Small-angle Scattering Of X-rays Research Articles

Study of Supported Metal Catalysts by the Methods of the Small-Angle Scattering of Neutrons and X-rays

Study of Supported Metal Catalysts by the Methods of the Small-Angle Scattering of Neutrons and X-rays

Temperature dependent self-organization of DMPC membranes promoted by intermediate amounts of the saponin aescin

The plant-derived biosurfactant aescin is naturally present in many plants and is used for treatment of disorders such as varicose veins and inflammation of veins. The hemolytic activity of this saponin is attributed to its interaction with cholesterol in the red blood cell membrane. This work investigates the phase and aggregation behavior of saponin-containing model membranes consisting of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The aescin concentrations studied range from 1 mol% to 7 mol% with respect to the total lipid content. The methods of choice to elucidate the structural picture are small-angle scattering of X-rays (SAXS) and neutrons (SANS) and cryogenic transmission electron microscopy (cryo-TEM). SANS and SAXS revealed that at lower aescin contents vesicular structures are conserved and vesicles tend to aggregate already at aescin contents of around 1 mol%. Aggregation and vesicle deformation effects are found to be stronger when the phospholipids are in the Lβ′ phase. With increasing aescin content, mixed structures, i.e. aggregated and deformed vesicles and solubilized bilayer fragments, are present. This was proven for a sample with 4 mol% aescin by cryo-TEM. An increasing aescin amount leads to membrane decomposition and free standing bilayers which tend to build stacks at high temperature. These stacks are characterized by SAXS using the modified Caillé theory. Analyses and model dependent fitting reveal formation of well-defined structures beginning at 7 mol% aescin.

Small-angle Scattering of X-rays and Neutrons as Structural Tools inNanoscience: Block Copolymer Micelles, Protein-detergent Complexes,Microgels and other Nanoparticles

Small-angle Scattering of X-rays and Neutrons as Structural Tools inNanoscience: Block Copolymer Micelles, Protein-detergent Complexes,Microgels and other Nanoparticles

Advanced Synchrotron Radiation and Neutron Scattering Techniques for Microstructural Characterization in Industrial Research

The rapid development of new materials and their application in an extremely wide variety of research and technological fields has lead to the request of increasingly sophisticated characterization methods. In particular residual stress measurements by neutron diffraction, small angle scattering of X-rays and neutrons, as well as 3D imaging techniques with spatial resolution at the micron or even sub-micron scale, like micro-and nano-computerized tomography, have gained a great relevance in recent years.Residual stresses are autobalancing stresses existing in a free body not submitted to any external surface force. Several manufacturing processes, as well as thermal and mechanical treatments, leave residual stresses within the components. Bragg diffraction of X-rays and neutrons can be used to determine residual elastic strains (and then residual stresses by knowing the material elastic constants) in a non-destructive way. Small Angle Scattering of neutrons or X-rays, complementary to Transmission Electron Microscopy, allows the determination of structural features such as volume fraction, specific surface and size distribution of inhomogeneities embedded in a matrix, in a huge variety of materials of industrial interest. X-ray microtomography is similar to conventional Computed Tomography employed in Medicine, allowing 3D imaging of the investigated samples, but with a much higher spatial resolution, down to the sub-micron scale. Some examples of applications of the experimental techniques mentioned above are described and discussed.

Structural Characterization of Highly Flexible Proteins by Small-Angle Scattering.

Intrinsically Disordered Proteins (IDPs) are fundamental actors of biological processes. Their inherent plasticity facilitates very specialized tasks in cell regulation and signalling, and their malfunction is linked to severe pathologies. Understanding the functional role of disorder requires the structural characterization of IDPs and the complexes they form. Small-angle Scattering of X-rays (SAXS) and Neutrons (SANS) have notably contributed to this structural understanding. In this review we summarize the most relevant developments in the field of SAS studies of disordered proteins. Emphasis is given to ensemble methods and how SAS data can be combined with computational approaches or other biophysical information such as NMR. The unique capabilities of SAS enable its application to extremely challenging disordered systems such as low-complexity regions, amyloidogenic proteins and transient biomolecular complexes. This reinforces the fundamental role of SAS in the structural and dynamic characterization of this elusive family of proteins.

