X-ray Fiber Diffraction Patterns Research Articles

Molecular packing and crystalline morphologies of biodegradable poly(alkylene dicarboxylate)s derived from 1,6-hexanediol

The crystalline structures of two aliphatic polyesters derived from 1,6-hexanediol and suberic or dodecanodioic acid are studied by means of X-ray and electron diffraction and real space electron microscopy. Chain-folded lamellar crystals have been obtained by isothermal crystallization of dilute n-hexanol or n-heptanol solutions and their crystalline habit have been studied. A regular folding habit is deduced from polyethylene decoration techniques. An orthorhombic unit cell containing four chain segments with a planar zig-zag conformation is determined for the two studied polyesters. A P2 1 ab space group symmetry can be assumed taking into account the detected systematic absences and the symmetry of diffraction patterns. In addition, evidence of a second non-orthorhombic structure is found for the suberic acid derivative. The orthorhombic crystal packing of these polymers is characterized by a setting angle close to 45°, showing a similarity with polyethylene. Refinement of the X-ray fiber diffraction pattern of polyester 6-12 allows to determine the c-chain axis shift between the molecular chains, being observed an alignment of the CH 2–O methylene groups with the CH 2–O oxygen atoms of neighbouring chains. Crystallization from the melt renders spherulites with a negative birefringence and a fibrillar texture, whereas banded morphologies constituted by multilayered single crystals can be obtained by evaporation of xylene solutions. Electron diffraction patterns of ultrathin samples allow the spherulitic radial growth direction to be correlated with the crystalline structure.

β-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

We have studied the core structure of amyloid fibers of yeast prion protein Sup35. We developed procedures to prepare straight fibers of relatively uniform diameters from three kinds of fragments; N (1–123), NMp (1–189), and NM (1–253). X-ray fiber diffraction patterns from dried oriented fibers gave common reflections in all three cases; a sharp meridional reflection at 4.7 Å, and a diffuse equatorial peak at around 9 Å, apparently supporting the typical “cross-β” structure with stacked β-sheets proposed for many different amyloid fibers. However, X-ray fiber diffraction from hydrated fibers showed the meridional reflection at 4.7 Å but no equatorial reflections at 9 Å in all three cases, indicating that the stack of β-sheets in dried fibers is an artifact produced by drying process. Thus, the core structure of these amyloid fibers made of the N domain is likely to be β-helix nanotube as proposed by Perutz et al.

The role of prefibrillar assemblies in the pathogenesis of amyloid diseases

The formation of amyloid fibrils is associated with a large number of major diseases including Alzheimer's disease, type 2 diabetes, prion diseases. Parkinson's disease and various famillal and systemic amyloidosis disorders. The formation of well-ordered fibrils of 7∼10 nm in diameter with a distinct X-ray fiber diffraction pattern and a β-sheet secondary structure is observed in all these amyloid diseases. The formation of the fibrils is correlated with cellular death that is co-localized with the fibrillar deposits. Nevertheless, recent studies have indicated that early prefibrillar assemblies, rather than large amyloid fibrils, may mediate the cytotoxicty that is associated with the fibrillization process. It was clearly demonstrated that unrelated amyloid-forming polypeptides form remarkably similar nanometric annular structures at the early stages of assembly. Those structures are kinetically transient and show strong membrane-interacting and -permeating abilities. The ultrastructure of the transient assemblies and their membrane activity are consistent with a membrane pore formation mechanism of cytotoxicity. The identification of prefibrillar structures as the key cytotoxic agents in amyloid disease suggests that therapeutic approaches to the treatment of amyloid diseases should be ases directed toward the early stages of molecular recognition that facilitate the formation of the early assemblies.

