Small-angle X-ray Scattering Results Research Articles

Chromatin structure revealed by X-ray scattering analysis and computational modeling

It remains unclear how the 2m of human genomic DNA is organized in each cell. The textbook model has long assumed that the 11-nm-diameter nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, is folded into a 30-nm chromatin fiber. One of the classical models assumes that the 30-nm chromatin fiber is further folded helically to form a larger fiber. Small-angle X-ray scattering (SAXS) is a powerful method for investigating the bulk structure of interphase chromatin and mitotic chromosomes. SAXS can detect periodic structures in biological materials in solution. In our SAXS results, no structural feature larger than 11nm was detected. Combining this with a computational analysis of “in silico condensed chromatin” made it possible to understand more about the X-ray scattering profiles and suggested that the chromatin in interphase nuclei and mitotic chromosomes essentially consists of irregularly folded nucleosome fibers lacking the 30-nm chromatin structure. In this article, we describe the experimental details of our SAXS and modeling systems. We also discuss other methods for investigating the chromatin structure in cells.

Electrical percolation, morphological and dispersion properties of MWCNT/PMMA nanocomposites

Nanocomposites of poly (methyl methacrylate) (PMMA) and carbon nanotubes have a high potential for applications where conductivity and low specific weight are required. This piece of work concerns investigations of the level of dispersion and morphology on the electrical properties of in situ polymerized nanocomposites in different concentrations of multi-walled carbon nanotubes (MWCNT) in a PMMA matrix. The electrical conductivity was measured by the four point probe. The morphology and dispersion was analyzed by Transmission Electron Microscopy (TEM) and Small Angle X-ray Scattering (SAXS). The correlation between electrical conductivity and the MWCNT amount, presented a typical percolation behavior, whose electrical percolation threshold determined by power law relationship was 0.2 vol. (%) The exponent t from the percolation power law indicated the formation of a 3D network of randomly arranged MWCNT. SAXS detected that the structures are intermediate to disks or spheres indicating fractal geometry for the MWCNT aggregates instead of isolated rods. HR-TEM images allowed us to observe the MWCNT individually dispersed into the matrix, revealing their distribution without preferential space orientation and absence of significant damage to the walls. The combined results of SAXS and HR-TEM suggest that MWCNT into the polymeric matrix might present interconnected aggregates and some dispersed single structures.

Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome

Ataxin-3 (AT3) is the protein that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It cleaves isopeptide bonds between ubiquitin monomers, an event involved in protein quality control mechanisms. AT3 has been implicated in the pathway that sorts aggregated protein to aggresomes via microtubules, in which dynein and histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of small angle X-ray scattering (SAXS) and surface plasmon resonance (SPR), we have investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS results, the AT3 oligomer, consisting of 6–7 subunits, tightly binds to the tubulin hexameric oligomer in a “parallel” fashion. By SPR analysis we have demonstrated that AT3 binds to tubulin dimer with a 50nM affinity. Binding fits with a Langmuir 1:1 model and involves a single binding interface. Nevertheless, the interaction surface consists of three distinct, discontinuous tubulin-binding regions (TBR), one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. In the absence of any of the three TBRs, the affinity is drastically reduced. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, with affinity in the range 0.1–1μM. These results shed light on the interactions among the components of the transport machinery that sorts aggregate protein to the aggresome, and pave the way to in vivo studies aimed at further clarifying their roles.

Crystal and Solution Structure Analysis of FhuD2 from Staphylococcus aureus in Multiple Unliganded Conformations and Bound to Ferrioxamine-B

