Small-angle X-ray Scattering Profiles Research Articles

Simultaneous Measurement of Two-Trace Two-Dimensional (2T2D) Near-Infrared (NIR) Asynchronous Correlation Spectra and Small-Angle X-ray Scattering (SAXS) to Characterize Thermally Aged Polypropylene (PP).

In this study, a new system was developed to carry out simultaneous near-infrared (NIR) and small-angle X-ray scattering (SAXS) measurements. Aged polypropylene (PP) was examined with the NIR-SAXS system to demonstrate how it can be utilized to derive pertinent information about the polymer structure. Pairs of SAXS profiles and NIR spectra of PP in its initial state and after aging were measured to derive an in-depth understanding of the aging phenomenon. The SAXS profiles of the PP samples showed a clear shift of the SAXS peak to the lower q direction induced by the thermal aging, indicating an increase in the length of the long-period structure. Two-trace two-dimensional (2T2D) asynchronous correlation spectra derived from NIR spectra clearly revealed that the aging treatment leads to a substantial increase in the spectral intensity of the regularity bands representing the longer helix present in a folded lamellar structure. In other words, it suggests that the long helix structure is more abundantly present than the short helix structure in the aged PP than in the initial PP. By combining the information derived from the SAXS profiles and NIR spectra, the details of the aging-induced variation were clearly determined. Namely, aging causes additional crystallization of the PP by developing more helical structures, which involves an increase in the lamellar thickness as well as a decrease in the amorphous region. The growth of the rigid crystalline phase restricts the elastic deformation in the amorphous structure, which eventually induces the deterioration of PP by making the polymer hard but brittle. Such observation, in turn, implies that retarding or accelerating the crystallized structure of PP substantially works to control the progress of aging.

Contrast dependence of scattering profiles for poly(ethylene glycol) in water: Investigation by small-angle neutron scattering with 3He spin filter and small-angle x-ray scattering.

The small-angle neutron scattering (SANS) and small-angle x-ray scattering (SAXS) measurements were performed for deuterated and non-deuterated poly(ethylene glycol) (d-PEG and h-PEG, respectively) in D2O and a D2O/H2O mixed solvent (Mix) to compare the scattering profiles. To determine the coherent scattering intensity of SANS, a 3He spin filter was utilized. The scattering profiles determined by the SANS measurements were analyzed in terms of the wormlike chain model with touched beads along the contour of the chain. However, the SAXS profiles were not explained by the same model with uniform beads but with beads each consisting of a core and a shell having different electron densities. To explore the chain thickness determined from the SANS profile, the scattering intensities for different combinations of d-PEG/D2O, d-PEG/Mix, h-PEG/D2O, and h-PEG/Mix were also examined.

Size-refocusing fitting of small-angle X-ray scattering from polydisperse nanoparticles for shape determination

Small-angle X-ray scattering (SAXS) which records reciprocal-space signals with characteristic Bessel-type oscillations is a powerful technique for studying nanoparticles. However, the size polydispersity (or size distribution) of nanoparticles in an ensemble sample smears the oscillational peaks and valleys in the SAXS profile, making it difficult to extract accurate real-space information (e.g. three-dimensional geometry) on the nanoparticles. In this work, a method capable of eliminating the size-distribution-induced smearing effect from SAXS profiles by taking the known size-distribution function into consideration has been developed. The method employs a penalized iterative regression to fit the pair distance distribution function (PDDF) derived from a SAXS profile, recovering the representative PDDF of the nanoparticles. The method has been evaluated with a series of nanoparticle systems of various shapes and size distributions, showing their PDDF profiles to have high fidelity to the reference ideal PDDF profiles. Inverse Fourier transformation of the recovered PDDF profiles gives SAXS profiles presenting the characteristic Bessel-type oscillations, enabling reconstruction of the representative three-dimensional geometry of the nanoparticles. This method will help in the use of SAXS to image synthesized colloidal nanoparticles where size polydispersity is inevitable.

Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations.