Alexander Rawson Stokes

Alexander Rawson Stokes

Time-Resolved Study of the Martensitic Phase Transition in Syndiotactic Polypropylene

The structural transformations occurring in fibers of syndiotactic polypropylene (sPP) during stretching have been studied by wide-angle X-ray diffraction measurements, using syncrotron radiation. sPP samples were cyclically stretched and relaxed at controlled rate, while recording X-ray fiber diffraction patterns and stress−strain curves. Clear evidences that the polymorphic transition between the helical form II and the trans-planar form III of sPP is a fast process and occurs on the same time scale as the rate the material is stretched, are provided. The transition of form II into form III starts during the stretching in correspondence to critical values of the stress−strain parameters. As the strain increases, crystals of form II transform into form III and the inverse transition occurs, releasing the tension. Below a critical strain, the sample is almost completely in the helical form, as in the initial unstrained state. A complete recovery of the initial dimensions of the specimen is observed, upon releasing the tension. Moreover, the total amount of crystallinity does not change during cyclic elengation and recovery. This suggests that the reversible phase transition between form II and form III does not involve the formation of any disordered, intermediate phase, i.e., it is a direct and cooperative process, implying conformational and structural rearrangements of large bundles of close neighboring chains and occurs instantaneously. These data indicate that while the driving force leading the conventional elastomers to recover the initial dimensions is merely entropic, in the case of sPP elasticity is also assisted by the enthalpic gain achieved when the sample is relaxed. When the tension is removed, both the enthalpic factor, due to the structural transition in the crystalline region, and an entropic factor, due to the conformational transition of the chains in the entangled amorphous phase, contribute to the elastic recovery of the fiber. A possible mechanism for the martensitic crystal−crystal phase transition of sPP is suggested.

Acetan:glucomannan interactions—a molecular modeling study

X-ray fiber diffraction patterns from deacylated acetan and glucomannan (konjac mannan) blends are diagnostic of good orientation and modest polycrystallinity. The meridional reflection on the sixth layer line suggests that the binary complex is a 6-fold helix of pitch 55.4 Å. A molecular modeling study incorporating this information reveals that a double helix in which one strand is acetan and the other glucomannan is stereochemically feasible. While the backbone and side groups are sufficiently flexible to allow the chains to associate with the same or opposite polarity, the parallel model is superior in terms of unit cell packing. The results are compatible with the observed synergy; namely the weak gelation behavior of the complex. The molecular model can be generalized for the binary system when acetan is replaced by xanthan or glucomannan by galactomannan.

A Systematic Investigation into the Effect of Protein Destabilisation on Beta 2-Microglobulin Amyloid Formation

Beta-2-microglobulin (β 2m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (∼1 μm), straight morphology are produced by incubation of the protein at pH≤3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of β 2m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.

Crystal Structure of Form I of Syndiotactic 1,2-Poly(4-methyl-1,3-pentadiene)

The crystal structure of the form I of syndiotactic 1,2-poly(4-methyl-1,3-pentadiene) has been determined studying the X-ray fiber diffraction pattern recorded onto a Fuji image plate. The chain conformation (T6G2T2G2), previously proposed by Meille et al. on the basis of the identity period, has been confirmed, but with deviations of chain torsion angles from the 60 and 180° values. The crystal system is triclinic, a = 17.513(8) Å, b = 9.117(6) Å, c = 11.251(14) Å, α = 95.20(4)°, β = 98.01(3)°, γ = 92.74(5)°, and V = 1768(3) Å3. The space group is P1̄, with two chains per cell, each formed by six nonequivalent CH2CH[CHC(CH3)2] chemical units. The crystal structure was determined and refined by the least-squares method, keeping bond lengths fixed and assuming that chemically equivalent bond angles are equal to each other. The refined model shows unrealistic values for some side group bond angles, in particular for the angle between methyl groups. Quite satisfactory results are obtained fixing this angle to the value of 122.7°, as suggested by searching the Cambridge Structural Database. Final disagreement indices are R1 = 14.3% and R2 = 15.0%.

