Use Of Small-angle X-ray Research Articles

Nanostructural changes in Polysaccharide-Casein Gel-Like structures upon in vitro gastrointestinal digestion

This work reports on the nanostructural changes taking place during the in vitro gastrointestinal digestion of polysaccharide-casein gel-like structures through the use of small angle X-ray scattering (SAXS). The results indicated that during the gastric phase, the hydrolysis of casein led to a swelling of the micellar structure, yielding peptide clusters. The presence of sulphated polysaccharides such as agar and κ-carrageenan was seen to limit the hydrolysis of casein during the gastric phase, hence decreasing the size of the formed clusters. After the intestinal phase, the produced peptidic fragments appeared to interact with the bile salts present in the digestion medium, yielding a mixture of bile salt lamellae/micelles and vesicular structures. However, in the presence of polysaccharides, which can interact with bile salts, the formation of vesicular structures was limited. Interestingly, the inclusion of casein within hybrid gel-like structures led to the formation of strong polysaccharide-protein interactions, especially in the case of κ-carrageenan. As a result, in some of the formulations, polysaccharide-peptide complexes were released towards the liquid medium, which formed larger vesicular structures. This was related to the greater protective effect of these particular gel-like structures. Furthermore, κ-carrageenan hindered the formation of bile salt lamellae/micelles. These results are of high relevance to understand the intestinal transport mechanism of the digestion products from protein-based ingredients and will allow a rational design of novel products with optimum nutritional and functional properties.

Loss of a membrane phase under soft confinement conditions imposed by a porous silica colloids network

Poly(ethylene glycol) mono-alkyl ether (CnEm) nonionic surfactants can form various liquid crystalline phases in water that are not altered by a change of pH nor by the presence inorganic materials. Their nonionic membrane phases were thus used as model system to mimic biological cell membranes and their behavior in the presence of inorganic colloids. In this study, a membrane phase formed by the triethylene glycol monodecyl ether (C10E3) nonionic surfactant was used as a model system to study the impact of topological constraints, and other confinement effects imposed / induced by aerosil gels. Smoked silica aerosil nanoparticles formed porous gels when aerosils interacted by siloxane bonds, in which both the pore volume and surface disorder were modulated by the concentration of nanoparticles with diameters of 7, 16, 20 & 40 nm. With the use of small angle X-ray scattering (SAXS) and polarized optical microscopy (POM) techniques as probes, the nonionic membranes could be stabilized at high temperatures, up to 8 °C higher than with bulk systems. These collapsed at high aerosil concentrations where, however drastic effects of confinement (finite size, interaction to the surface) altered the rigidity of the membranes. These effects led to a slowing down of the dynamics and a change in the elastic properties of the membranes that were not able to adapt within the aerosil gels, leading to their collapse into micellar phases. Since aerosils gels can be aligned by weak stimuli, one of a research prospects may concern the understanding of the confinement of nonionic membrane phases formed by the C10E3 as well as other CnEm surfactants in these anisotropic soft confinement matrixes.

Hierarchical Conformational Dynamics Confers Thermal Adaptability to preQ1 RNA Riboswitches

Single-stranded noncoding regulatory RNAs, as exemplified by bacterial riboswitches, are highly dynamic. The conformational dynamics allow the riboswitch to reach maximum switching efficiency under appropriate conditions. Here we characterize the conformational dynamics of preQ1 riboswitches from mesophilic and thermophilic bacterial species at various temperatures. With the integrative use of small-angle X-ray scattering, NMR, and molecular dynamics simulations, we model the ensemble-structures of the preQ1 riboswitch aptamers without or with a ligand bound. We show that the preQ1 riboswitch is sufficiently dynamic and fluctuating among multiple folding intermediates only near the physiological temperature of the microorganism. The hierarchical folding dynamics of the RNA involves the docking of 3′-tail to form a second RNA helix and the helical stacking to form an H-type pseudoknot structure. Further, we show that RNA secondary and tertiary dynamics can be modulated by temperature and by the length of an internal loop. The coupled equilibria between RNA folding intermediates are essential for preQ1 binding, and a four-state exchange model can account for the change of ligand-triggered switching efficiency with temperature. Together, we have established a relationship between the hierarchical dynamics and riboswitch function, and illustrated how the RNA adapts to high temperature.

