Time-resolved SAXS Research Articles

Frontispiece: Monitoring the Discontinuous Dodecamer–Icosamer Transition of a Calix[4]arene‐Derived Surfactant by Time‐Resolved Small‐Angle X‐ray Scattering

Frontispiece: Monitoring the Discontinuous Dodecamer–Icosamer Transition of a Calix[4]arene‐Derived Surfactant by Time‐Resolved Small‐Angle X‐ray Scattering

Frontispiz: Monitoring the Discontinuous Dodecamer–Icosamer Transition of a Calix[4]arene‐Derived Surfactant by Time‐Resolved Small‐Angle X‐ray Scattering

Frontispiz: Monitoring the Discontinuous Dodecamer–Icosamer Transition of a Calix[4]arene‐Derived Surfactant by Time‐Resolved Small‐Angle X‐ray Scattering

Protein Immobilization onto Cationic Spherical Polyelectrolyte Brushes Studied by Small Angle X-ray Scattering.

The immobilization of bovine serum albumins (BSA) onto cationic spherical polyelectrolyte brushes (SPB) consisting of a solid polystyrene (PS) core and a densely grafted poly(2-aminoethyl methacrylate hydrochloride) (PAEMH) shell was studied by small-angle X-ray scattering (SAXS). The observed dynamics of adsorption of BSA onto SPB by time-resolved SAXS can be divided into two stages. In the first stage (tens of milliseconds), the added proteins as in-between bridge instantaneously caused the aggregation of SPB. Then BSA penetrated into the brush layer driven by electrostatic attractions, and reached equilibrium in the second stage (tens of seconds). The amount of BSA immobilized onto brush layer reached the maximum when pH was increased to about 6.1 and BSA concentration to 10 g/L. The cationic SPB were confirmed to provide stronger adsorption capacity for BSA compared to anionic ones.

PHLIP Peptide Interaction with a Membrane Monitored by SAXS.

pH (Low) Insertion Peptides (pHLIP peptides) find application in studies of membrane-associated folding because spontaneous insertion of these peptides is conveniently triggered by varying pH. Here, we employed small-angle X-ray scattering (SAXS) to investigate a wild-type (WT) pHLIP peptide oligomeric state in solution at high concentrations and monitor changes in the liposome structure upon peptide insertion into the bilayer. We established that even at high concentrations (up to 300 μM) the WT pHLIP peptide at pH 8.0 does not form oligomers larger than tetramers (which exhibit concentration-dependent transfer to the monomeric state, as was shown previously). This finding has significance for medical applications when high concentration of the peptide is injected into blood and diluted in blood circulation. The interaction of WT pHLIP peptide with liposomes does not alter the unilamellar vesicle structure upon peptide adsorption by the lipid bilayer at high pH or upon insertion across the bilayer at low pH. At the same time, SAXS data clearly demonstrate the insertion of the peptide into the membrane at low pH, which opens the possibility of investigating the kinetic process of polypeptide insertion and exit from the membrane in real time by time-resolved SAXS.

Impact of aluminum on the structure of geopolymers from the early stages to consolidated material

The aim of this study is to assess the effects of the addition of aluminum (NaAlO2) into the activating solution on the geopolymerization reactions and the characteristics of the porous network. It was established by liquid state NMR that the speciation of the aluminosilicate oligomers and then the mean degree of polymerization were modified by the addition of sodium aluminate in the activating solution. Time-resolved SAXS and rheology results on geopolymer pastes showed that the molar ratios Si/Al and (Si+Al)/Na2O in the activating solution play a major role in the geopolymer properties at liquid and solid state. Indeed, the geopolymer percolating network appeared sooner and the properties of the porous network were dramatically changed with the addition of the NaAlO2. On the other hand, two geopolymers having identical final chemical composition but a different activating solution composition, showed to the same setting time and the same porous network characteristics.

Structural Evolution of Metastable Protein Aggregates in the Presence of Trivalent Salt Studied by (V)SANS and SAXS.