Iron acquisition is a central process for virtually all organisms. In Staphylococcus aureus, FhuD2 is a lipoprotein that is a high-affinity receptor for iron-bound hydroxamate siderophores. In this study, FhuD2 was crystallized bound to ferrioxamine-B (FXB), and also in its ligand-free state; the latter structures are the first for hydroxamate-binding receptors within this protein family. The structure of the FhuD2-FXB conformation shows that residues W197 and R199 from the C-terminal domain donate hydrogen bonds to the hydroxamate oxygens, and a ring of aromatic residues cradles the aliphatic arms connecting the hydroxamate moieties of the siderophore. The available ligand-bound structures of FhuD from Escherichia coli and YfiY from Bacillus cereus show that, despite a high degree of structural conservation, three protein families have evolved with critical siderophore binding residues on either the C-terminal domain (S. aureus), the N-terminal domain (E. coli), or both (B. cereus). Unliganded FhuD2 was crystallized in five conformations related by rigid body movements of the N- and C-terminal domains. Small-angle X-ray scattering (SAXS) indicates that the solution conformation of unliganded FhuD2 is more compact than the conformations observed in crystals. The ligand-induced conformational changes for FhuD2 in solution are relatively modest and depend on the identity of the siderophore. The crystallographic and SAXS results are used to discuss roles for the liganded and unliganded forms of FhuD2 in the siderophore transport mechanism.

Mechanical investigation of confined amorphous phase in semicrystalline polymers: Case of PET and PLA

The effect of confinement onto the mechanical properties of the amorphous phase of Polyethylene terephthalate (PET) and poly(lactic acid) (PLA) was investigated. These polymers have the advantage of being in bulk amorphous or in semicrystalline state allowing mechanical and physical investigation of the amorphous phase on bulk and confined configuration. Based on small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA) experiments, the micro-structural arrangement of the amorphous and crystalline phase, the rigid amorphous fraction, and the visco-elastic mechanical properties of the different semicrystalline samples were investigated. DSC results help quantifying the rigid amorphous fraction dependence on the crystallinity. DMA measurements lead to quantify the viscoelastic properties of the free and confined amorphous phases for PET and PLA polymers. Indeed, based on the DMA tests, where the maximum of tan(δ) peak is usually related to the glass transition temperature, shifts upon crystallization, the mechanical properties of the restricted and mobile amorphous phase were determined. This result was correlated along with the amorphous phase thickness distribution determined by SAXS results. This observation was bolstered based on literature results about geometrical confinement configurations and their effect on the glass transition temperature of polymeric materials. POLYM. ENG. SCI., 55:397–405, 2015. © 2014 Society of Plastics Engineers

SAXS and TEM investigation of ion tracks in neodymium-doped yttrium aluminium garnet

Neodymium-doped yttrium aluminium garnet (Nd:Y3Al5O12 or Nd:YAG) crystals were irradiated with 2.2GeV 197Au ions. Ion track formation was investigated using synchrotron based small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). Cylindrical ion tracks consisted of an amorphous core with sharp boundaries within the crystalline matrix. The SAXS results, modelled as long cylindrical tracks, provided a constant track density that is 0.6±0.3% different to that of the surrounding matrix. The average track radii determined by both techniques were in excellent agreement, 4.4±0.1nm from SAXS and 4.4±0.5nm from TEM. A comparison with previous results of ion tracks in YAG indicates that the track radius is not affected by the presence of 1mol% Nd dopant.

Fabrication and Characterization of Polyimide/Aln Nanocomposite Films

Polyimide/AlN (PI/AlN) nanocomposite films were prepared by in-situ polymerization method with different doping concentrations and their microstructures were investigated by small angle X-ray scattering (SAXS) technique. Analyzed SAXS results indicate the average diameters of the nanoparticles in hybrid films distribute from 55 nm to 60 nm, which are consistent with the observation from Scanning Electron Microscope (SEM). The Fourier Transform Infrared Spectroscopy (FTIR) results confirm the formation of AlN particles in the PI matrix. The effects of content on the dielectric constant, loss tangent, breakdown strength and corona ageing were studied. The corona resistant time of PI/AlN with 20 wt.% doping concentration is 58.5 hours, which is twenty times more than that of pure PI. The results show that the corona resistance of PI/AlN has been improved significantly.