Martini bead form factors for nucleic acids and their application in the refinement of protein–nucleic acid complexes against SAXS data

The use of small-angle X-ray scattering (SAXS) in combination with molecular dynamics simulation is hampered by its heavy computational cost. The calculation of SAXS from atomic structures can be speeded up by using a coarse-grain representation of the structure. Following the work of Niebling, Björling & Westenhoff [J. Appl. Cryst. (2014), 47, 1190–1198], the Martini bead form factors for nucleic acids have been derived and then implemented, together with those previously determined for proteins, in the publicly available PLUMED library. A hybrid multi-resolution strategy has also been implemented to perform SAXS restrained simulations at atomic resolution by calculating the virtual positions of the Martini beads on the fly and using them for the calculation of SAXS. The accuracy and efficiency of the method are demonstrated by refining the structure of two protein–nucleic acid complexes. Instrumental for this result is the use of metainference, which allows the consideration and alleviation of the approximations at play in the present SAXS calculations.

DNA Sequence and Histone Core Composition Control the Unwrapping of DNA from Nucleosome Core Particles

As controlling access to DNA is a major component of transcriptional regulation, understanding the effects of interactions between core proteins and the DNA is crucial to gaining an overall understanding of transcription. We report on the use of small angle X-ray scattering with contrast variation (CV-SAXS) to analyze DNA unwrapping from nucleosome core particles. Interactions between the histone core and the DNA are destabilized using increasing concentrations of neutral salt, lowering the strength of electrostatic interactions that bind the components together. As the complex is destabilized many unwrapping states are accessible and the DNA explores possible unwrapping pathways. The CV-SAXS data reports on full range of structures present in the sample. They are fit using sequence-dependent models of unwrapped structures with thermal motions, selected by an ensemble optimization method [1]. Different combinations of DNA sequence and histone composition also alter their interactions, and the unwrapped structures reflect biophysical differences in the nucleosome compositions. 1. Mauney A., Tokuda J.M., Gloss L.M., Gonzalez O., Pollack L. Local DNA sequence controls the cooperativity and asymmetry of DNA unwrapping from nucleosome core particles. Biophys J. 2018 Sep 4;115(5):773-781

Characterisation of structured and functionalised particles by small-angle X-ray scattering (SAXS)

In this work we provided an overview about the use of small-angle X-ray scattering (SAXS) technology for non-invasive characterisation of nanostructured and functionalised particles. Firstly, we demonstrated our modular-designed laboratory SAXS camera with different options and possibilities for quick in-situ measuring various disperse particulate systems including powders and suspensions as well as aerosol nanoparticles which can be deposited on a certain substrate. The detection angle reaches 90°, allowing simultaneous wide-angle X-ray scattering (WAXS) measurement on the crystalline structures. Further extension of the sample-to-detector distance enables multi-level structural characterisation of particle systems ranging from about one nanometer to several hundred nanometers. The use of an on-line X-ray detector makes on-line in-situ analysis possible. Secondly, different data processing methods and models have been introduced for analyzing the acquired scattering data and retrieving the structural parameters of the investigated particle systems. Finally, we presented some selected experimental results obtained by SAXS–WAXS, including commercial nanoparticles, and laboratory-synthesized nanostructured and functionalised particles such as silica multiplets, silica-magnetite core–shell nanocomposites, metallic nanoparticles and catalyst nanodots on oxide support particles in different liquid- and gas-phase synthesis processes as well as nanostructured particles in particle handling processes like high pressure dispersion and high temperature calcination.

Composite material characterisation using an advanced small angle x-ray (SAXS) technique

Materials development in the field of composite material spurs the use of advanced characterization technique. As the fillers become in the nanoscale range in size, the effect of agglomeration become apparent and cannot be avoided. The use of Small Angle X-Ray (SAXS) Scattering technique revealed the information on agglomeration based on the value of specific surface (m2/g). Thermoplastic natural rubber composite was found isotropic based on 2D saxs scattering pattern. As the amount of fillers increased from 2-10% wt., the value of specific surface dropped accordingly. This indicated the higher the amount of filler used, the higher the degree of agglomeration. The SAXS system was also tested by Alumina (BAM) powder and yield result which was in good agreement with BET technique.