We present a study of the structural evolution of protein aggregates formed in solutions of a globular protein, β-lactoglobulin (BLG), in the presence of YCl3. These aggregates are often observed before crystallization starts and they are metastable with respect to the crystalline phase. Here we focus on the characterization of the hierarchical structure of this intermediate phase and its temperature dependent structure evolution using a combination of (very) small angle neutron and X-ray scattering (VSANS, SANS, and SAXS). Results show that the hierarchical structure ranges from nanometer scale protein monomer, dimer and compact protein clusters to micrometer scale fractal protein aggregates. Upon cooling, the overall hierarchical structure is preserved, but the evolution of the internal structure within the aggregates is clearly visible: the monomer-monomer correlation peak reduces its intensity and disappears completely at lower temperatures, whereas the cluster-cluster correlation is enhanced. At a larger length scale, the fractal dimension of protein aggregates increases. The kinetics of the structure change during a temperature ramp was further investigated using time-resolved SAXS. The time dependent SAXS profiles show clear isosbestic points and the kinetics of the structural evolution can be well described using a two-state model. These dynamic properties of protein aggregates on a broad length scale may be essential for being the precursors of nucleation.

Kinetics of Photocontrollable Micelles: Light-Induced Self-Assembly and Disassembly of Azobenzene-Based Surfactants Revealed by TR-SAXS.

The kinetics of micelles involving photosensitive surfactants is still not well understood. In this work, we unravel the mechanistic pathways involved in the micelle formation and dissolution of photocontrollable micelles. We focus on the fast self-assembly processes of photosensitive cationic azobenzene-containing surfactants (AzoTMA) that display a change in hydrophobicity induced by a reversible cis-trans conformational transition upon exposure to light. By combining both in situ time-resolved small-angle X-ray scattering (SAXS) and light scattering, we characterized the detailed structure and phase behavior of AzoTMA in mixtures of water and dimethylformamide (DMF). Time-resolved synchrotron SAXS with monochromatic light as a trigger enabled us to observe the nonequilibrium formation and dissolution process of micelles (demicellization) directly on the nanoscale with a time resolution starting from milliseconds. The structural results show that in pure water UV-light illumination leads to a 12% reduction of the aggregation number of the micelles and more than a 50% increase in the critical micelle concentration (CMC). Close to the CMC, adjusted by the addition of DMF, UV light illumination leads to a complete dissolution of the micelles, while shining blue light reverses the process and leads to the reformation of micelles. The UV-triggered dissolution follows a two-step mechanism; the first and rapid (second time scale) release of unimers is followed by a slower decomposition of the micelles (over tens of seconds) as a result of an increase in temperature due to optical absorption. Similarly, the reverse process, i.e., micelle formation, occurs rapidly upon photoconversion to trans conformers under blue light, and micelles are disrupted at long exposure time due to the optical absorption and corresponding increase in temperature. Interestingly, the coexistence of unimers with regular micelles is found at all times, and no other transient assemblies could be detected by SAXS.

Ferritin Assembly Revisited: A Time-Resolved Small-Angle X-ray Scattering Study.

The assembly reaction of Escherichia coli ferritin A (EcFtnA) was studied using time-resolved small-angle X-ray scattering (TR-SAXS). EcFtnA forms a cagelike structure that consists of 24 identical subunits and dissociates into dimers at acidic pH. The dimer maintains nativelike secondary and tertiary structures and is able to reassemble into a 24-mer when the pH is increased. The reassembly reaction was induced by pH jump, and reassembly was followed by TR-SAXS. Time-dependent changes in the forward scattering intensity and in the gyration radius suggested the existence of a significant population of intermediate oligomers during the assembly reaction. The initial reaction was a mixture of second- and third-order reactions (formation of tetramers and hexamers) from the protein concentration dependence of the initial velocity. The time-dependent change in the SAXS profile was roughly explained by a simple model in which only tetramers, hexamers, and dodecamers were considered as intermediates.