Formation of liquid‐crystalline morphologies in dilute solutions of a charged random terpolymer

Formation of micellar morphologies in aqueous solutions of charged random copolymers has been reported in several studies. This paper reports on liquid crystallization of the cationic random acrylic terpolymer i.e., poly[(dimethylaminoethyl methacrylate)‐co‐(methyl methacrylate)‐co‐(butyl methacrylate)] containing no mesogenic groups. The viscometric behaviour of the terpolymer solutions deviated in several ways from ordinary behaviour of polyelectrolytes supporting the possibility of self‐assembly of this cationic random terpolymer in a solvent mixture containing acetone, ethanol and 1‐propanol or each of these solvents separately. The existence of nano‐sized liquid‐crystalline structures was demonstrated using small‐angle X‐ray scattering (SAXS) analysis of the terpolymer solutions. Morphology and sizes of the liquid‐crystalline structures were determined according to the SAXS results and confirmed by dynamic light scattering and atomic force microscopy findings. Liquid crystallization was reasoned on the basis of the existence of a segregated chain microstructure as for polyelectrolytes containing folded parts connected to each other by monomer strings. In a random amphiphilic copolymer or the terpolymer under investigation, the folded parts and strings must be hydrophilic and hydrophobic, respectively. The role of alcohol aggregates should be considered in self‐assembly of the terpolymer chains. The alcohol aggregates can act as physical crosslinkers leading to the formation of more compact liquid‐crystalline structures. The deviations observed in the viscometric behaviour of the terpolymer solutions are suggested as the result of the ability of terpolymer chains to self‐assemble. © 2013 Society of Chemical Industry

Comparative study of n-dodecyl tetraethylene monoether lyotropic liquid crystals incorporated with graphene and graphene oxide.

Two kinds of carbon materials, i.e., graphene and graphene oxide (GO), were successfully incorporated into a lyotropic liquid crystal (LLC) matrix formed by n-dodecyl tetraethylene monoether (C12E4). The properties of graphene-C12E4 and GO-C12E4 LLC composites were characterized by UV-vis absorption, transmission electron microscopy (TEM) observations, polarized optical microscopy (POM) observations, small-angle X-ray scattering (SAXS) and rheological measurements. SAXS results indicate that both graphene and GO are well-dispersed in the C12E4 LLC matrix and some interactions occur between the C12E4 LLC matrix and graphene (or GO) sheets. Moreover, it is demonstrated that graphene interacts with the hydrophobic part of C12E4 LLC while GO mainly interacts with the hydrophilic part of C12E4 LLC because of the different properties of graphene and GO. Integration of graphene and GO into C12E4-PEG systems by a spontaneous phase separation method reveals the different interaction mechanisms of graphene and GO with C12E4 LLC. It can be concluded that the mechanical and electrical properties of the C12E4 LLC have been largely improved by the incorporation of graphene and GO, which opens the door for wide applications in nanotechnology, electrochemical and biochemical areas.

Effect of multi walled carbon nanotube on the crystalline structure of polypropylene fibers

Polypropylene fibers containing varying amounts of multi walled carbon nanotube (MWCNT) have been spun using a conventional melt spinning and drawing apparatus. Changes in morphology and crystalline structure of composite fibers induced by addition of MWCNT were studied by small angle X-ray scattering (SAXS), wide angle X-ray scattering (WAXS), Fourier transform infrared spectroscopy (FTIR) and birefringence measurements. The results of SAXS experiments showed an increase in lamellar thickness, long period and crystallinity of the composite fibers in comparison to pure polypropylene fibers. Molecular orientation and helical content of the fibers were increased due to the addition of MWCNT to the polypropylene matrix. WAXS results, being in agreement with the SAXS results, also showed an increase in crystallinity of the composite fibers due to the increase in MWCNT content. This is probably because of nucleating effect of nanotubes in the fiber matrix, causing more crystallization and orientation of molecules to take place around them.

