated with the shortening of a hydrogen bond between a hydrogensulfate ion and an oxonium ion. The structure of form II remains elusive although at ambient temperature it is stable (or metastable) at pressures as low as 0.42 GPa, perhaps indicating that it could be recoverable to ambient-pressure at low temperature.     

Structure of human low-density lipoprotein subfractions determined by X-ray small-angle scattering

The structure of low-density lipoprotein (LDL) particles from three different density ranges (LDL-1: d = 1.006−1.031 g/ml; LDL-3: d = 1.034−1.037 g/ml; LDL-6: d = 1.044−1.063 g/ml) was determined by X-ray small-angle scattering. By using a theoretical particle model, which accounted for the polydispersity of the samples, we were able to obtain fits of the scattering intensity that were inside the noise interval of the measured intensity. The assumption of deviations from radial symmetry is not supported by our data. This implies a spread-out conformation of the apolipoprotein B (apoB) molecule, which appears to be localized in the outer surface shell. A globular structure is not consistent with our data. Furthermore, different models exist concerning the structure of the cholesterol ester core below the phase transition temperature. The electron density data suggest an arrangmeent in which the steroid moieties are localized at average radii of 3.2 and 6.4 nm. Model calculations show that packing problems can only be avoided if approximately half of the acyl chains of each shell are pointing towards the center of the particle, the other half towards the surface. This arrangement of the acyl chains has never been proposed before. The LDL particles of different density classes differ mainly with respect to the size of the core but also with respect to the width of the surface shells. Model calculations show that the size of different LDL particles can be accurately predicted from the compositional data.     

Structure of tumour necrosis factor by X-ray solution scattering and preliminary studies by single crystal X-ray diffraction

The structure of tumour necrosis factor has been investigated by X-ray small-angle scattering and X-ray diffraction using synchrotron radiation. The overall radius of gyration is 25.5 A. A plausible model accounting for the scattering curves consists of an elongated trimer with an axial ratio of 3 to 4 and a maximal chord with a lower limit of 80 A. Tumour necrosis factor has been crystallized in a trigonal space group. Our results are in favour of a single trimer in the asymmetric unit. The diffraction extends to 3.5 A.     

Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering

The structure of low-density lipoprotein (LDL) particles from three different density ranges (LDL-1: d = 1.006-1.031 g/ml; LDL-3: d = 1.034-1.037 g/ml; LDL-6: d = 1.044-1.063 g/ml) was determined by X-ray small-angle scattering. By using a theoretical particle model, which accounted for the polydispersity of the samples, we were able to obtain fits of the scattering intensity that were inside the noise interval of the measured intensity. The assumption of deviations from radial symmetry is not supported by our data. This implies a spread-out conformation of the apolipoprotein B (apoB) molecule, which appears to be localized in the outer surface shell. A globular structure is not consistent with our data. Furthermore, different models exist concerning the structure of the cholesterol ester core below the phase transition temperature. The electron density data suggest an arrangement in which the steroid moieties are localized at average radii of 3.2 and 6.4 nm. Model calculations show that packing problems can only be avoided if approximately half of the acyl chains of each shell are pointing towards the center of the particle, the other half towards the surface. This arrangement of the acyl chains has never been proposed before. The LDL particles of different density classes differ mainly with respect to the size of the core but also with respect to the width of the surface shells. Model calculations show that the size of different LDL particles can be accurately predicted from the compositional data.     

Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles

Quantitative structures of the fully hydrated fluid phases of dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) were obtained at 30 degrees C. Data for the relative form factors F(q(z)) for DMPC were obtained using a combination of four methods. 1), Volumetric data provided F(0). 2), Diffuse x-ray scattering from oriented stacks of bilayers provided relative form factors |F(q(z))| for high q(z), 0.22 < q(z) < 0.8 A(-1). 3), X-ray scattering from extruded unilamellar vesicles with diameter 600 A provided |F(q(z))| for low q(z), 0.1 < q(z) < 0.3 A(-1). 4), Previous measurements using a liquid crystallographic x-ray method provided |F(2 pi h/D)| for h = 1 and 2 for a range of nearly fully hydrated D-spacings. The data from method 4 overlap and validate the new unilamellar vesicles data for DMPC, so method 4 is not required for DLPC or future studies. We used hybrid electron density models to obtain structural results from these form factors. Comparison of the model electron density profiles with that of gel phase DMPC provides areas per lipid A, 60.6 +/- 0.5 A(2) for DMPC and 63.2 +/- 0.5 A(2) for DLPC. Constraints on the model provided by volume measurements and component volumes obtained from simulations put the electron density profiles rho(z) and the corresponding form factors F(q(z)) on absolute scales. Various thicknesses, such as the hydrophobic thickness and the steric thickness, are obtained and compared to literature values.     

Molecular and crystal structures of chitosan/HI type I salt determined by X-ray fiber diffraction

The three-dimensional structure of chitosan/HI type I salt was determined by the X-ray fiber diffraction technique and linked-atom least-squares refinement method. Two polymer chains and four iodide ions (I(-)) crystallized in a monoclinic unit cell with dimensions a = 9.46(2), b = 9.79(2)], c (fiber axis)=10.33(2)A, beta = 105.2(2) degrees and a space group P2(1). Chitosan chains adopted an extended twofold helical conformation that was stabilized by O-3...O-5 hydrogen bonds, and the O-6 atom adopted nearly gt orientation. Polymer chains zigzag along the b-axis and directly connect to each other by N-2...O-6 hydrogen bonds. Two columns of iodide ions were shown to pack at the bending points of the zigzag sheets, and their locations are closely related to those of water columns in the hydrated chitosan. The iodide ions stabilized the salt structure by forming hydrogen bonds with the N-2 and O-6 atoms of the polymer chains together with an electrostatic interaction between N-2 and the iodide ions.     