This study combines molecular dynamics (MD) simulations with small angle x-ray scattering (SAXS) measurements to investigate the range of conformations that can be adopted by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations in solution. To explore how the conformational distribution of proteins may be modified in the environmental niches of biological media, we focus on the periplasmic ferric binding protein A (FbpA) from Haemophilus influenzae involved in the mechanism by which bacteria capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment. While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms, detection of conformational changes due to the point mutation D52A and changes in ionic strength (IS) from SAXS scattering profiles have been challenging. Here, to reach conclusions, statistical analyses with SAXS profiles and results from different techniques were combined in a complementary fashion. The SAXS data complemented by size exclusion chromatography point to multiple and/or alternative conformations at physiological IS, whereas they are well-explained by single crystallographic structures in low IS buffers. By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates. We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

Extracting time series matching a small-angle X-ray scattering profile from trajectories of molecular dynamics simulations

Solving structural ensembles of flexible biomolecules is a challenging research area. Here, we propose a method to obtain possible structural ensembles of a biomolecule based on small-angle X-ray scattering (SAXS) and molecular dynamics simulations. Our idea is to clip a time series that matches a SAXS profile from a simulation trajectory. To examine its practicability, we applied our idea to a multi-domain protein ER-60 and successfully extracted time series longer than 1 micro second from trajectories of coarse-grained molecular dynamics simulations. In the extracted time series, the domain conformation was distributed continuously and smoothly in a conformational space. Preferred domain conformations were also observed. Diversity among scattering curves calculated from each ER-60 structure was interpreted to reflect an open-close motion of the protein. Although our approach did not provide a unique solution for the structural ensemble of the biomolecule, each extracted time series can be an element of the real behavior of ER-60. Considering its low computational cost, our approach will play a key role to identify biomolecular dynamics by integrating SAXS, simulations, and other experiments.

Temperature dependence of the casein micelle structure in the range of 10–40 °C: An in-situ SAXS study

Milk is used and processed under various environmental temperature, and its physicochemical properties are also strongly affected by temperature. Therefore, it is important to reveal the structure of milk at variable temperatures. In this study, the temperature dependence of the inner structure of bovine casein micelles in the temperature range of 10–40 °C was investigated by in-situ small-angle X-ray scattering (SAXS) method. The micelle size calculated from the SAXS profiles using a micelle model including water domains was almost independent of temperature. The water domain expanded and the distance between the colloidal calcium phosphates (CCP) decreased with increasing temperature. The number of CCPs in a micelle increased, because CCPs were newly formed by the transfer of calcium and inorganic phosphate from serum into the micelle. These structural changes occurred during the cooling process. Therefore, in the temperature range of 10–40 °C, the structure of the casein micelle varied sensitively with the temperature, and these structural changes were thermoreversible in nature.

Kinetics of denaturation and renaturation processes of double-stranded helical polysaccharide, xanthan in aqueous sodium chloride

Circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements were made for three xanthan samples, a double helical polysaccharide, in 5 or 10 mM aqueous NaCl after rapid temperature change to investigate the kinetics of the conformational change between the ordered and disordered states. After the rapid heating, the CD signal mainly reflecting the carbonyl groups on the side chains quickly changed (<150 s) while the scattering intensity from SAXS around q (magnitude of the scattering vector) = 1 nm−1 changed more gradually, reflecting the main-chain conformation. The difference between CD and SAXS implies us the intermediate conformation which can be regarded as a loose double helix. The SAXS profile in the rapid cooling process showed that the loose double helical structure was constructed within 150 s, but the CD signal slowly changed with around 2 days to recover the native tight double helix.

Structural insights into rice SalTol QTL located SALT protein

Salinity is one of the major stresses affecting rice production worldwide, and various strategies are being employed to increase salt tolerance. Recently, there has been resurgence of interest to characterize SalTol QTL harbouring number of critical genes involved in conferring salt stress tolerance in rice. The present study reports the structure of SALT, a SalTol QTL encoded protein by X-ray crystallography (PDB ID: 5GVY; resolution 1.66 Å). Each SALT chain was bound to one mannose via 8 hydrogen bonds. Compared to previous structure reported for similar protein, our structure showed a buried surface area of 900 Å2 compared to only 240 Å2 for previous one. Small-angle X-ray scattering (SAXS) data analysis showed that the predominant solution shape of SALT protein in solution is also dimer characterized by a radius of gyration and maximum linear dimension of 2.1 and 6.5 nm, respectively. The SAXS profiles and modelling confirmed that the dimeric association and relative positioning in solution matched better with our crystal structure instead of previously reported structure. Together, structural/biophysical data analysis uphold a tight dimeric structure for SALT protein with one mannose bound to each protein, which remains novel to date, as previous structures indicated one sugar unit sandwiched loosely between two protein chains.