The native-like conformation of Ure2p in fibrils assembled under physiologically relevant conditions switches to an amyloid-like conformation upon heat-treatment of the fibrils

The [URE3] phenotype in the yeast Saccharomyces cerevisiae is inherited by a prion mechanism involving self-propagating Ure2p aggregates. It is believed that assembly of intact Ure2p into fibrillar polymers that bind Congo Red and show yellow-green birefringence upon staining and are resistant to proteolysis is the consequence of a major change in the conformation of the protein. We recently dissected the assembly process of Ure2p and showed the protein to retain its native α-helical structure upon assembly into protein fibrils that are similar to amyloids in that they are straight, bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis ( Bousset et al., 2002). Here we further show using specific ligand binding, FTIR spectroscopy and X-ray fiber diffraction that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-β core. The X-ray fiber diffraction pattern of these fibrils reveals their well-defined axial supramolecular order. By analyzing the effect of heat-treatment on Ure2p fibrils we bring evidences for a large conformational change that occurs within the fibrils with the loss of the ligand binding capacity, decrease of the α helicity, the formation of a cross-β core and the disappearance of the axial supramolecular order. The extent of the conformational change suggests that it is not limited to the N-terminal part of Ure2p polypeptide chain. We show that the heat-treated fibrils that possess a cross-β core are unable to propagate their structural characteristic while native-like fibrils are. Finally, the potential evolution of native-like fibrils into amyloid fibrils is discussed.

Crystallization and properties of poly(ethylene terephthalate) copolymers containing 5- tert-butyl isophthalic units

The influence of incorporating 5- tert-butyl isophthalic units ( t BI) in the polymer chain of poly(ethylene terephthalate) (PET) on the crystallization behavior, crystal structure, and tensile and gas transport properties of this polyester was evaluated. Random poly(ethylene terephthalate- co-5- tert-butyl isophthalate) copolyesters (PET t BI) containing between 5 and 40 mol% of t BI units were examined. Isothermal crystallization studies were performed on amorphous glassy films at 120 °C and on molten samples at 200 °C by means of differential scanning calorimetry. Furthermore, the non-isothermal crystallization behavior of the copolyesters was investigated. It was observed that both crystallinity and crystallization rate of the PET t BI copolyesters tend to decrease largely with the comonomeric content, except for the copolymer containing 5 mol% of t BI units, which crystallized faster than PET. Fiber X-ray diffraction patterns of the semicrystalline PET t BI copolyesters proved that they adopt the same triclinic crystal structure as PET with the comonomeric units being excluded from the crystalline phase. Although PET t BI copolyesters became brittle for higher contents in t BI, the tensile modulus and strength of PET were barely affected by copolymerization. The incorporation of t BI units slightly increased the permeability of PET, but copolymers containing up to 20 mol% of the comonomeric units were still able to present barrier properties.

Distinct Structural Changes Detected by X-Ray Fiber Diffraction in Stabilization of F-Actin by Lowering pH and Increasing Ionic Strength

Lowering pH or raising salt concentration stabilizes the F-actin structure by increasing the free energy change associated with its polymerization. To understand the F-actin stabilization mechanism, we studied the effect of pH, salt concentration, and cation species on the F-actin structure. X-ray fiber diffraction patterns recorded from highly ordered F-actin sols at high density enabled us to detect minute changes of diffraction intensities and to precisely determine the helical parameters. F-actin in a solution containing 30 mM NaCl at pH 8 was taken as the control. F-actin at pH 8, 30 to 90 mM NaCl or 30 mM KCl showed a helical symmetry of 2.161 subunits per turn of the 1-start helix (12.968 subunits/6 turns). Lowering pH from 8 to 6 or replacing NaCl by LiCl altered the helical symmetry to 2.159 subunits per turn (12.952/6). The diffraction intensity associated with the 27-Å meridional layer-line increased as the pH decreased but decreased as the NaCl concentration increased. None of the solvent conditions tested gave rise to significant changes in the pitch of the left-handed 1-start helix (∼59.8 Å). The present results indicate that the two factors that stabilize F-actin, relatively low pH and high salt concentration, have distinct effects on the F-actin structure. Possible mechanisms will be discussed to understand how F-actin is stabilized under these conditions.