The electrostatic potential for the phosphodiester group determined from X-ray diffraction.

The charge density distribution in the crystal structure of ammonium dimethylphosphate at 123 K has been determined from x-ray diffraction data (MoK alpha) using 8437 reflections with sin theta/lambda less than 1.33 A-1 [NH4+.(CH3)2PO4-, M(r) = 143.08, monoclinic, P2(1)/c, a = 10.007(1), b = 6.926(1), c = 9.599(2) A, beta = 105.40(1) degrees, V = 641.4(3) A3, Z = 4, F000 = 304, Dx = 1.4815 g.cm-3, mu = 3.726 cm-1]. Least-squares structure refinement assuming Stewart's rigid pseudoatom model (variables including Slater-type radial exponents and electron populations for multipole terms extending to octapoles for C, N, O, and P, and dipoles for H) gave R(F2) = 0.039 for all reflections. The dimethylphosphate anion is in the gauche-gauche conformation and has approximate twofold symmetry. One phosphoryl O atom forms three hydrogen bonds and the other forms one. Neither of the ester O atoms is hydrogen bonded. For the dimethylphosphate anion isolated from the crystal structure, a map of the electrostatic potential obtained using the pseudoatom charge parameters shows that the phosphoryl O atoms are considerably more electronegative than the ester O atoms. The electrostatic potential distribution obtained in this way has been fitted by least squares to a system of atom-centered point charges. The potential calculated from these point charges agrees with the experimental result. It also agrees reasonably well with potentials obtained from three other systems of point charges that are widely used as part of the semiempirical force field for molecular mechanics and molecular dynamics calculations involving nucleic acids.     

Structure of the filamentous bacteriophage,Pf3, by X-ray fiber diffraction

Fiber diffraction data have been obtained for the filamentous bacteriophage Pf3. The virus crystallizes on a hexagonal net with lattice constants a = b = 5.61 nm and c = 7.50 nm at 0% relative humidity and a = 6 = 6.20 nm and c = 7.99 nm at 98% relative humidity. The X-ray diffraction pattern resembles those of two other bacteriophages, Pf 1 and Xf, and therefore belongs to class II. The data are consistent with a 27₅ helix symmetry with an axial rise of 0.277 (dry) to 0.296 (wet) nm.     

Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction

The structure of native poly(3-hydroxybutyrate) (PHB) granules of Alcaligenes eutrophus was characterized in wet cells or wet granules by analysis of X-ray diffraction. The PHB granules in intact cells were completely amorphous, but became crystalline after treatment with alkali or sodium hypochlorite. The native PHB granules were isolated from the cells by treatment with enzymes and sonic oscillation. The isolated PHB granules remained amorphous in suspension. The PHB granules were crystallized by various treatments with aqueous acetone, alkaline solution (of either NaOH or sodium hypochlorite), and lipase in an aqueous environment. These results suggest that crystallization of PHB molecules is started by the removal of a lipid component from native granules by various treatments.     

Polymorphic phase behavior of cardiolipin derivatives studied by 31P NMR and X-ray diffraction

The polymorphic phase behavior of cardiolipin (diphosphatidylglycerol) analogues with two to five chains per phospholipid head group, namely, dilysocardiolipin, monolysocardiolipin, cardiolipin, and acylcardiolipin, respectively, has been studied by 31P NMR and X-ray diffraction. Dilysocardiolipin dispersions at low salt concentration are micellar, and a transition to a lamellar phase takes place between 1 and 2 M NaCl. From light-scattering measurements, it is also found that a transition takes place from the micellar state with a midpoint at 5.2 mM CaCl2, 0.95 M HClO4, and 1.5 M NaCl. Monolysocardiolipin dispersions are lamellar throughout the concentration range from zero to saturated NaCl. Cardiolipin dispersions undergo a transition from a lamellar to an inverted hexagonal phase between 1 and 2 M NaCl. Acylcardiolipin dispersions are in an inverted hexagonal phase throughout the concentration range from zero to saturated NaCl. The chemical shift anisotropies of both phosphate groups in dilysocardiolipin and of one of the phosphate groups in monolysocardiolipin are drastically reduced in the lamellar phase, indicating a different conformation of the phosphatidyl head group from that normally found in diacyl phospholipid bilayers. The results provide strong support for the "shape" concept of lipid polymorphism when viewed in its most general form including configurational entropy, hydrophobic effects, etc. and indicate the importance of head-group interactions in determining the lipid phase behavior.     

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

High resolution (6 Å) 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. Low-angle 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.     

Water-silica gel interactions,X-ray diffraction study at room and low temperature

X-ray diffraction experiments on water confined in silica gel powder hydrated at about 20% are presented and analyzed at room temperature and down to 77 K. The structural modification of confined water observed at second neighbors is due to the competition between the confinement effect and the water-silica interaction.