Small-angle X-ray Scattering Experiments Research Articles

Combination of acoustic levitation with small angle scattering techniques and synchrotron radiation circular dichroism. Application to the study of protein solutions

BackgroundThe acoustic levitation technique is a useful sample handling method for small solid and liquids samples, suspended in air by means of an ultrasonic field. This method was previously used at synchrotron sources for studying pharmaceutical liquids and protein solutions using x-ray diffraction and small angle x-ray scattering (SAXS). MethodsIn this work we combined for the first time this containerless method with small angle neutron scattering (SANS) and synchrotron radiation circular dichroism (SRCD) to study the structural behavior of proteins in solutions during the water evaporation. SANS results are also compared with SAXS experiments. ResultsThe aggregation behavior of 45μl droplets of lysozyme protein diluted in water was followed during the continuous increase of the sample concentration by evaporating the solvent. The evaporation kinetics was followed at different drying stage by SANS and SAXS with a good data quality. In a prospective work using SRCD, we also studied the evolution of the secondary structure of the myoglobin protein in water solution in the same evaporation conditions. ConclusionsAcoustic levitation was applied for the first time with SANS and the high performances of the used neutron instruments made it possible to monitor fast container-less reactions in situ. A preliminary work using SRCD shows the potentiality of its combination with acoustic levitation for studying the evolution of the protein structure with time. General significanceThis multi-techniques approach could give novel insights into crystallization and self-assembly phenomena of biological compound with promising potential applications in pharmaceutical, food and cosmetics industry. This article is part of a Special Issue entitled “Science for Life” Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Regulation of Microtubule Assembly by Tau and not by Pin1

The molecular mechanism by which the microtubule-associated protein (MAP) tau regulates the formation of microtubules (MTs) is poorly understood. The activity of tau is controlled via phosphorylation at specific Ser/Thr sites. Of those phosphorylation sites, 17 precede a proline, making them potential recognition sites for the peptidyl-prolyl isomerase Pin1. Pin1 binding and catalysis of phosphorylated tau at the AT180 epitope, which was implicated in Alzheimer's disease, has been reported to be crucial for restoring tau's ability to promote MT polymerization in vitro and in vivo [1]. Surprisingly, we discover that Pin1 does not promote phosphorylated tau-induced MT formation in vitro, refuting the commonly accepted model in which Pin1 binding and catalysis on the A180 epitope restores the function of the Alzheimer's associated phosphorylated tau in tubulin assembly [1, 2].Using turbidity assays, time-resolved small angle X-ray scattering (SAXS), and time-resolved negative stain electron microscopy (EM), we investigate the mechanism of tau-mediated MT assembly and the role of the Thr231 and Ser235 phosphorylation on this process. We discover novel GTP-tubulin ring-shaped species, which are detectable in the earliest stage of tau-induced polymerization and may play a crucial role in the early nucleation phase of MT assembly. Finally, by NMR and SAXS experiments, we show that the tau molecules must be located on the surface of MTs and tubulin rings during the polymerization reaction. The interaction between tau and tubulin is multipartite, with a high affinity interaction of the four tubulin-binding repeats, and a weaker interaction with the proline-rich sequence and the termini of tau.

Robustness of the fractal regime for the multiple-scattering structure factor

In the single-scattering theory of electromagnetic radiation, the fractal regime is a definite range in the photon momentum-transfer q, which is characterized by the scaling-law behavior of the structure factor: S(q)∝1/qdf. This allows a straightforward estimation of the fractal dimension df of aggregates in Small-Angle X-ray Scattering (SAXS) experiments. However, this behavior is not commonly studied in optical scattering experiments because of the lack of information on its domain of validity. In the present work, we propose a definition of the multiple-scattering structure factor, which naturally generalizes the single-scattering function S(q). We show that the mean-field theory of electromagnetic scattering provides an explicit condition to interpret the significance of multiple scattering. In this paper, we investigate and discuss electromagnetic scattering by three classes of fractal aggregates. The results obtained from the TMatrix method show that the fractal scaling range is divided into two domains: (1) a genuine fractal regime, which is robust; (2) a possible anomalous scaling regime, S(q)∝1/qδ, with exponent δ independent of df, and related to the way the scattering mechanism uses the local morphology of the scatterer. The recognition, and an analysis, of the latter domain is of importance because it may result in significant reduction of the fractal regime, and brings into question the proper mechanism in the build-up of multiple-scattering.