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure.

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-beta reflections characteristic of all amyloid fibrils. In addition to these cross-beta reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1--93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide.

Novel image analysis of two-dimensional X-ray fiber diffraction patterns: example of a polypropylene fiber drawing study

On-line studies of the structural development during continuous drawing of a polypropylene fiber were carried out using synchrotron wide-angle X-ray diffraction. A unique image analysis method was introduced to extract quantitative information on the crystal, amorphous and mesomorphic fractions. In addition, the unit-cell parameters and the crystal orientation were obtained under various draw conditions. It was found that the mesophase remained about constant during drawing. At draw ratios less than 6.0, the crystallinity increased and the crystal density decreased as a result of the stress-induced crystallization of crystals with a large degree of disordering. At draw ratios above 6.0, the crystallinity increased slowly and the decrease in crystal density was also retarded because of the draw-induced melting. The crystal orientation increased with increasing draw ratio up to a ratio of 6.0 and then decreased, probably because of chain breakage.

Glycosaminoglycan conformation: do aqueous molecular dynamics simulations agree with x-ray fiber diffraction?

Glycosaminoglycan-protein interactions are biologically important and require an appreciation of glycan molecular shape in solution, which is presently unavailable. In previous studies we found strong similarity between aqueous molecular dynamics (MD) simulations and published x-ray diffraction refinements of hyaluronan. We have applied a similar approach here to chondroitin and dermatan, attempting to clarify some of the issues raised by the x-ray diffraction literature relating to chondroitin and dermatan sulfate. We predict that chondroitin has the same beta(1-->4) linkage conformation as hyaluronan, and that their average beta(1-->3) conformations differ. This is explained by changes in hydrogen-bonding across this linkage, resulting from its axial hydroxyl, causing a different sampling of left-handed helices in chondroitin (2.5- to 3.5-fold) as compared with hyaluronan (3.0- to 4.0-fold). Few right-handed helices, which lack intramolecular hydrogen-bonds, were sampled during our MD simulations. Thus, we propose that the 8-fold helix observed in chondroitin-6-sulfate, represented in the literature as an 8(3) helix (right-handed), though it has never been refined, is more likely to be 8(5) (left-handed) helix. Molecular dynamics simulations implied that (4)C(1) and (2)S(O), but not (1)C(4), forms of iduronate could be used in refinements of dermatan x-ray fiber diffraction patterns. Current models of 8-fold dermatan sulfate chains containing (4)C(1) iduronate refine to right-handed helices, which possess no intramolecular hydrogen-bonds. However, MD simulations predict that models containing (2)S(O) iduronate could provide better (8(5) helix) starting structures for refinement. Thus, the 8-fold dermatan sulfate refinement (8(3) helix) could be in error.

Analysis of X-ray diffraction from fibres of pf1 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered

X-ray fibre diffraction patterns of well-aligned Pf1 filamentous bacteriophage show sharp layer-lines attributable to an ordered helical array of protein subunits. Electron density maps calculated from the intensity on these layer-lines show no evidence for DNA following the symmetry of the protein, nor is there evidence on the diffraction patterns for the additional layer-lines expected if ordered DNA follows a symmetry different from that of the protein. We conclude that the interactions between DNA and protein in the Pf1 virion, like those in the Ff virion, are delocalized rather than specific, and the DNA structure in the virion is less regular than the protein structure. This conclusion has implications for the process of virion assembly, and we suggest a possible model for the change in the viral DNA symmetry as the DNA is passed to the virion from the intracellular complex with the viral gene 5 single-stranded DNA-binding protein.

Iterative Low-Pass Filtering for Estimation of the Background in Fiber Diffraction Patterns

Disorientation of particles in fibrous specimens results in diffraction patterns in which the layer-line intensity is arced about the center, causing layer lines to overlap at larger radii and, as a consequence, making correction of the background problematic. Taking advantage of the fact that the background is typically made up of spatial frequencies that are substantially lower than those constituting the diffracted intensities, estimates of background in fiber diffraction patterns were made by applying an iterative low-pass filter to the diffraction data. This procedure has been applied to X-ray fiber diffraction patterns from filamentous bacteriophages to calculate the intensities along layer lines as well as to correct for the background.