Domain Organization in Plant Blue-Light Receptor Phototropin2 of Arabidopsis thaliana Studied by Small-Angle X-ray Scattering.

Phototropin2 (phot2) is a blue-light (BL) receptor protein that regulates the BL-dependent activities of plants for efficient photosynthesis. Phot2 is composed of two light-oxygen-voltage sensing domains (LOV1 and LOV2) to absorb BL, and a kinase domain. Photo-activated LOV domains, especially LOV2, play a major role in photo-dependent increase in the phosphorylation activity of the kinase domain. The atomic details of the overall structure of phot2 and the intramolecular mechanism to convert BL energy to a phosphorylation signal remain unknown. We performed structural studies on the LOV fragments LOV1, LOV2, LOV2-linker, and LOV2-kinase, and full-length phot2, using small-angle X-ray scattering (SAXS). The aim of the study was to understand structural changes under BL irradiation and discuss the molecular mechanism that enhance the phosphorylation activity under BL. SAXS is a suitable technique for visualizing molecular structures of proteins in solution at low resolution and is advantageous for monitoring their structural changes in the presence of external physical and/or chemical stimuli. Structural parameters and molecular models of the recombinant specimens were obtained from SAXS profiles in the dark, under BL irradiation, and after dark reversion. LOV1, LOV2, and LOV2-linker fragments displayed minimal structural changes. However, BL-induced rearrangements of functional domains were noted for LOV2-kinase and full-length phot2. Based on the molecular model together with the absorption measurements and biochemical assays, we discuss the intramolecular interactions and domain motions necessary for BL-enhanced phosphorylation activity of phot2.

SAS-cam: a program for automatic processing and analysis of small-angle scattering data

Small-angle X-ray scattering (SAXS) is a widely used method for investigating biological macromolecules in structural biology, providing information on macromolecular structures and dynamics in solution. Modern synchrotron SAXS beamlines are characterized as high-throughput, capable of collecting large volumes of data and thus demanding fast data processing for efficient beamline operations. This article presents a fully automated and high-throughput SAXS data analysis pipeline, SAS-cam, primarily based on the SASTBX package. Five modules are included in SAS-cam, encompassing the data analysis process from data reduction to model interpretation. The model parameters are extracted from SAXS profiles and stored in an HTML summary file, ready for online visualization using a web browser. SAS-cam can provide the user with the possibility of optimizing experimental parameters based on real-time feedback and it therefore significantly improves the efficiency of beam time. SAS-cam is installed on the BioSAXS beamline at the Shanghai Synchrotron Radiation Facility. The source code is available upon request.

Insights into the supramolecular structure and techno-functional properties of starch isolated from oat rice kernels subjected to different processing treatments

Oat rice kernels were subjected to decortication (DOR), decortication and enzyme deactivation (DDOR), decortication and cooking (DCOR), as well as combined decortication, enzyme deactivation and cooking (DDCOR). The starch fractions were isolated and their structural features were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, solid-state 13C nuclear magnetic resonance, small angle X-ray scattering (SAXS), and scanning electron microscope. In the cooked oat rice samples (DCOR and DDCOR), in addition to losing a significant amount of the A-type crystalline structure, there was an enhancement in the proportion of V-type crystallinity. The cooking process completely destroyed the periodic lamellar structure of oat starch on the SAXS profile. The Mw values (1.195 × 107–1.459 × 107 g/mol) were in the following order: DOR > DDOR > DCOR > DDCOR. The data was in line with the results for crystallinity, double helix content, degree of order, melting enthalpy, and those obtained for textural parameters, resistant starch content, and bile acid binding capacity.

Morphological changes of hydrophobic matrix and hydrophilic ionomers in water-swollen perfluorinated sulfonic acid membranes detected using small-angle X-ray scattering

Time-resolved small-angle X-ray scattering (SAXS) was employed to examine the hydration dynamics of perfluorinated sulfonic acid membranes in contact with liquid water. Following thermal pretreatment of the membranes, a peak corresponding to the fluorocarbon polymer backbones was clearly observed in the SAXS profiles, in addition to a peak corresponding to the ionomer clusters consisting of hydrophilic side chains and water molecules. The variation of these two peaks was monitored with a frame time as low as 21 ms, which permitted the successful capture of the subsecond dynamics of water uptake by 25 and 50 μm thick membranes. The dynamics of the initial rapid water uptake were well reproduced using Fick's equation, although the thinner sample exhibited a smaller diffusion coefficient. This result suggests that the surface resistance influences the diffusion of water into the membrane. The initial rapid water absorption was followed by the slow and continuous reconstruction of the polymer structure. The measured dynamics of the two peaks indicated that, during this long-term reconstruction, the increase in the ionomer volume became slower and the fusion of clusters was the dominant factor responsible for increasing the ionomer spacing.