Folding of Protein L with Implications for Collapse in the Denatured State Ensemble.

A fundamental question in protein folding is whether the coil to globule collapse transition occurs during the initial stages of folding (burst phase) or simultaneously with the protein folding transition. Single molecule fluorescence resonance energy transfer (FRET) and small-angle X-ray scattering (SAXS) experiments disagree on whether Protein L collapse transition occurs during the burst phase of folding. We study Protein L folding using a coarse-grained model and molecular dynamics simulations. The collapse transition in Protein L is found to be concomitant with the folding transition. In the burst phase of folding, we find that FRET experiments overestimate radius of gyration, Rg, of the protein due to the application of Gaussian polymer chain end-to-end distribution to extract Rg from the FRET efficiency. FRET experiments estimate ≈6 Å decrease in Rg when the actual decrease is ≈3 Å on guanidinium chloride denaturant dilution from 7.5 to 1 M, thereby suggesting pronounced compaction in the protein dimensions in the burst phase. The ≈3 Å decrease is close to the statistical uncertainties of the Rg data measured from SAXS experiments, which suggest no compaction, leading to a disagreement with the FRET experiments. The transition-state ensemble (TSE) structures in Protein L folding are globular and extensive in agreement with the Ψ-analysis experiments. The results support the hypothesis that the TSE of single domain proteins depends on protein topology and is not stabilized by local interactions alone.

Computational Analysis of SAXS Data Acquisition.

Small-angle x-ray scattering (SAXS) is an experimental biophysical method used for gaining insight into the structure of large biomolecular complexes. Under appropriate chemical conditions, the information obtained from a SAXS experiment can be equated to the pair distribution function, which is the distribution of distances between every pair of points in the complex. Here we develop a mathematical model to calculate the pair distribution function for a structure of known density, and analyze the computational complexity of these calculations. Efficient recursive computation of this forward model is an important step in solving the inverse problem of recovering the three-dimensional density of biomolecular structures from their pair distribution functions. In particular, we show that integrals of products of three spherical-Bessel functions arise naturally in this context. We then develop an algorithm for the efficient recursive computation of these integrals.

Accounting for observed small angle X-ray scattering profile in the protein-protein docking server ClusPro.

The protein-protein docking server ClusPro is used by thousands of laboratories, and models built by the server have been reported in over 300 publications. Although the structures generated by the docking include near-native ones for many proteins, selecting the best model is difficult due to the uncertainty in scoring. Small angle X-ray scattering (SAXS) is an experimental technique for obtaining low resolution structural information in solution. While not sufficient on its own to uniquely predict complex structures, accounting for SAXS data improves the ranking of models and facilitates the identification of the most accurate structure. Although SAXS profiles are currently available only for a small number of complexes, due to its simplicity the method is becoming increasingly popular. Since combining docking with SAXS experiments will provide a viable strategy for fairly high-throughput determination of protein complex structures, the option of using SAXS restraints is added to the ClusPro server. © 2015 Wiley Periodicals, Inc.

Model for neutron total cross-section at low energies for nuclear grade graphite

At subthermal neutron energies, polycrystalline graphite shows a large total cross-section due to small angle scattering processes. In this work, a new methodology to determine pore size distributions through the neutron transmission technique at subthermal energies is proposed and its sensitivity is compared with standard techniques. A simple model based on the form factor for spherical particles, normally used in the Small Angle Neutron Scattering technique, is employed to calculate the contribution of small angle effect to the total scattering cross-section, with the width and center of the radii distributions as free parameters in the model. Small Angle X-ray Scattering experiments were performed to compare results as a means to validate the method. The good agreement reached reveals that the neutron transmission technique is a useful tool to explore small angle scattering effects. This fact can be exploited in situations where large samples must be scanned and it is difficult to investigate them with conventional methods. It also opens the possibility to apply this method in energy-resolved neutron imaging. Also, since subthermal neutron transmission experiments are perfectly feasible in small neutron sources, the present findings open new possibilities to the work done in such kind of facilities.

