Small-angle X-ray Scattering Experiments Research Articles

Determination of upper limit of Guinier approximation on dilute polydisperse system in SAXS

For a dilute polydisperse system in small-angle X-ray scattering (SAXS), Guinier’s law can be used to perform an approximate analysis to determine the mean radius of gyration of scatterers in a sample and extend the scattering curve to the lower angle region. The main idea of the law is that the Guinier plot appears linear in the vicinity of the scattering vector equaling zero. In this short contribution, a differential method is proposed to uniquely determine the upper limit of the effective range of the Guinier approximation on dilute polydisperse systems. The theoretical simulation is mainly performed on the typical spherical scatterers in several dilute polydisperse systems to explore the relationship between the upper limit and the shape and size of the scatterers. The SAXS experiment on a sample of silicon dioxide colloid is carried out to verify the feasibility of the new method.

Crystal Structure of the Brugia malayi Thymidylate Kinase-dTMP Complex and Small Angle X-ray Scattering Experiments Identifies Changes in the Dimeric Association Compared to the Human Homolog

Crystal Structure of the Brugia malayi Thymidylate Kinase-dTMP Complex and Small Angle X-ray Scattering Experiments Identifies Changes in the Dimeric Association Compared to the Human Homolog

Elucidating the impact of microstructure on mechanical properties of phase-segregated polyurea: Finite element modeling of molecular dynamics derived microstructures

Phase-segregated polyureas (PU) have received considerable interest due to their use as tough, impact-resistant coatings. Polyureas are favored for these applications due to their mechanical strain rate sensitivity and energy dissipation. Predicting and tailoring the mechanical response of PU remains challenging due to the complex interaction between its elastomeric and glassy phases. To elucidate the role of PU microstructure on its mechanical properties, we developed a finite element modeling framework in which each phase is represented by a volume fraction within a representative volume element (RVE). Critically, we used separate constitutive models to describe the elastomeric and glassy phases. We developed a plasticity-driven breakdown process in which we model the glassy phase disaggregating into a new phase. The overall contribution of each phase at a material point is determined by their respective volume fractions within the RVE. We applied our modeling methods to two compositions of PU with differing elastomeric segment lengths derived from oligoether diamines, Versalink P650 and P1000. Our simulations show that a combination of microstructural differences and elastomeric phase properties accounts for the difference in mechanical response between P650 and P1000. We show our model’s ability to predict PU behavior in various loading conditions, including low-rate cyclic loading and monotonic loading over a wide range of strain rates. Our model produces microstructure transformations that mirror those indicated by small-angle X-ray scattering (SAXS) experiments. Fourier transform analysis of our RVEs reveals glassy phase fibrillation due to deformation, a finding consistent with SAXS experiments.

Pore Characterization of Sandy Silty Soil in Metro Surrounding Rock: A Synchrotron Small-Angle X-ray Scattering Experiment

Pore Characterization of Sandy Silty Soil in Metro Surrounding Rock: A Synchrotron Small-Angle X-ray Scattering Experiment

Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS)

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

PNIPAM-stabilized cubosomes as fusogenic delivery nanovectors for anticancer applications

In recent years, lipid cubic nanoparticles have emerged as promising nanocarriers for drug delivery, due to the several advantages they exhibit with respect to other lipid systems. Here, we report on lipid cubic nanoparticles stabilized by PNIPAM-based amphiphilic block copolymers, specifically, poly(N, N-dimethylacrylamide)-block-poly(N-isopropylacrylamide) (PDMA-b-PNIPAM), as a new class of drug delivery systems (DDS). In vitro studies on the internalization efficiency of the DDS towards two types of human cancer cells (colon HCT-116 and bladder T24 cells), carried out employing a set of sensitive techniques (confocal laser scanning microscopy (CLSM), flow cytometry, scanning electron microscopy (SEM), fluorescence spectroscopy), highlight a prominent role of PDMA-b-PNIPAM stabilizer in enhancing the uptake of cubosomes, compared to the standard Pluronic F127-based formulations. The drug delivery potential of cubosomes, tested by encapsulating a chemotherapeutic drug, camptothecin (CPT), and conducting cytotoxicity studies against 2D plated cells and 3D spheroids, confirm that PDMA-b-PNIPAM-stabilized cubosomes improve the efficacy of treatment with CPT. The origin of this effect lies in the higher lipophilicity of the stabilizer, as we confirm by studying the interaction between the cubosomes and biomimetic membranes of lipid vesicles with Small Angle X-Ray Scattering (SAXS) and CLSM experiments. These results corroborate our fundamental understanding of the interaction between cubosomes and cells, and on the role of polymer to formulate lipid cubic nanoparticles as DDS.