Multiple recrystallization behavior of poly(1,1-dimethysilacyclobutane): A combined calorimetric and small angle X-ray scattering study

The crystallization and melting behavior of a series of poly(1,1-dimethysilacyclobutane) (PDMSB) samples accessible via living anionic polymerization protocols with different molar masses were studied by differential scanning calorimetry (DSC), small angle X-ray scattering (SAXS) and X-ray diffraction (XRD). It has been found that in the cooling process only one crystallization exotherm was observed by using DSC, but in subsequent heating two clear endotherms appeared. DSC measurements additionally revealed that there will be two melting peaks when the isothermal crystallization temperature, Tc, is low enough, but only one melting peak could be obtained when Tc is high. The value of the lower melting peak Tm1 increased with Tc, showing the increase of lamella thickness due to increase of Tc, which was also evidenced by using SAXS measurements. However, the value of the higher melting peak Tm2 stayed almost constant with Tc. XRD analysis showed that there is only one crystal form independent of Tc. While SAXS results revealed only one long range order, indicating this multiple endotherms phenomenon was caused by the recrystallization process during heating, but not isothermal thickening and thinning process as observed for e.g. poly(ethylene oxide)s (PEOs) with low molar masses. Heating rate dependent DSC experiments showed that there are two recrystallization processes with different time scales. The recrystallization at low temperature always showed up independent of heating rate while the recrystallization at high temperature showed up only when the heating rate was very low. This interesting phenomenon was explained by the different energy barrier for the recrystallization process at low and high temperature as shown by in situ SAXS measurements during heating.

New Phases Found in Reverse Micelle Systems with High Concentrations of AOT

This paper discusses the phase behavior, rheology, and structure of self-assembled sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle systems at high AOT concentrations. When the amount of AOT and w(o) (the molar ratio of water to AOT) were changed, many different phases were found, a fact which is not discussed in the literature. Opaque gel-like phase (phase separation) occurred with high concentrations of AOT in organic solvents without water. When the AOT concentration and w(o) were increased to 18-72 m and 2, respectively, the samples were gel-like and translucent. Dynamic rheological results indicate that the viscoelastic transition agreed with a multirelaxation time model. Small-angle X-ray scattering (SAXS) results imply that these samples showed a hexagonally close-packed cylindrical structure in which the diameter of a cylinder was ~2.5-3.0 nm, depending on the water contents. Moreover, these AOT cylinders self-assembled into fiber bundles with a diameter of 1-10 μm, as determined using a polarized optical microscope. As w(o) was increased to 2-6 in 72 m AOT samples, similar rheological and SAXS results were obtained. However, a different type of viscoelastic transition occurred, from multirelaxation to single-relaxation, when w(o) was increased to 7-11. The samples were in the transparent gel-like phase, and the structures determined by SAXS were a combination of hexagonally packed cylindrical and lamellar structure. The close-packed cylindrical structures had larger radii and shorter lengths with increasing w(o). Furthermore, when w(o) was increased to 12, the gel-like phase disappeared and a highly viscous solution was observed. This is because all the cylindrical structures collapsed and transformed into lamellar structures when the amount of water was further increased.

Effects of pressurized hot water extraction on the nanoscale structure of birch sawdust

Pressurized hot water extraction with a flow-through system was used to extract hemicelluloses and lignin from birch sawdust. The structure of the extraction residue was studied on various levels. Molecular mass distributions were determined with gel permeation chromatography and the crystal structure of cellulose was characterized using wide-angle X-ray scattering (WAXS). Information on the short-range order of cellulose microfibrils and on the nanoscale pore structure was obtained with small-angle X-ray scattering (SAXS), and the micrometre scale cellular morphology was imaged with X-ray microtomography. The pressurized hot water treatment was observed to increase the lateral width of cellulose crystallites, determined with WAXS, whereas a possible small decrease in the crystallinity of cellulose compared to native wood was detected. The molecular mass of cellulose remained at a relatively high level. According to the SAXS results, a tighter lateral association of cellulose microfibrils was observed in the extracted samples, which possibly led to opening of pores between bundles of microfibrils, as indicated by an increased specific surface area. A reduction in the thickness of the fibre cell walls was evidenced by X-ray microtomography.