Resolving Individual Components in Protein-RNA Complexes Using Small-Angle X-ray Scattering Experiments.

Small-angle X-ray scattering (SAXS) of protein-RNA complexes has developed into an efficient and economical approach for determining low-resolution shapes of particles in solution. Here, we demonstrate a mutliphase volumetric modeling approach capable of resolving individual components within a low-resolution shape. Through three case studies, we describe the SAXS data collecting strategies, premodeling analysis, and computational methods required for deconstructing complexes into their respective components. This chapter presents an approach using the programs ScÅtter and MONSA and custom scripts for averaging and aligning of multiple independent modeling runs. The method can image small (7kDa) masses within the context of complex and is capable of visualizing ligand-induced conformational changes. Nevertheless, computational algorithms are not without error, and we describe specific considerations during SAXS data reduction and modeling to mitigate possible false positives.

Small Angle Neutron and X-Ray Scattering Reveal Conformational Differences in Detergents Affecting Rhodopsin Activation

Understanding G-protein-coupled receptor (GPCR) activation plays a crucial role in the development of novel improved molecular drugs. During photoactivation, the retinal chromophore of the visual GPCR rhodopsin isomerizes from the 11-cis conformation to the all-trans conformation, yielding an equilibrium between inactive Meta-I and active Meta-II states [1]. The principal goals of this work are to address whether the activation of rhodopsin leads to a single state or a conformational ensemble, and how the protein organizational structure changes with the detergent environment. We used small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) to answer the above questions. Both SANS and SAXS are powerful techniques to study the macromolecular structures in solution within the length scale from angstroms to several nanometers. In our experiments, rhodopsin is solubilized in CHAPS detergent, which favors the inactive Meta-I state. By contrast, dodecylmaltoside (DDM) detergent stabilizes the active Meta-II state [2]. Notably SANS with contrast-variation enables the separate study of the protein structure within the detergent assembly [3], and suggests a looser structure of rhodopsin in DDM versus CHAPS micelles. Such results are consistent with the SAXS data fitted by either a core-shell ellipsoid or core-shell cylindrical model, describing a monolayer of detergent molecules surrounding the rhodopsin molecule. Moreover, the SAXS experiments with different rhodopsin to detergent ratios delineate the role of the detergent in stabilization of the protein in solution. Our combined approach of SANS and SAXS studies reveals the protein structural changes associated with GPCR activation in the case of visual rhodopsin. [1] A. V. Struts et al. (2011) PNAS 108, 8263-8268. [2] A. V. Struts et al. (2014) Meth. Mol. Biol. in press. [3] R. K. Le et al. (2014) Arch. Biochem. Biophys. 550-551, 50-57.

Liposomal suspension of DSPC/cholesterol with polyethylene glycol: a study by light and X-ray scattering

To study the structure and dynamic properties of liposomal suspension with polyethylene glycol (PEG), we used small-angle X-ray scattering (SAXS) and dynamic light scattering techniques. Light scattering experiment revealed that increase in PEG concentration decreased diffusion coefficient of liposomal structure. Moreover, cholesterol addition contributed to reduction of collective diffusion coefficient in the presence of PEG polymer. The SAXS experiment showed that the size and interaction of the liposome nanoparticle increased with an increase in PEG and cholesterol concentration.