Chaotic advection mixer for capturing transient states of diverse biological macromolecular systems with time-resolved small-angle X-ray scattering.

Advances in time-resolved structural techniques, mainly in macromolecular crystallography and small-angle X-ray scattering (SAXS), allow for a detailed view of the dynamics of biological macromolecules and reactions between binding partners. Of particular promise, are mix-and-inject techniques, which offer a wide range of experimental possibility as microfluidic mixers are used to rapidly combine two species just prior to data collection. Most mix-and-inject approaches rely on diffusive mixers, which have been effectively used within crystallography and SAXS for a variety of systems, but their success is dependent on a specific set of conditions to facilitate fast diffusion for mixing. The use of a new chaotic advection mixer designed for microfluidic applications helps to further broaden the types of systems compatible with time-resolved mixing experiments. The chaotic advection mixer can create ultra-thin, alternating layers of liquid, enabling faster diffusion so that even more slowly diffusing molecules, like proteins or nucleic acids, can achieve fast mixing on timescales relevant to biological reactions. This mixer was first used in UV-vis absorbance and SAXS experiments with systems of a variety of molecular weights, and thus diffusion speeds. Careful effort was also dedicated to making a loop-loading sample-delivery system that consumes as little sample as possible, enabling the study of precious, laboratory-purified samples. The combination of the versatile mixer with low sample consumption opens the door to many new applications for mix-and-inject studies.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

Amphiphilic polymer conetworks with ideal and non-ideal swelling behavior demonstrated by small angle X-ray scattering

Amphiphilic polymer conetworks (APCNs) combine two incompatible properties within one material by featuring two interconnected independently swelling nanophases. To simultaneously address both properties, the APCNs need to be swellable in orthogonal solvents without changing their nanostructure. This has not been demonstrated yet. Two novel APCN families applying the macromeric cross-linker approach have been synthesized by cross-linking the hydrophilic poly(2-hydroxyethyl acrylate) (PHEA) or poly(N,N-dimethylacrylamide) (PDMA), respectively, with the hydrophobic poly(2-(1-ethylpentyl)-2-oxazoline) (PEPOx). For the first time, the APCN PHEA-l-PEPOx could be proven to swell in two orthogonal solvents, water and n-heptane, retaining its nanostructure in a broad range of compositions by using small-angle X-ray scattering (SAXS). PDMA-l-PEPOx seems to show a similar behavior according to swelling experiments, but SAXS revealed that particularly the PDMA phase reversibly changes its nanostructure upon swelling. Thus, the structural integrity of APCNs upon swelling depends on the topology as well as the chemical nature of the polymer phases. Altogether, SAXS experiments are required and well suited to judge changes in nanostructure upon swelling of APCNs.

Taurine Stabilizing Effect on Lysozyme.

Taurine is an important organic osmolyte in mammalian cells, and it weakens inflammation and oxidative stress mediated injuries in some diseases. Recently, taurine has been demonstrated to play a therapeutic role against neurodegenerative disorders, although its parallel involvement in several biochemical mechanisms makes not clear taurine specific role in these diseases. Furthermore, the stabilizing effect of this molecule in terms of protein stability is known, but not deeply investigated. In this work we explore by Circular Dichroism the stabilizing impact of taurine in lysozyme thermal denaturation and its influence in lysozyme aggregation into amyloid fibrils. Taurine even at low concentration modifies protein-protein interactions in lysozyme native state, as revealed by Small Angle X-ray Scattering experiments, and alters the amyloid aggregation pattern without completely inhibiting it, as confirmed by UV/Vis spectroscopy with Congo Red and by Atomic Force Microscopy. Evaluation of the cytotoxicities of the amyloid fibrils grown in presence or in absence of taurine is investigated on SH-SY5Y neuroblastoma cells.