Melting and freezing temperatures of confined Bi nanoparticles over a wide size range

The size dependences of the melting and freezing temperatures, T m and T f, respectively, of spherical Bi nanoparticles embedded in a sodium borate glass were determined by applying a new experimental procedure based on the combined and simultaneous use of small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). This experimental procedure is particularly useful for materials in which a widely polydisperse set of nanoparticles are embedded. The results provide additional and stronger evidence supporting the main previous conclusions: (i) the melting and freezing temperatures both decrease linearly for increasing reciprocal radius (1/R); and (ii) the effect of undercooling is suppressed for Bi nanoparticles with radii smaller than a critical value equal to 1.8 nm. These results confirm a previously proposed low-resolution structural model for Bi nanocrystals below their melting temperature and with radius R > 1.8 nm, which consists of a crystalline core surrounded by a disordered shell. In the present work, a number of samples with different and partially overlapping radius distributions were studied, allowing the determination of T m(R) and T f(R) functions over a wide range of radii (1 < R < 11 nm). Comparison of the experimentally determined T m(R) and T f(R) functions corresponding to different samples indicates good reproducibility of the experimental results. This allowed the verification of the robustness of the experimental procedure based on in situ combined use of SAXS and WAXS for determination of the radius dependence of the melting and freezing temperatures of spherical nanoparticles in dilute solution.

Use of small-angle X-ray scattering to resolve intracellular structure changes of Escherichia coli cells induced by antibiotic treatment

The application of small-angle X-ray scattering (SAXS) to whole Escherichia coli cells is challenging owing to the variety of internal constituents. To resolve their contributions, the outer shape was captured by ultra-small-angle X-ray scattering and combined with the internal structure resolved by SAXS. Building on these data, a model for the major structural components of E. coli was developed. It was possible to deduce information on the occupied volume, occurrence and average size of the most important intracellular constituents: ribosomes, DNA and proteins. E. coli was studied after treatment with three different antibiotic agents (chloramphenicol, tetracycline and rifampicin) and the impact on the intracellular constituents was monitored.

Fabrication and Structural Characterization of Module-Assembled Amphiphilic Conetwork Gels

Structural analysis of inhomogeneity-free poly(ethylene glycol)–poly(dimethylsiloxane) (PEG–PDMS) amphiphilic conetwork gels has been performed by the complementary use of small-angle X-ray and neutron scattering. Because of the hydrophobicity of PDMS units, the PEG–PDMS gels exhibit a microphase-separated structure in water. Depending on the volume fraction of PDMS, the microphase-separated structure varies from core–shell to lamellar. The obtained X-ray and neutron scattering profiles are reproduced well using a core–shell model together with a Percus–Yevick structure factor when the volume fraction of PDMS is small. The domain size is much larger than the size of individual PEG and PDMS unit, and this is explained using the theory of block copolymers. Reflecting the homogeneous dispersion conditions in the as-prepared state, scattering peaks are observed even at a very low PDMS volume fraction (0.2%). When the volume fraction of PDMS is large, the microphase-separated structure is lamellar and is demon...

Improving the fracture toughness of epoxy with nanosilica-rubber core-shell nanoparticles

Nanocomposites from epoxy and nanosilica-rubber core–shell nanoparticles were fabricated. The soft shell was a copolymer synthesized from caprolactone and meso-lactide and was designed compatible with the cured epoxy system. The core–shell nanoparticles achieved a reasonably good dispersion in the matrix with minor aggregations. With the addition of the nanosilica based core–shell nanoparticles, the Young's modulus, maximum strength, and fracture toughness of the epoxy were all improved. The impact strength of the epoxy was improved by 39.4% with a loading of 2.0wt% nanofillers. The glass transition temperature of the epoxy was slightly decreased with the addition of nanofillers. The toughening mechanisms were investigated with the use of small angle X-ray scattering together with morphological observations. It was confirmed that crazing, microcracks formation and debonding of nanoparticles from the matrix were the main causes leading to the improvement of fracture toughness.