The Coherent X-ray Scattering Beamline at Taiwan Photon Source

The coherent X-ray scattering beamline is one of the phase I beamlines designed for the Taiwan Photon Source, a new 3 GeV ring under construction at the National Synchrotron Radiation Research Center in Taiwan. By using a pair of 2m-long in-vacuum undulators, this beamline will provide a highly coherent beam for X-ray photon correlation spectroscopy principally; moreover, it will share a part of beamtime for small-angle X-ray scattering (SAXS) experiments with similar setup of the beamline. The operating photon energy is designed within the range of 5-20 keV. In vertical direction, the beam spot size at sample position is 1 μm with focusing mirror and by using 1D compound refractive lenses (CRLs) the beam spot size is 10 μm. The horizontal beam spot size is in the range of 1 to 10 μm with a two-stage focusing design. The vertical and horizontal transverse coherence lengths of the 10 μm2 beam spot size at the photon energy of 5 KeV are 212 and 6 μm at sample position respectively. Beside XPCS the beamline configuration can cope with the requirements of most SAXS experiments, including anomalous measurements and micro-beam mapping. In addition, the increasing biological SAXS demand is also considered and the on-line fast performance liquid chromatography (FPLC) will be enclosed for biological users.

Automation for BioSAXS experiments at BM29 of the ESRF

Small Angle X-ray Scattering (SAXS) experiments of macromolecules (proteins, nucleic acids, ...) in solution can provide a wealth of information concerning structure, dynamics and therefore function. However, correct interpretation of results depends critically on data quality. To achieve this, well defined acquisition protocols and immediate feedback are essential. At beamline BM29 at ESRF, BioSAXS data are acquired in a temperature controlled flow through capillary. Samples and buffers can be loaded into the capillary via a robotic liquid handling sample changer (SC) [1] or by connecting the capillary to the outlet of a size exclusion chromatography (SEC) setup [2]. Samples loaded by the SC can be passed through the beam to avoid unwanted effects caused by radiation damage. All recorded data are immediately analyzed to give user-oriented feedback on estimated molecular weight, size, possible radiation damage and inter-particle effects, quality of background correction, etc. Subsequent downstream analysis, also carried out autmatically, results in the calculation of molecular envelopes for solution structures. The information management system ISPyBB (Information System for Protein crystallography Beamlines and BioSAXS) logs and presents all information on data acquisition and analysis and allows users to easily compare the results of different data acquisitions and to decide whether further measurements or changes of acquisition parameters are necessary. The combination of these tools allows non-expert users to carry out BioSAXS experiments autonomously, thereby making the technique accessible to a broader community.

On the supramacromolecular structure of core–shell amphiphilic macromolecules derived from hyperbranched polyethyleneimine

The supramacromolecular structure of core–shell amphiphilic macromolecules (CAMs) with hyperbranched polyethyleneimine (HPEI) cores and fatty acid chain shells (HPEI-Cn) for different chain lengths was investigated both, in colloidal suspension, solid phase and at the air–water interface using Small Angle X-ray Scattering (SAXS), Wide Angle X-ray Scattering (WAXS), X-ray Reflectometry (XRR) and Langmuir isotherms.At low temperatures colloidal toluene suspensions of the HPEI-Cn polymers form, as evidenced by peaks arising in the structure factor of the system showing mean particle-to-particle distances correlated with the length of the aliphatic chains forming the shells of HPEI-Cn unimicelles. The CAM sizes as found from the SAXS experiments also display a clear dependence on shell thickness suggesting that the aliphatic chains adopt a brush-like configuration.After solvent extraction, HPEI-Cn adopts ordered structures with hexagonal packing of the aliphatic chains.Submitted to lateral pressure Π at the air–water interface, HPEI-Cn undergoes a disorder–order transition with increasing transition pressure for increasing chain lengths. The CAMs show different behaviors in-plane and out-of-plane. While out-of-plane the aliphatic chains behave as a brush remaining almost fully unfolded, whereas parallel to the air–water interface the chains fold down in a mushroom way with increasing lateral pressure Π.

Replacement of CTAB with peptidic ligands at the surface of gold nanorods and their self-assembling properties