Upgrade of the BioMUR beamline at the Kurchatov synchrotron radiation source for serial small-angle X-ray scattering experiments in solutions

Technical and methodological work was carried out to improve the quality of the experiments at the small-angle X-ray scattering (SAXS) beamline ”BioMUR” at the Kurchatov synchrotron radiation source, commissioned in 2018. Scatterless slits (JJ X-ray company, Denmark) were installed and tested, and the beam adjustment scheme of the “BioMUR” modified accordingly. Now outdated and not very effective mylar windows were replaced by mica vacuum windows providing significantly lower parasitic scattering. SAXS experiments on the solutions of weakly scattering lysozyme and bovine serum albumin proteins confirmed the significant improvement in the signal-to-noise ratio achieved due to the upgrade of the “BioMUR”.

Small-angle X-ray scattering for the proteomics community: current overview and future potential

Introduction: Proteins are biological nanoparticles. For structural proteomics and hybrid structural biology, complementary methods are required that allow both high throughput and accurate automated data analysis. Small-angle X-ray scattering (SAXS) is a method for observing the size and shape of particles, such as proteins and complexes, in solution. SAXS data can be used to model both the structure, oligomeric state, conformational changes, and flexibility of biomolecular samples. Areas covered: The key principles of SAXS, its sample requirements, and its current and future applications for structural proteomics are briefly reviewed. Recent technical developments in SAXS experiments are discussed, and future potential of the method in structural proteomics is evaluated. Expert opinion: SAXS is a method suitable for several aspects of integrative structural proteomics, with current technical developments allowing for higher throughput and time-resolved studies, as well as the analysis of complex samples, such as membrane proteins. Increasing automation and streamlined data analysis are expected to equip SAXS for structure-based screening workflows. Originally, structural genomics had a heavy focus on folded, crystallizable proteins and complexes – SAXS is a method allowing an expansion of this focus to flexible and disordered systems.

Injectable thermoresponsive gels offer sustained dual release of bupivacaine hydrochloride and ketorolac tromethamine for up to two weeks

Bupivacaine and ketorolac are commonly used in combination to reduce perioperative pain. This study aimed to develop and characterize an injectable system that offers simultaneous and prolonged release of bupivacaine and ketorolac. Formulations were prepared using poloxamer 407 with increasing concentrations of poloxamer 188 and sodium chloride. Small Angle X-ray Scattering (SAXS) experiments demonstrated that the poloxamers form gels with a cubic lattice arrangement regardless of the matrix composition, whereas the system porosity is driven by poloxamers concentration. Drug loading slightly reduced the intermicellar spacing. Fourier transform infrared spectroscopy and thermal analysis suggested electrostatic interactions between the loaded drugs and poloxamers. Mechanical and rheological studies confirmed the formulations exhibit Newtonian-like flow at room temperature followed by a transition to a viscous gel at body temperature. Importantly, the developed formulations demonstrated steady and sustained release of both bupivacaine and ketorolac over two weeks. Sodium chloride reduced the initial burst release over the first six hours for BH, from 8.6 ± 0.18% to 1.6 ± 0.11%, and KT, from 7.7 ± 0.27% to 1.5 ± 0.10%. Hence, poloxamer-based thermoresponsive gelling systems are promising delivery platforms for the sustained delivery of bupivacaine and ketorolac, with potential clinical benefits for managing perioperative pain.

One-Pot Synthesis of Supertough, Sustainable Polyester Thermoplastic Elastomers Using Block-Like, Gradient Copolymer as Soft Midblock

It remains challenging to synthesize supertough thermoplastic elastomers (TPEs) since the stretchability and tensile strength are mutually exclusive. Here, we report a one-pot strategy for the prep...