SAXS/SANS on Supercharged Proteins Reveals Residue-Specific Modifications of the Hydration Shell

Water molecules in the immediate vicinity of biomacromolecules, including proteins, constitute a hydration layer characterized by physicochemical properties different from those of bulk water and play a vital role in the activity and stability of these structures, as well as in intermolecular interactions. Previous studies using solution scattering, crystallography, and molecular dynamics simulations have provided valuable information about the properties of these hydration shells, including modifications in density and ionic concentration. Small-angle scattering of x-rays (SAXS) and neutrons (SANS) are particularly useful and complementary techniques to study biomacromolecular hydration shells due to their sensitivity to electronic and nuclear scattering-length density fluctuations, respectively. Although several sophisticated SAXS/SANS programs have been developed recently, the impact of physicochemical surface properties on the hydration layer remains controversial, and systematic experimental data from individual biomacromolecular systems are scarce. Here, we address the impact of physicochemical surface properties on the hydration shell by a systematic SAXS/SANS study using three mutants of a single protein, green fluorescent protein (GFP), with highly variable net charge (+36, −6, and −29). The combined analysis of our data shows that the hydration shell is locally denser in the vicinity of acidic surface residues, whereas basic and hydrophilic/hydrophobic residues only mildly modify its density. Moreover, the data demonstrate that the density modifications result from the combined effect of residue-specific recruitment of ions from the bulk in combination with water structural rearrangements in their vicinity. Finally, we find that the specific surface-charge distributions of the different GFP mutants modulate the conformational space of flexible parts of the protein.

Small-Angle Scattering Analysis of Empty or Loaded Hierarchical Porous Materials

Small-angle scattering of X-rays (SAXS) or neutrons (SANS) is one of the few experimental methods that can in principle be used for the in situ study at the mesoscopic scale of physicochemical phenomena occurring inside nanoporous solids. However, the potential of the method is often limited by the lack of suitable data analysis methods to convert scattering data into real-space structural information. This is notably the case for most porous materials of practical interest, which exhibit a hierarchical structure with micro, meso, and macropores, with often a secondary material confined in the pores, such as in supported catalysts, as well as fuel-cell and battery materials. In the present paper, we propose a general analysis of X-ray scattering by this type of material. Assuming that each structural level is statistically independent from the others and has a distinct characteristic length scale, compact mathematical expressions are derived for the scattering of the entire hierarchical structure. The res...

Structural Characterization of Heparin-induced Glyceraldehyde-3-phosphate Dehydrogenase Protofibrils Preventing α-Synuclein Oligomeric Species Toxicity

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.

Three-dimensional reconstruction of liquid phases in disordered mesopores usingin situsmall-angle scattering

Small-angle scattering of X-rays (SAXS) or neutrons is one of the few experimental methods currently available for thein situanalysis of phenomena in mesoporous materials at the mesoscopic scale. In the case of disordered mesoporous materials, however, the main difficulty of the method lies in the data analysis. A stochastic model is presented, which enables one to reconstruct the three-dimensional nanostructure of liquids confined in disordered mesopores starting from small-angle scattering data. This so-called plurigaussian model is a multi-phase generalization of clipped Gaussian random field models. Its potential is illustrated through the synchrotron SAXS analysis of a gel permeated with a critical nitrobenzene/hexane solution that is progressively cooled below its consolute temperature. The reconstruction brings to light a wetting transition whereby the nanostructure of the pore-filling liquids passes from wetting layers that uniformly cover the solid phase of the gel to plugs that locally occlude the pores. Using the plurigaussian model, the dewetting phenomenon is analyzed quantitatively at the nanometre scale in terms of changing specific interface areas, contact angle and specific length of the triple line.

Conformational Analysis of a Genetically Encoded FRET Biosensor by SAXS

Genetically encoded FRET (Foerster resonance energy transfer) sensors are exciting tools in modern cell biology. Changes in the conformation of a sensor lead to an altered emission ratio and provide the means to determine both temporal and spatial changes in target molecules, as well as the activity of enzymes. FRET sensors are widely used to follow phosphorylation events and to monitor the effects of elevated calcium levels. Here, we report for the first time, to our knowledge, on the analysis of the conformational changes involved in sensor function at low resolution using a combination of in vitro and in cellulo FRET measurements and small-angle scattering of x rays (SAXS). The large and dynamic structural rearrangements involved in the modification of the calcium- and phosphorylation-sensitive probe CYNEX4 are comprehensively characterized. It is demonstrated that the synergistic use of SAXS and FRET methods allows one to resolve the ambiguities arising due to the rotation of the sensor molecules and the flexibility of the probe.