Herein, we describe the self-assembling of gold nanorods (GNRs) induced during the ligand exchange at their surface. An exchange reaction between tricysteine PEGylated peptidic ligands and cetyltrimethylammonium bromide (CTAB)-protected gold nanorods is conducted. We demonstrated that the terminal group charge (positively or negatively charged) and the hydrophobicity of the peptidic ligands (bearing or not an undecanoyl chain) strongly affects the self-organization of the GNRs occurring in solution. Adjusting the amount of short PEGylated peptides causes a self-organization of the gold nanorods in solution, resulting in a red- or blue-shift of the plasmon bands. The decrease of their surface charge and the self-assembling in solution were first shown by zetametry, by Dynamic Light Scattering and UV-spectroscopy. Thanks to Small Angle X-ray Scattering experiments and Transmission Electron Microscopy images, the self-organization of the nanorods in solution was clearly demonstrated and correlated to the spectroscopic change in absorbance. Conversely, in the case of longer PEGylated peptidic ligands including an undecanoyl chain, the GNRs are particularly stable against aggregation for several days after purification. By controlled drying on a substrate, we showed their ability to self-organize into well-defined ordered structures making them very attractive as building blocks to design optical materials.

Co-condensation of nonane and D2O in a supersonic nozzle.

We study the unary and binary nucleation and growth of nonane-D2O nanodroplets in a supersonic nozzle. Fourier Transform Infrared spectroscopy measurements provide the overall composition of the droplets and Small Angle X-ray Scattering experiments measure the size and number density of the droplets. The unary nucleation rates Jmax of nonane, 9.4 × 10(15) < Jmax /cm(-3) s(-1) < 2.0 × 10(16), and those of D2O, 2.4 × 10(17) < Jmax /cm(-3) s(-1) < 4.1 × 10(17), measured here agree well with previous results. In most of the binary condensation experiments new particle formation is dominated by D2O, but the observed nucleation rates are decreased by up to a factor of 6 relative to the rates measured for pure D2O, an effect that can be partly explained by non-isothermal nucleation theory. The subsequent condensation of D2O is inhibited both by the increased temperature of the binary droplets relative to the pure D2O droplets, and because the binary droplet surface is expected to be comprised largely of nonane. For the one case where nonane appears to initiate condensation, we find that the nucleation rate is about 50% higher than that observed for pure nonane at comparable pv0, consistent with significant particle formation driven by D2O.

Formation of Dynamic Soluble Surfactant-Induced Amyloid Beta Peptide Aggregation Intermediates

The 40-42 residue Amyloid β (Aβ) peptide forms β-structured oligomers on-pathway to amyloid fibril formation and are linked to neuronal damage characteristic for Alzheimer's disease. Surfactants such as SDS induce relatively stable β-structured Aβ co-aggregates and may be considered as a model system for lipids. We have characterized different intermediate aggregation states appearing during the Aβ aggregation process, as well as the kinetics of their formation and dynamic exchange between free and bound peptide. A broad range of biophysical techniques were used, including small angle X-ray scattering (SAXS) and NMR spectroscopy, particularly 15N-CPMG relaxation dispersion experiments. Aβ shows a three-state secondary structure transition depending on surfactant concentration, from random coil-like, via β-structure to α-helix at high surfactant concentration. Structural information on the β-structured co-aggregates was obtained by SAXS experiments that at the beginning showed a large fraction of globular co-aggregates (diameter ∼ 75 Å). This fraction gradually vanished on a min to hr timescale and elongated co-aggregated fibrils were formed (diameter ∼ 60 Å and length > 350 Å), in line with transmission electron microscopy images. A fast dynamic exchange process (kex ∼ 1100 s-1) between free and co-aggregate bound peptide takes place, as monitored by NMR relaxation dispersion experiments. This study of the surfactant-induced Aβ aggregation may serve as a model for aggregation of Aβ alone or in the presence of lipids. Reference: 1. Abelein, A. et al., J. Biol. Chem. (2013), 288, 23518-23528.

Ru-core/Cu-shell bimetallic nanoparticles with controlled size formed in one-pot synthesis.

Suspensions of bimetallic nanoparticles (NPs) of Ru and Cu have been synthesized by simultaneous decomposition of two organometallic compounds in an ionic liquid. These suspensions have been characterized by Anomalous Small-Angle X-ray Scattering (ASAXS) at energies slightly below the Ru K-edge. It is found that the NPs adopt a Ru-core, a Cu-shell structure, with a constant Ru core diameter of 1.9 nm for all Ru : Cu compositions, while the Cu shell thickness increases with Cu content up to 0.9 nm. The formation of RuCuNPs thus proceeds through rapid decomposition of the Ru precursor into RuNPs of constant size followed by the reaction of the Cu precursor and agglomeration as a Cu shell. Thus, the different decomposition kinetics of precursors make possible the elaboration of core-shell NPs composed of two metals without chemical affinity.