The effect of comonomer content on structure and property relationship of propylene-1-octene copolymer during uniaxial stretching

The structure and property relationships of propylene-1-octene random copolymer having different octene comonomer concentrations were investigated. In specific, the crystal structure evolution in these copolymers during uniaxial stretching at 60 °C was characterized by in-situ synchrotron wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS) techniques. With high octene content, the copolymer behaved like an elastomer with small elastic modulus and yield stress, low crystallinity and low crystal orientation under stretching. Furthermore, step-cycle tensile test showed that the copolymer with high octene content had a high recovery ratio. With low octene content, the copolymer behaved like a plastomer with large elastic modulus and yield stress, high crystallinity and high crystal orientation under stretching. From 2D SAXS results, it was found that in low octene content sample, lamellar fragmentation occurred resulting in a significant decrease in lamellar lateral size. In contrast, in high octene content sample, stress might be mainly concentrated on the amorphous matrix, leading to an inter-lamellar slip and a small decrease in lamellar lateral size. Schematic structural changes of propylene-1-octene copolymer under tensile deformation were illustrated to explain the different elasticity behavior in these copolymers.

Microstructure and dielectric property study of polyimide/BaTiO3 nanocomposite films

Polyimide/BaTiO3 composite films with different BaTiO3 doping concentrations have been successfully fabricated by in-situ polymerization method and their microstructures were studied by synchrotron radiation small angle X-ray scattering (SAXS) technique. Analyzed SAXS results indicate that the average radius of the nanoparticles in nanocomposite films distribute from 30nm to 42nm, which are consistent with the observation from a Scanning Electron Microscope. The positive deviation of SAXS results indicates that there is a fluctuation of electron density in the materials and the distribution of inorganic is not uniform. The dielectric constant and loss tangent increase with the BaTiO3 doping concentration increases, and the breakdown strength decreases.

Structural and thermal properties of polystyrene nanocomposites containing hydrophilic and hydrophobic layered double hydroxides

Zn–Al layered double hydroxide (LDH) was synthesized and modified using sodium dodecyl benzene sulfonate (DBS). Hydrophilic and hydrophobic LDHs were applied as fillers in a free-emulsion polymerization of styrene monomer, which enhance their thermal stability, and strengthen their structure. In order to increase structural stability we modified the surface of LDH with anionic surfactant and examined its effect on the exfoliation in the polystyrene (PS) matrix. The structure of polystyrene–LDH nanocomposites was investigated by X-ray diffraction pattern technique (XRD), and small angle X-ray scattering (SAXS). Furthermore, thermal analysis (TGA and DSC) was evaluated and the prepared nanocomposites show significantly improved thermal stability at higher temperature. Both XRD and SAXS results confirmed the totally delaminated state of the LDH in the polymer matrix. It was established that the hydrophilic filler has a better exfoliation effect than the hydrophobic LDH in the hydrophobic polymer structure.

Interactions of a cationic surfactant – (benzyloxymethyl) dodecyldimethylammonium chloride with model biomembrane systems

Phospholipids are the main components of biological membranes. The aim of the present study was to determine the influence of a cationic surfactant on phospholipid structure and dynamics. Fourier transform infrared (FTIR) and dielectric relaxation (DRS) spectroscopies as well as small-angle X-ray scattering (SAXS) with synchrotron radiation have been used to analyse the structure of fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in the presence of a quaternary ammonium surfactant: (benzyloxymethyl) dodecyldimethylammoniumchloride (BzMDDAC). The presence of the surfactant caused changes in the temperature of the DMPC phase transition, as revealed using FTIR and DRS measurements. This change results from the disappearance of the multilamellar phase of DMPC and the formation of the unilamellar (most likely bicellar) phase, as indicated by the SAXS results.