Self-Assembly of Amphiphilic Amylose Derivatives in Aqueous Media.

Six amylose derivative (C12CMA) samples with hydrophobic dodecyl ether groups and hydrophilic sodium carboxymethyl groups were synthesized from an enzymatically synthesized amylose for which the weight-average molar mass is 50 kg mol-1 to realize amylose-based amphiphilic polymer micelles. The degree of substitution of hydrophobic (DSC12) and hydrophilic (DSCM) groups ranges between 0.076 and 0.39 and between 0.35 and 1.83, respectively. Static and dynamic light scattering, small-angle X-ray scattering (SAXS), and fluorescence measurements with pyrene as a probe were carried out for the samples in 150 mM aqueous NaCl to characterize the higher-order structure in solution. The fluorescence from pyrene showed that all six samples have hydrophobic environment, while the hydrophobicity tends to increase with rising DSC12. All six samples have high scattering intensity owing to the relatively large concentrated droplets ranging in the hydrodynamic radius from 50 to 110 nm, whereas the weight fraction of such large particles is substantially small except for the highest DSC12 sample. Most polymer chains for relatively low DSC12 of 0.076 were molecularly dispersed with a very small amount of large droplets. The dispersed chain has a slightly smaller helix pitch per residue and a more rigid main chain than those for amylose in dimethyl sulfoxide, suggesting that the amylosic main chain of C12CMA has a helical structure with dodecyl groups at least locally. On the other hand, an anisotropic shaped micelle-like structure is only found for relatively high DSC12 (0.23 and 0.39) samples, which was detected by the SAXS profile at a high scattering vector range. The micelle structure for high DSC12 samples is consistent with the high chain stiffness.

Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

Study on the structure of polymer electrolyte membrane using small angle X-ray scattering and positron annihilation spectroscopy

The structure of poly (styrenesulfonic acid)- grafted poly(ethylene-co- tetrafluoroethylene) polymer electrolyte membrane (ETFE-PEM) was investigated by comparison with those of precursor original ETFE film and styrene-grafted films (grafted-ETFE) using positron annihilation lifetime spectroscopy (PALS) and small angle Xray scattering (SAXS). PALS of these samples indicated that there were two lifetime components, which were assigned to the annihilation of ortho- Positronium (o-Ps). The two types of o-Ps relate to the free volume at sub nanoscale locating in the crystalline and amorphous phases. SAXS profiles of these samples indicated the presence of lamellar stacks andlamellar grains. Sizes of lamellar stacks and lamellar grains varied significantly during the graftingprocess, whereas only change a little bit at sulfonation process and at wet state. A compination of PALS and SAXS allowed us to investigate the structures of ETFEPEMs with scale ranging from several Ångströms to micrometer.

Modeling Structure and Dynamics of Protein Complexes with SAXS Profiles.

Small-angle X-ray scattering (SAXS) is an increasingly common and useful technique for structural characterization of molecules in solution. A SAXS experiment determines the scattering intensity of a molecule as a function of spatial frequency, termed SAXS profile. SAXS profiles can be utilized in a variety of molecular modeling applications, such as comparing solution and crystal structures, structural characterization of flexible proteins, assembly of multi-protein complexes, and modeling of missing regions in the high-resolution structure. Here, we describe protocols for modeling atomic structures based on SAXS profiles. The first protocol is for comparing solution and crystal structures including modeling of missing regions and determination of the oligomeric state. The second protocol performs multi-state modeling by finding a set of conformations and their weights that fit the SAXS profile starting from a single-input structure. The third protocol is for protein-protein docking based on the SAXS profile of the complex. We describe the underlying software, followed by demonstrating their application on interleukin 33 (IL33) with its primary receptor ST2 and DNA ligase IV-XRCC4 complex.