Quasielastic Light Scattering and Structure of Nanodroplets Mixed with Polycaprolactone

The interaction of polycaprolactone (PCL) with droplets of a microemulsion is studied with quasielastic light scattering and small angle X-ray scattering At constant droplet size we vary the PCL concentration and there is clear evidence for an increasing attractive interaction of the droplets from structural investigations with small-angle X-ray scattering (SAXS). The collective diffusion coefficient (Dc) of the droplets is monitored with quasielastic light scattering (QELS). We mainly focus on the variation of the dynamic behavior as a function of the PCL concentration and length scale (M.W. = 5000 and 10000) in microemulsion. With increasing PCL concentration and length scale the dynamics of the system slow down. A hard sphere model with depletion potential can fit well the SAXS experiment of microemulsion mixed with PCL. The results show with increase of PCL on microemulsion the size of droplets is constant at 83Å but the size ratio of polymer to droplets is changing.

Magnetic and elastic anisotropy in magnetorheological elastomers using nickel-based nanoparticles and nanochains

Nickel (Ni) based nanoparticles and nanochains were incorporated as fillers in polydimethylsiloxane (PDMS) elastomers and then these mixtures were thermally cured in the presence of a uniform magnetic field. In this way, macroscopically structured-anisotropic PDMS-Ni based magnetorheological composites were obtained with the formation of pseudo-chains-like structures (referred as needles) oriented in the direction of the applied magnetic field when curing. Nanoparticles were synthesized at room temperature, under air ambient atmosphere (open air, atmospheric pressure) and then calcined at 400 °C (in air atmosphere also). The size distribution was obtained by fitting Small Angle X-ray Scattering (SAXS) experiments with a polydisperse hard spheres model and a Schulz-Zimm distribution, obtaining a size distribution centered at (10.0 ± 0.6) nm with polydispersivity given by σ = (8.0 ± 0.2) nm. The SAXS, X-ray powder diffraction, and Transmission Electron Microscope (TEM) experiments are consistent with single crystal nanoparticles of spherical shape (average particle diameter obtained by TEM: (12 ± 1) nm). Nickel-based nanochains (average diameter: 360 nm; average length: 3 μm, obtained by Scanning Electron Microscopy; aspect ratio = length/diameter ∼ 10) were obtained at 85 °C and ambient atmosphere (open air, atmospheric pressure). The magnetic properties of Ni-based nanoparticles and nanochains at room temperature are compared and discussed in terms of surface and size effects. Both Ni-based nanoparticles and nanochains were used as fillers for obtaining the PDMS structured magnetorheological composites, observing the presence of oriented needles. Magnetization curves, ferromagnetic resonance (FMR) spectra, and strain-stress curves of low filler's loading composites (2% w/w of fillers) were determined as functions of the relative orientation with respect to the needles. The results indicate that even at low loadings it is possible to obtain magnetorheological composites with anisotropic properties, with larger anisotropy when using nanochains. For instance, the magnetic remanence, the FMR field, and the elastic response to compression are higher when measured parallel to the needles (about 30% with nanochains as fillers). Analogously, the elastic response is also anisotropic, with larger anisotropy when using nanochains as fillers. Therefore, all experiments performed confirm the high potential of nickel nanochains to induce anisotropic effects in magnetorheological materials.

Anomalous small-angle scattering as a way to solve the Babinet principle problem

X-ray absorption spectra (XAS) have been used to determine the absorption edges of atoms present in a sample under study. A series of small-angle X-ray scattering (SAXS) measurements using different monochromatic X-ray beams at different wavelengths near the absorption edges is performed to solve the Babinet principle problem. The sizes of clusters containing atoms determined by the method of XAS were defined in SAXS experiments. In contrast to differential X-ray porosimetry, anomalous SAXS makes it possible to determine sizes of clusters of different atomic compositions.