Structural analysis of intrinsically disordered proteins by small-angle X-ray scattering

Small-angle scattering of X-rays (SAXS) is an established method to study the overall structure and structural transitions of biological macromolecules in solution. For folded proteins, the technique provides three-dimensional low resolution structures ab initio or it can be used to drive rigid-body modeling. SAXS is also a powerful tool for the quantitative analysis of flexible systems, including intrinsically disordered proteins (IDPs), and is highly complementary to the high resolution methods of X-ray crystallography and NMR. Here we present the basic principles of SAXS and review the main approaches to the characterization of IDPs and flexible multidomain proteins using SAXS. Together with the standard approaches based on the analysis of overall parameters, a recently developed Ensemble Optimization Method (EOM) is now available. The latter method allows for the co-existence of multiple protein conformations in solution compatible with the scattering data. Analysis of the selected ensembles provides quantitative information about flexibility and also offers insights into structural features. Examples of the use of SAXS and combined approaches with NMR, X-ray crystallography, and computational methods to characterize completely or partially disordered proteins are presented.

Density minimum of confined water at low temperatures: a combined study by small-angle scattering of X-rays and neutrons

A simple explanation is given for the low-temperature density minimum of water confined within cylindrical pores of ordered nanoporous materials of different pore size. The experimental evidence is based on combined data from in-situ small-angle scattering of X-rays (SAXS) and neutrons (SANS), corroborated by additional wide-angle X-ray scattering (WAXS). The combined scattering data cannot be described by a homogeneous density distribution of water within the pores, as was originally suggested from SANS data alone. A two-step density model reveals a wall layer covering approximately two layers of water molecules with higher density than the residual core water in the central part of the pores. The temperature-induced changes of the scattering signal from both X-rays and neutrons are consistent with a minimum of the average water density. We show that the temperature at which this minimum occurs depends monotonically on the pore size. Therefore we attribute this minimum to a liquid-solid transition of water influenced by confinement. For water confined in the smallest pores of only 2 nm in diameter, the density minimum is explained in terms of a structural transition of the surface water layer closest to the hydrophilic pore walls.

Structure and permeability of magnetoliposomes loaded with hydrophobic magnetic nanoparticles in the presence of a low frequency magnetic field

In this paper we describe the effect of a low frequency alternating magnetic field (LF-AMF) on the structure and permeability of magnetoliposomes, i.e. liposomes formulated in the presence of magnetic nanoparticles. Hydrophobic cobalt ferrite nanoparticles (CoFe2O4) coated with a shell of oleic acid were prepared, characterized and employed in the preparation of magnetoliposomes. The stability of the lipid bilayer after the application of an oscillating magnetic field was studied by means of Dynamic Light Scattering (DLS), Small Angle Scattering of X-rays (SAXS) and Differential Scanning Calorimetry (DSC). The enhancement of liposome permeability upon LF-AMF exposure was measured as the self-quenching decrease of the fluorescent molecule carboxyfluorescein (CF) entrapped in the liposome pool. Carboxyfluorescein leakage from magnetoliposomes was investigated as a function of field frequency, time of exposure to the magnetic field, and cobalt ferrite nanoparticles concentration. Kinetics of CF release from LF-AMF treated magnetoliposomes, monitored through the fluorescence intensity increase during time, highlights a slow release of CF during the first hours, followed by a faster release a few hours after the field treatment which leads to a complete leakage of CF. DSC provides insights about the effect of the LF-AMF treatment, showing that the first few hours correspond to a complete loss of the transition peak from the lamellar gel (Lβ) phase to the liquid crystalline (Lα) phase of the PC bilayers. These results suggest that the slow release takes place through the formation of local pores or defects at the membrane level, while the fast release corresponds to an increased permeability of the membrane that can be related to a structural change of the bilayer.

Structural characterization of proteins and complexes using small-angle X-ray solution scattering

Small-angle scattering of X-rays (SAXS) is an established method for the low-resolution structural characterization of biological macromolecules in solution. The technique provides three-dimensional low-resolution structures, using ab initio and rigid body modeling, and allow one to assess the oligomeric state of proteins and protein complexes. In addition, SAXS is a powerful tool for structure validation and the quantitative analysis of flexible systems, and is highly complementary to the high resolution methods of X-ray crystallography and NMR. At present, SAXS analysis methods have reached an advanced state, allowing for automated and rapid characterization of protein solutions in terms of low-resolution models, quaternary structure and oligomeric composition. In this communication, main approaches to the characterization of proteins and protein complexes using SAXS are reviewed. The tools for the analysis of proteins in solution are presented, and the impact that these tools have made in modern structural biology is discussed.