Structural Insights of Shigella Translocator IpaB and Its Chaperone IpgC in Solution.

Bacterial Type III Secretion Systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or deliver Type III Secretion effectors directly into eukaryotic host cell cytoplasm. Shigella, the causing agent of bacillary dysentery or shigellosis, bears a set of T3SS proteins termed translocators that form a pore in the host cell membrane. IpaB, the major translocator of the system, is a key factor in promoting Shigella pathogenicity. Prior to secretion, IpaB is maintained inside the bacterial cytoplasm in a secretion competent folding state thanks to its cognate chaperone IpgC. IpgC couples T3SS activation to transcription of effector genes through its binding to MxiE, probably after the delivery of IpaB to the secretion export gate. Small Angle X-ray Scattering experiments and modeling reveal that IpgC is found in different oligomeric states in solution, as it forms a stable heterodimer with full-length IpaB in contrast to an aggregation-prone homodimer in the absence of the translocator. These results support a stoichiometry of interaction 1:1 in the IpgC/IpaB complex and the multi-functional nature of IpgC under different T3SS states.

In situ Grazing Incidence Small Angle X-ray Scattering on Roll-To-Roll Coating of Organic Solar Cells with Laboratory X-ray Instrumentation.

We present an in-house, in situ Grazing Incidence Small Angle X-ray Scattering (GISAXS) experiment, developed to probe the drying kinetics of roll-to-roll slot-die coating of the active layer in organic photovoltaics (OPVs), during deposition. For this demonstration, the focus is on the combination of P3HT:O-IDTBR and P3HT:EH-IDTBR, which have different drying kinetics and device performance, despite their chemical structure only varying slightly by the sidechain of the small molecule acceptor. This article provides a step-by-step guide to perform an in situ GISAXS experiment and demonstrates how to analyze and interpret the results. Usually, performing this type of in situ X-ray experiments to investigate the drying kinetics of the active layer in OPVs relies on access to synchrotrons. However, by using and further developing the method described in this paper, it is possible to perform experiments with a coarse temporal and spatial resolution, on a day-to-day basis to gain fundamental insight in the morphology of drying inks.

Integrative structural biology of the penicillin-binding protein-1 from Staphylococcus aureus, an essential component of the divisome machinery.

The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis ofPBP1 ofStaphylococcus aureus(saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the "penicillin-binding protein and serine/threonine kinase associated" (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domainswas determined at 1.5Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains ofsaPBP1 as conformationally mobile and separated from the transpeptidasedomain.This conclusion was confirmed by SAXS experiments on the full-length protein in solution.A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain insaPBP1 could play a crucial role forin vivointeraction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.

Applying joint theoretical experimental research to aptamer modeling

The aim of the research. In this work we studied the structure of LC-18 DNA aptamer, which exhibits specifi c binding to lung adenocarcinoma cells. Obtaining the 3D structure of the aptamer is necessary for understanding the mechanism of binding of the aptamer to the target. Th erefore, the aim of the research was modeling of the LC-18 aptamer spatial structure using combination of theoretical methods: DNA folding tools, quantum-chemical calculations and molecular dynamic simulations. Material and methods. Th e secondary structure of the LC-18 aptamer was predicted by using OligoAnalyzer and MFold online soft ware under the conditions typical small-angle X-ray scattering (SAXS) experiment. Th e molecular modeling of the aptamer was carried out using the Avogadro program. For prediction of the structure two computational methods were used: quantum-mechanical method with third-order density-functional tight-binding (DFTB3) and molecular dynamics (MD) with force fi elds. Results. In this paper it was shown that molecular simulations can predict structures from the SAXS experiments. OligoAnalyzer and MFold web servers have been used to generate a set of several likely models. However, more accurate calculations have showed that these models do not predict the relative importance of isomers. Meanwhile, application of quantum-chemical and molecular dynamics calculations have showed reliable molecular structures which have a small deviations from the experimental SAXS curves. Conclusion. Th is study demonstrates the approach for modeling 3D structures of DNA-aptamers in solution using both experimental and theoretical methods. It could be very helpful in designing more effi cient aptamers based on results obtained from molecular simulations.