Identification of a major intermediate along the self-assembly pathway of an icosahedral viral capsid by using an analytical model of a spherical patch.

Viruses are astonishing edifices in which hundreds of molecular building blocks fit into the final structure with pinpoint accuracy. We established a robust kinetic model accounting for the in vitro self-assembly of a capsid shell derived from an icosahedral plant virus by using time-resolved small-angle X-ray scattering (TR-SAXS) data at high spatiotemporal resolution. By implementing an analytical model of a spherical patch into a global fitting algorithm, we managed to identify a major intermediate species along the self-assembly pathway. With a series of data collected at different protein concentrations, we showed that free dimers self-assembled into a capsid through an intermediate resembling a half-capsid. The typical lifetime of the intermediate was a few seconds and yet the presence of so large an oligomer was not reported before. The progress in instrumental detection along with the development of powerful algorithms for data processing contribute to shedding light on nonequilibrium processes in highly complex systems such as viruses.

Structural Evolution of Aqueous Zirconium Acetate by Time-Resolved Small-Angle X-ray Scattering and Rheology

The nanostructure and viscosity of aqueous zirconium acetate solutions was investigated by combining small-angle X-ray scattering (SAXS) and rheology measurements. The structural evolution at concentrations of 22, 43, and 220 mM was furthermore investigated by time-resolved SAXS at a moderate temperature of 85 °C. Modeling of the SAXS data revealed that zirconium acetate exists as extended oligomer chains with an average contour length of 100 ± 15 A and radius of 4.9 ± 0.9 A. This chain radius corresponds to a radius of gyration of 3.8 A for a sphere-like object, which is similar to that for individual cyclic tetramers, [Zr(OH)2·4H2O]48+, suggesting that the chains consist of connected tetramers. Their suspension in the aqueous solution is stabilized by their charge as corroborated by addition of NaCl, which lowers the stabilizing potential and leads to chain growth and aggregation upon aging at room temperature. The time-resolved SAXS measurements allow a detailed characterization of the polymerization t...

The supramolecular chemistry of gold and l -cysteine: Formation of photoluminescent, orange-emitting assemblies with multilayer structure

The protein mediated approach is a common method for the synthesis of photoluminescent gold quantum clusters (GQCs), where proteins, acting as reducing and stabilizing agents, react with gold salts through cysteine residues. For the better understanding of the phenomenon, the aqueous phase reaction of HAuCl4 and l-cysteine has been investigated at the supramolecular level by various experimental techniques and molecular mechanics simulations. We have observed the formation of a novel photoluminescent product, (AuCys)nβ, which shows emission in the orange region of the spectrum. Small- and wide-angle X-ray scattering (SWAXS) measurements have revealed the presence of nanosized lamellae, which have an internal multilayer superlattice structure with a characteristic periodic distance of 1.3 nm. Based on the results, the layers are built up by zigzag shaped (AuCys)n polymer chains connected through aurophilic bonds. The aurophilic network is stabilized via salt bridges and hydrogen bonds, which are also responsible for the interlayer interactions. Here, the evolution of the multilayer structure has been monitored by the combined application of photoluminescence spectroscopy and time-resolved SAXS. It has been concluded that there is a strong correlation between the emission and the scattering intensity, which suggests that the two- and three-dimensional aggregation of the building blocks to form sheets and multilayers are simultaneous processes. Furthermore, we have revealed that the formation and behavior of (AuCys)nβ show significant differences to that of Au-l-glutathione compounds described earlier despite the similarity of l-cysteine and l-glutathione. These results evidence that l-cysteine and gold species form building blocks that can be applied expansively in supramolecular and cluster chemistry.