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Nano-confinement of water (“water pocket”) in room-temperature ionic liquid (RTILs) was experimentally proved by a complementary use of small angle X-ray and neutron scattering. The size of the water pocket was estimated to be 2 ~ 3 nm. The water pocket is loosely packed and size-tunable in the RTIL. Hydrogen bonding water of the water pocket is different from that of bulk water. The water pocket is modified by intrinsic nanodomains in the RTILs. With reflecting slow dynamic fluctuations on the nanoscale, the polar/non-polar nanodomains in the RTILs are utilized for loosely packed nano-confinement in bioscience.

Nanostructures in self-organizing supramolecular systems: SAXS and WAXS studies

The methodology of studying nonequilibrium and equilibrium supramolecular systems via SAXS and WAXS methods is examined in detail. The problem of phase factors in an expression for structural factors is solved through minimization of the fourth moment of electron-density distribution maps in a unit cell and through construction of nodal periodic surfaces and calculation of scattering from model structures. The possibility of direct application of the WAXS method to the data obtained with the use of small-angle X-ray scattering is discussed. Artifacts primarily related to the incorrect determination of lattice symmetry are studied for real examples. The results of the calculation of the electron-density profile in the unit cell are analyzed in more detail. The essence of the first-order phase transition of the ordered lamellar phase to the disordered lamellar phase in polyelectrolyte complexes with oppositely charged surfactants is clarified. This transition is quite analogous to a similar thermotropic transition in the hexagonal phase.

The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly

Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the β subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8β4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.

Transformation from Multi- to Single-lamellar Vesicle by Addition of a Cationic Lipid to 1,2-Dilauroyl-sn-glycero-3-phosphocholine Explored with SAXS and TEM

Abstract 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC) is one of the major lipids constituting cellular membranes. When DLPC itself is dispersed in aqueous solutions, multilamellar vesicles are formed, although the cellular membranes are made of single bilayers. We found that the addition of a calix[4]arene derivative (CaL[4]C3) bearing four amine groups as the headgroup to the DLPC multilamellar vesicle induced the structural transformation to a single-lamellar vesicle, by use of small-angle X-ray scattering and electron microscopy, indicating that the highly condensed ionic groups of CaL[4]C3 are useful to control membrane morphology.

Curcumin-Encapsulated Hexagonal Liquid Crystalline Formed by Brij 97/NaDC Mixtures

The phase diagram of a five-components Brij 97-NaDC/IPM-PEG 400/H2O system was determined at 25°C. The hexagonal liquid crystalline phase (H1) was found in this system. By use of small-angle x-ray scattering (SAXS), polarization microscopy, and rheology techniques, the influence of composition, temperature, and addition of curcumin on H1 phase was studied. It is shown that: 1) the investigated hexagonal liquid crystals exhibit a strong shear thinning behavior and viscoelasticity and the strength of the network of H1 phase becomes weaker with increasing oil content; 2) the frequency-dependent moduli of H1 samples decreases as the temperature increases and the steady-state limiting viscosity of the hexagonal samples shows an Arrhenius-like dependence on temperature; and 3) samples in H1 phase containing curcumin retained their organized hexagonal structure. The SAXS results show that the curcumin molecules may be solubilized both into the apolar core of cylinders together with IPM and in the polar domain coexisting with PEG 400 between the cylinders. When curcumin is encapsulated in samples with low oil content, there is a significant decrease in the frequency-dependent moduli. The tendency of frequency behavior for samples incorporating curcumin as a function of temperature is weakened.

Interaction of bovine serum albumin (BSA) with novel gemini surfactants studied by synchrotron radiation scattering (SR-SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR).