Common core structure of amyloid fibrils by synchrotron X-ray diffraction

Tissue deposition of normally soluble proteins as insoluble amyloid fibrils is associated with serious diseases including the systemic amyloidoses, maturity onset diabetes, Alzheimer’s disease and transmissible spongiform encephalopathy. Although the precursor proteins in different diseases do not share sequence homology or related native structure, the morphology and properties of all amyloid fibrils are remarkably similar. Using intense synchrotron sources we observed that six different ex vivo amyloid fibrils and two synthetic fibril preparations all gave similar high-resolution X-ray fibre diffraction patterns, consistent with a helical array of β-sheets parallel to the fibre long axis, with the strands perpendicular to this axis. This confirms that amyloid fibrils comprise a structural superfamily and share a common protofilament substructure, irrespective of the nature of their precursor proteins.

X-ray study of beijeran sodium salts, a new galacturonic acid-containing exo-polysaccharide

X-Ray fiber diffraction patterns were obtained from oriented films of sodium salts of a new uronic acid-containing polysaccharide (beijeran) both in its native, poly [ → 3)- α- d-GalA-(1 → 3)- β- l-Rha-(1 → 3)- α- d-Glc- O6Ac-(1 → ], and deacetylated forms. Initially the stretched films of both polysaccharides were amorphous, but the crystallinity was much improved by annealing at high temperature. The deacetylated specimen had higher crystallinity than the native. Both films showed similar X-ray fiber patterns indicating that these polysaccharides had similar unit cell dimensions and that the O-acetyl groups in the native beijeran chain did not disturb the regular array in the crystal having space group P2 1. All the visible reflections could be indexed in terms of a monoclinic unit cell with dimensions a = 1.277, b = 1.611, c (fiber axis) = 2.437 nm, and γ = 96.79°. The fiber axis length and the presence of (002) and (006) reflections indicated that the conformation was made up of two trisaccharide residues, in an extended two-fold helix.

Polymorphism of syndiotactic poly( p-methylstyrene): oriented samples

Possible routes for obtaining oriented fibres of the various known crystalline forms of syndiotactic poly( p-methylstyrene) are described. These routes include solvent diffusion and/or annealing on mesomorphic fibres. The X-ray fibre diffraction patterns show that form I, form II and all the clathrate forms are characterized by chains with helical s(2/1)2 conformation and repeating distance of ≈7.8 Å, while the mesomorphic form (form IV), form III as well as a new crystalline form (form V), are characterized by chains with trans planar conformation and repeating distance of ≈5.1 Å.

Diffraction study of crystalline structure of random copolymers of poly(ethylene terephthalate) and poly(ethylene naphthalene-2,6-dicarboxylate)

Abstract A recent study has indicated that random copolymers of poly(ethylene terephthalate) and poly(ethylene naphthalene-2,6-dicarboxylate) (PET/PEN) can crystallize over the full composition range under the condition of hot-drawing, and that the crystallites formed are based on random sequences of PET and PEN units. This paper is an extension of this study. Wide-angle fibre X-ray diffraction patterns from PET/PEN random copolymers have been recorded using a charge coupled device based three-circle transmission diffractometer, from which reflections on higher layer lines can be observed quantitatively without distortion. These reflections provide additional indications regarding the tilt of the c -axis away from the fibre axis, the crystallite thickness and the nature of the non-periodic layer crystallites themselves. The long period has also been measured as a function of composition by small-angle X-ray diffraction. Diffraction modelling of the crystallites using the CERIUS package confirms the random nature of polymeric chains in the PET/PEN crystallites and enables the level of register between them to be compared with the ‘plane start’ and the non-periodic layer (npl) models. The most appropriate model is an ‘npl’, but containing defects in the otherwise perfect sequence register.