Structural insights into the membrane receptor ShuA in DDM micelles and in a model of gram-negative bacteria outer membrane as seen by SAXS and MD simulations

Successful crystallization of membrane proteins in detergent micelles depends on key factors such as conformational stability of the protein in micellar assemblies, the protein-detergent complex (PDC) monodispersity and favorable protein crystal contacts by suitable shielding of the protein hydrophobic surface by the detergent belt. With the aim of studying the influence of amphiphilic environment on membrane protein structure, stability and crystallizability, we combine molecular dynamics (MD) simulations with SEC-MALLS and SEC-SAXS (Size Exclusion Chromatography in line with Multi Angle Laser Light Scattering or Small Angle X-ray Scattering) experiments to describe the protein-detergent interactions that could help to rationalize PDC crystallization. In this context, we compare the protein-detergent interactions of ShuA from Shigella dysenteriae in n-Dodecyl-β-D-Maltopyranoside (DDM) with ShuA inserted in a realistic model of gram-negative bacteria outer membrane (OM) containing a mixture of bacterial lipopolysaccharide and phospholipids. To evaluate the quality of the PDC models, we compute the corresponding SAXS curves from the MD trajectories and compare with the experimental ones. We show that computed SAXS curves obtained from the MD trajectories reproduce better the SAXS obtained from the SEC-SAXS experiments for ShuA surrounded by 268 DDM molecules. The MD results show that the DDM molecules form around ShuA a closed belt whose the hydrophobic thickness appears slightly smaller (~22 Å) than the hydrophobic transmembrane domain of the protein (24.6 Å) suggested by Orientations of Proteins in Membranes (OPM) database. The simulations also show that ShuA transmembrane domain is remarkably stable in all the systems except for the extracellular and periplasmic loops that exhibit larger movements due to specific molecular interactions with lipopolysaccharides (LPS). We finally point out that this detergent behavior may lead to the occlusion of the periplasmic hydrophilic surface and poor crystal contacts leading to difficulties in crystallization of ShuA in DDM.

Quantification of pore modification in coals due to pulverization using synchrotron small angle X-ray scattering

Small angle X-ray scattering (SAXS) has been widely employed to characterize the pore structure of coal and other geomaterials. However, in the many previous experimental investigations by SAXS, the probed samples were not standardized in terms of shape and particle size, even for the same geomaterial. The influence of pulverization on the pore structure of coal is generally ignored by the researchers using SAXS for structure characterization. In this study, SAXS is used to investigate the effect of pulverizing on pore modification of virgin coal. Two kinds of coal with different ranks were selected and pulverized for SAXS measurements. Both powder samples (five particle size fractions) obtained by grinding and slice samples were investigated. The results show that the nano-pore structure of coal has been significantly modified due to pulverization. The sample pore volume per unit mass increases with the decrease of sample particle size. Pulverizing the slice sample into the minimum particle sample particle size of 0.075–0.125 mm leads to an increase in the pore volume per unit mass of coal by 39.53% and 18.29% for tested medium volatile bituminous coal and anthracite, respectively. Pulverization can reduce the volume of smaller pores (<20 nm) and increase the volume of larger pores (>20 nm). After grinding the original sample into the minimum particle size of 0.075–0.125 mm, the fractal dimension of the tested medium volatile bituminous coal and anthracite decreased by 0.266 and 0.179, respectively. Surface fractal dimension of pore decreases with the reduction in coal particle size. In the case of the powder coal sample, the increase in pore volume (especially for pore greater than 20 nm) and the reduction of fractal dimension are beneficial to the diffusion of gas in coal and promoting the desorption of residual gas. This study may provide a quantitative insight into the effect of pulverization on pore structure of coal sample. Moreover, it is of great significance for understanding the mechanism of coalbed methane storage and migration and is helpful for the selection of the sample size in SAXS experiments.