Structural Comparability Between Recombinant FVIII-Fc and Its Isolated FVIII and Fc Constituents

AbstractAbstract 1135rFVIIIFc is a recombinant fusion protein comprising human B domain-deleted (BDD) coagulation factor VIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1). rFVIIIFc is currently in a phase 3 clinical study as a novel long-lasting FVIII molecule designed to potentially reduce dosing frequency and provide prolonged protection from bleeds for patients with hemophilia A receiving prophylaxis or on-demand treatment, or undergoing surgery. In this study, we evaluated the structural comparability of BDD rFVIII and rFVIIIFc using small angle X-ray scattering (SAXS) and hydrogen/deuterium exchange (HDX) mass spectrometry. SAXS analysis of BDD rFVIII and rFVIIIFc, performed at the Advanced Light Source Synchrotron, provided the electron pair radius distribution of each molecule and accurate but inherently low resolution structural information. Both BDD rFVIII and rFVIIIFc were monomeric in solution. BDD rFVIII had a radius of gyration (Rg) of 39.7 Å, compared with a Rg of 38 Å calculated from its published atomic coordinates (2R7E.pdb). rFVIIIFc had a larger Rg of 51 Å, which was consistent with a simple computational model of BDD rFVIII tethered to Fc that predicted an Rg of 50 Å. HDX mass spectrometric analysis yielded 94% coverage of the BDD rFVIII component and 87% coverage of the Fc component. Rates of deuterium exchange within peptides derived from each of these components in the context of the rFVIIIFc fusion protein were very similar to those of the isolated BDD rFVIII and Fc constituents. Taken together, the results of SAXS and HDX analyses support the conclusion that fusion of the Fc moiety to BDD rFVIII does not alter the higher order structure of either component. Disclosures:Kulman:Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Berkowitz:Biogen Idec: Employment. Light:biogenidec: Employment. Mezo:Biogen Idec: Employment. Wang:Biogen Idec: Employment. Whitlow:Colabrativ Inc.: Employment; Biogen Idec: Consultancy. Culyba:Biogen Idec: Employment. Peters:Biogen Idec: Employment.

Towards understanding structural changes of photoperiod-sensitive sorghum biomass during sulfuric acid pretreatment

Wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS) were employed to investigate the structural changes of photoperiod-sensitive sorghum biomass in both crystalline and amorphous domains after sulfuric acid pretreatment. WAXD results suggested that the crystalline cellulose melted at 120°C without significant polymer degradation and cellulose went through a simultaneous hydrolysis and crystallization at 160°C. Both the cellulose crystallinity and the crystal size increased as the temperature increased, which might lower cellulose digestibility. The efficiency of enzymatic hydrolysis (EEH) increased because the cellulose protective structure was compromised and most hemicellulose was removed, resulting in an increase in total surface area as suggested by SAXS results. The radius of gyration of the polymer structure decreased and the lamellar structure was destroyed after pretreatment. Both the EEH and cellulose degradation increased as the temperature increased. The total glucose yield increased to 79.7% as the temperature increased to 160°C.

Nitric oxide synthases in motion

To determine a dynamic functional model of full-length nitric oxide synthase (NOS), we are coupling Small Angle X-ray Scattering (SAXS) with our crystal structures of individual NOS components. SAXS is a solution scattering method that is well suited to capturing protein conformational changes. Our lab has solved crystal structures of all three parts of NOS enzymes: NOS oxygenase, NOS reductase and NOS calmodulin (CaM) peptide with CaM bound. Based on our crystal structures and biochemical data, we propose that the NOS FMN domain swings from the electron-accepting “IN” to an electron-donating “OUT” conformation. In the electron-accepting conformation observed in our crystal structure, the FMN is buried and positioned to accept an electron from redox partner FAD. We propose that the entire FMN domain swings “OUT” from the remainder of the reductase domain to transfer electrons to the active site heme. We are combining scattering data with atomic resolution crystal structures and molecular dynamics simulations to identify snapshots of NOS in solution. We analyze and compare FMN domain movement in different isozymes, making use of overlapping truncated constructs and post-translationally modified proteins. Our SAXS results are consistent with a large swinging movement in the FMN domain that could enable electron transfer from FMN to heme.