Structure Analysis of Synaptic Vesicles by Solution Small-Angle Scattering of X-Rays

The release of neurotransmitters from neurons, in response to stimulation, forms the basis of communication in the nervous system. Neurotransmitters are stored in small membraneous organelles, synaptic vesicles (SVs), within the presynaptic terminal. These vesicles undergo an elaborate cycle of fusion with the plasma membrane (releasing neurotransmitter), followed by retrieval and reformation and transport back to the plasma membrane for further rounds of fusion [1].In recent years there has been enormous progress in our knowledge of the molecular composition and structure of synaptic vesicles [2]. However, we still lack a detailed view of the physical properties of this trafficking organelle as it proceeds through its life-cycle.Here we use small-angle x-ray scattering (SAXS) to determine the average radial density profile rho(r) and the size polydispersity of SVs [3]. We show that SAXS can be used to study the supra-molecular structure of an entire functional organelle under physiological conditions. The profile rho(r) of SVs including structural parameters of the protein layers, as well as the polydispersity function p(R), are derived with no free prefactors on an absolute scale. The measured SV structure on length scales between the constituent biomolecules and the SV size confirms the main aspects of recent numerical modeling [2], which was based on the crystal structures of the constituent proteins and stoichiometric knowledge from biochemical studies. In addition, we present first evidence of a laterally anisotropic structure, indicative of larger protein clusters.[1] T. Sudhof, Annu. Rev. Neurosci. 27 (2004) 509.[2] S. Takamori et al, Cell 127 (2006) 831.[3] S. Castorph et al, in preparation.

Portrait of an Enzyme, a Complete Structural Analysis of a Multimodular β-N-Acetylglucosaminidase from Clostridium perfringens

Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens micro-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.

Structural Characterization of the Bradyzoite Surface Antigen (BSR4) from Toxoplasma gondii, a Unique Addition to the Surface Antigen Glycoprotein 1-related Superfamily

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects nearly one-third of the human population. The success of T. gondii is based on its complex life cycle; a lytic tachyzoite form disseminates infection, whereas an encysted bradyzoite form establishes a latent, chronic infection. Persistence and transmissibility is central to the survival of the parasite and is, in part, mediated by a family of antigenically distinct surface antigen glycoprotein (SAG)-related sequences (SRS) adhesins that play a dual role in host cell attachment and host immune evasion. More than 160 members of the SRS family have been identified with only the tachyzoite-expressed SAG1 structurally characterized. Here we report the first structural description of the bradyzoite adhesin BSR4 using x-ray crystallography and small angle x-ray scattering. The 1.90-A crystal structure of BSR4 reveals an architecture comprised of tandem beta sandwich domains organized in a head to tail fashion with the N-terminal domain responsible for dimer formation. A restructured topology in BSR4 results in a ligand-binding site that is significantly reorganized in both structure and chemistry relative to SAG1, consistent with BSR4 binding a distinct physiological ligand. The small angle x-ray scattering solution structure of BSR4 highlights a potentially important structural role for the interdomain polymorphic linker that imparts significant flexibility that may promote structural adaptation during ligand binding. This study reveals an unexpected level of structural diversity within the SRS superfamily and provides important insight into the role of these virulence factors.

SAXS study of chain-like structures formed by magnetic nanoparticles

Nanoparticles consisting of a magnetic core (γ-Fe2O3) and an organic shell were prepared by thermal decomposition of an iron carbonyl complex in the presence of oleic acid. The nanoparticles were then dispersed in cyclohexane to form a stable ferrofluid. These dispersions were investigated by means of Small Angle Scattering of X-rays (SAXS) and the data were interpreted according to a “pearl necklace” model, opportunely modified to account for the core-shell structure of the magnetic nanoparticles. The results of the fittings show that nearly monodisperse spherical particles with a radius of the magnetic core of about 3.9 nm and a shell of about 2 nm were obtained. These nanoparticles self-assemble in chain-like structures, even in the absence of an applied magnetic field.

Neutron Scattering Reveals the Dynamic Basis of Protein Adaptation to Extreme Temperature

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 A) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.

Structure and order in lamellar phases determined by small-angle scattering

Multilamellar phases can be identified and characterized by small-angle scattering of X-rays (SAXS) or neutrons (SANS). Equidistant peaks are the typical signature and their spacing allows the fast determination of the repeat distance,i.e.the mean distance between the midplane of neighbouring bilayers. The scattering function can be described as the product of a structure factor and a form factor. The structure factor is related to the ordering of the bilayers and is responsible for the typical equidistant peaks, but it also contains information about the bilayer flexibility and the number of coherently scattering bilayers. The form factor depends on the thickness and the internal structure (scattering length density distribution) of a single bilayer. The recently developed generalized indirect Fourier transformation (GIFT) method is extended to such systems. This method allows the simultaneous determination of the structure factor and the form factor of the system, including the correction of instrumental broadening effects. A few-parameter model is used for the structure factor, while the determination of the form factor is completely model-free. The method has been tested successfully with simulated scattering data and by application to experimental data sets.