Frank–Kasper σ phase in polybutadiene-poly(ε-caprolactone) diblock copolymer/polybutadiene blends

Microphase-separated structures in a polybutadiene-poly(ε-caprolactone) diblock copolymer (PB-PCL)/polybutadiene homopolymer (PB) blend were investigated by small-angle x-ray scattering (SAXS). Non-equilibrium spherical micelles were observed at temperatures ranging between 60 and 100 °C. An SAXS profile with >60 scattering peaks was recorded at 140 °C. All the peak positions were in good agreement with theoretical Frank–Kasper σ phase peak positions. Thus, these results indicate the formation of a Frank–Kasper σ phase in the PB-PCL/PB blend at 140 °C.

Characterization of Nanocellulose Using Small-Angle Neutron, X-ray, and Dynamic Light Scattering Techniques.

Nanocellulose extracted from wood pulps using TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-mediated oxidation and sulfuric acid hydrolysis methods was characterized by small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS) techniques. The dimensions of this nanocellulose (TEMPO-oxidized cellulose nanofiber (TOCN) and sulfuric acid hydrolyzed cellulose nanocrystal (SACN)) revealed by the different scattering methods were compared with those characterized by transmission electron microscopy (TEM). The SANS and SAXS data were analyzed using a parallelepiped-based form factor. The width and thickness of the nanocellulose cross section were ∼8 and ∼2 nm for TOCN and ∼20 and ∼3 nm for SACN, respectively, where the fitting results from SANS and SAXS profiles were consistent with each other. DLS was carried out under both the VV mode with the polarizer and analyzer parallel to each other and the HV mode having them perpendicular to each other. Using rotational and translational diffusion coefficients obtained under the HV mode yielded a nanocellulose length qualitatively consistent with that observed by TEM, whereas the length derived by the translational diffusion coefficient under the VV mode appeared to be overestimated.

Blue Light-excited Light-Oxygen-Voltage-sensing Domain 2 (LOV2) Triggers a Rearrangement of the Kinase Domain to Induce Phosphorylation Activity in Arabidopsis Phototropin1

Phototropin1 is a blue light (BL) receptor in plants and shows BL-dependent kinase activation. The BL-excited light-oxygen-voltage-sensing domain 2 (LOV2) is primarily responsible for the activation of the kinase domain; however, the molecular mechanism by which conformational changes in LOV2 are transmitted to the kinase domain remains unclear. Here, we investigated BL-induced structural changes of a minimum functional fragment of Arabidopsis phototropin1 composed of LOV2, the kinase domain, and a linker connecting the two domains using small-angle x-ray scattering (SAXS). The fragment existed as a dimer and displayed photoreversible SAXS changes reflected in the radii of gyration of 42.9 Å in the dark and 48.8 Å under BL irradiation. In the dark, the molecular shape reconstructed from the SAXS profiles appeared as two bean-shaped lobes in a twisted arrangement that was 170 Å long, 80 Å wide, and 50 Å thick. The molecular shape under BL became slightly elongated from that in the dark. By fitting the crystal structure of the LOV2 dimer and a homology model of the kinase domain to their inferred shapes, the BL-dependent change could be interpreted as the positional shift in the kinase domain relative to that of the LOV2 dimer. In addition, we found that lysine 475, a functionally important residue, in the N-terminal region of LOV2 plays a critical role in transmitting the structural changes in LOV2 to the kinase domain. The interface between the domains is critical for signaling, suitably changing the structure to activate the kinase in response to conformational changes in the adjoining LOV2.

SAXS Data Alone can Generate High-Quality Models of Protein-Protein Complexes

Modeling driven by small-angle X-ray scattering (SAXS) combines low-resolution data with computational modeling to predict the structure of biomolecular assemblies. A new protocol, ATTRACT-SAXS, has been developed and tested on a large protein-protein docking benchmark with simulated SAXS data. For 88% of cases, high-quality solutions were generated using SAXS data alone without a physiochemical force field (interface-RMSD ≦ 2Å or ligand-RMSD ≦ 5Å; and more than 30% native contacts). ATTRACT-SAXS gave significant improvements compared with previous approaches that filter by SAXS a posteriori. When combining SAXS and interface properties for scoring, the protocol placed high-quality models in 70% of cases among the top-ranked 100 clusters. ATTRACT-SAXS also gave good results when tested on experimental data if the native complex structure was compatible with the SAXS profile. Our results show that, in principle, SAXS on its own can contain enough information to generate high-quality models of protein-protein complexes.