The interaction of three dicationic (gemini) surfactants-3,3'-[1,6-(2,5-dioxahexane)]bis(1-dodecylimidazolium) chloride (oxyC2), 3,3'-[1,16-(2,15-dioxahexadecane)]bis(1-dodecylimidazolium) chloride (oxyC12), and 1,4-bis(butane)imidazole-1-yl-3-dodecylimidazolium chloride (C4)--with bovine serum albumin (BSA) has been studied by the use of small-angle X-ray scattering (SAXS), circular dichroism (CD), and (1)H nuclear magnetic resonance diffusometry. The results of CD studies show that the conformation of BSA was changed dramatically in the presence of all studied surfactants. The greater decrease (from 56 to 24%) in the α-helical structure of BSA was observed for oxyC2 surfactant. The radii of gyration estimated from SAXS data varied between 3 and 26 nm for the BSA/oxyC2 and BSA/oxyC12 systems. The hydrodynamic radius of the BSA/surfactant system estimated from NMR diffusometry varies between 5 and 11 nm for BSA/oxyC2 and 5 and 8 nm for BSA/oxyC12.

Direct Evidence of Confined Water in Room-Temperature Ionic Liquids by Complementary Use of Small-Angle X-ray and Neutron Scattering.

The direct evidence of confined water ("water pocket") inside hydrophilic room-temperature ionic liquids (RTILs) was obtained by complementary use of small-angle X-ray scattering and small-angle neutron scattering (SAXS and SANS). A large contrast in X-ray and neutron scattering cross-section of deuterons was used to distinguish the water pocket from the RTIL. In addition to nanoheterogeneity of pure RTILs, the water pocket formed in the water-rich region. Both water concentration and temperature dependence of the peaks in SANS profiles confirmed the existence of the hidden water pocket. The size of the water pocket was estimated to be ∼3 nm, and D2O aggregations were well-simulated on the basis of the observed SANS data.

Use of small-angle X-ray scattering to investigate the structure and function of dengue virus NS3 and NS5.

Small-angle X-ray scattering (SAXS) is a powerful reemerging biophysical technique that can be used to directly analyze many properties related to the size and shape of a macromolecule in solution. For example, the radius of gyration and maximum diameter of a macromolecule can be readily extracted from SAXS data, as can information regarding how well folded a protein is. Similarly, the molecular weight of macromolecular complexes can be directly determined from the complex's scattering profile, providing insight into the oligomeric state and stoichiometry of the assembly. Furthermore, recently developed procedures for ab initio shape determination can provide low-resolution (~20 Å) molecular envelopes of proteins/complexes in their native state. In conjunction with high-resolution structural data, more sophisticated analysis of SAXS data can help address questions regarding conformational change, molecular flexibility, and populations of states within molecular ensembles. Because SAXS samples are easy to prepare and SAXS data is relatively easy to collect, the technique holds great promise for investigating the structure of macromolecules and their assemblies as well as monitoring and modeling their conformational changes. Here we describe typical steps in SAXS sample preparation and data collection and analysis and provide examples of SAXS analysis to investigate the structure and function of dengue virus NS3 and NS5.

Procollagen C-proteinase enhancer grasps the stalk of the C-propeptide trimer to boost collagen precursor maturation

Tight regulation of collagen fibril deposition in the extracellular matrix is essential for normal tissue homeostasis and repair, defects in which are associated with several degenerative or fibrotic disorders. A key regulatory step in collagen fibril assembly is the C-terminal proteolytic processing of soluble procollagen precursors. This step, carried out mainly by bone morphogenetic protein-1/tolloid-like proteinases, is itself subject to regulation by procollagen C-proteinase enhancer proteins (PCPEs) which can dramatically increase bone morphogenetic protein-1/tolloid-like proteinase activity, in a substrate-specific manner. Although it is known that this enhancing activity requires binding of PCPE to the procollagen C-propeptide trimer, identification of the precise binding site has so far remained elusive. Here, use of small-angle X-ray scattering provides structural data on this protein complex indicating that PCPE binds to the stalk region of the procollagen C-propeptide trimer, where the three polypeptide chains associate together, at the junction with the base region. This is supported by site-directed mutagenesis, which identifies two highly conserved, surface-exposed lysine residues in this region of the trimer that are essential for binding, thus revealing structural parallels with the interactions of Complement C1r/C1s, Uegf, BMP-1 (CUB) domain-containing proteins in diverse biological systems such as complement activation, receptor signaling, and transport. Together with detailed kinetics and interaction analysis, these results provide insights into the mechanism of action of PCPEs and suggest clear strategies for the development of novel antifibrotic therapies.