Time-Resolved SAXS experiments with low sample consumption

Using Time-Resolved Small Angle X-ray Scattering (TR-SAXS) on the millisecond time scale to investigate biological systems is an extremely powerful tool to detect possible intermediate states that form during the course of the reaction and gain detailed insight into the sequence of events along the reaction pathway. However the use of TR-SAXS has often been difficult due to the large amount of sample typically required for such experiments. Here we report on our recent development of a fast TR-SAXS setup using a customized stopped-flow mixer. This new setup allows us to obtain a TR-SAXS data set from as little as 30ul of sample volume. It also eliminates the sample consuming priming of the tubing inside the stopped flow and thus substantially reduces the sample amount required for such experiments. An automatic wash cycle for the sample cell (capillary) is employed after every single shot to ensure that the capillary is clean and no residues remain in the cell from the previous measurement.

Revealing transient structures of nucleosomes as DNA unwinds.

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation.

Coaggregation of Two Anionic Azo Dyestuffs: A Combined Static Light Scattering and Small-Angle X-ray Scattering Study.

The formation of azo dyestuff aggregates in dilute aqueous solution induced by the addition of Mg2+, Ca2+, Sr2+, or Ba2+ ions is followed by time-resolved static light scattering (SLS) and time-resolved small-angle X-ray scattering (SAXS). Time-dependent molar mass data of the growing aggregates is interpreted by means of a kinetic model introduced by Lomakin et al. ( Proc. Natl. Acad. Sci. U.S.A. 1996 , 93 , 1125 ) for the description of β-amyloid aggregation. This interpretation reveals significant trends within the homologous series of alkaline earth cations. The trends refer to the nucleation and the growth rate of the dyestuff fibers. Time-resolved SAXS experiments indicate that these first two stages are followed by a third one during which a network forms by partial lateral alignment of fibers. At high enough dyestuff concentrations, this network formation even leads to a gel-like phase. Anomalous SAXS (ASAXS) on such a gel phase formed upon the addition of Sr2+ revealed the extent of neutralization of the dyestuff molecules within the gel by the specifically interacting alkaline earth cations.

Silver substrates for surface enhanced Raman scattering: Correlation between nanostructure and Raman scattering enhancement

The fabrication of substrates for Surface Enhanced Raman Scattering (SERS) applications matching the needs for high sensitive and reproducible sensors remains a major scientific and technological issue. We correlate the morphological parameters of silver (Ag) nanostructured thin films prepared by sputter deposition on flat silicon (Si) substrates with their SERS activity. A maximum enhancement of the SERS signal has been found at the Ag percolation threshold, leading to the detection of thiophenol, a non-resonant Raman probe, at concentrations as low as 10−10M, which corresponds to enhancement factors higher than 7 orders of magnitude. To gain full control over the developed nanostructure, we employed the combination of in-situ time-resolved microfocus Grazing Incidence Small Angle X-ray Scattering with sputter deposition. This enables to achieve a deepened understanding of the different growth regimes of Ag. Thereby an improved tailoring of the thin film nanostructure for SERS applications can be realized.

Time-Resolved Small-Angle X-ray Scattering Study on the Growth Behavior of Silver Nanoparticles

Silver nanoparticles were prepared with a chemical reduction method in the presence of polyvinylpyrrolidone (PVP) used as a stabilizing agent. During the synthesis of Ag nanoparticles, aqueous silver nitrate solutions were used as the precursors and sodium borohydride was used as the reducing agent. Four Ag-nanoparticle suspensions were prepared with the initial Ag+ concentrations of 2, 4, 6, and 8 mM in the corresponding precursors. The in-situ time-resolved small-angle X-ray-scattering (SAXS) technique was used to monitor the nucleation and growth processes of Ag nanoparticles. The particle-size change with time was obtained by analyzing the SAXS data with a tangent-by-tangent method. The SAXS results demonstrate that the Ag nanoparticle growth behaves as a linear relation in the initial growth stage (<1 s), which was used to evaluate the critical particle size. The growth of Ag nanoparticles experienced a fast stage and then a slow stage in their whole formation process. A diffusion–coalescence model has been proposed to describe the growth behavior. The particle size change with growth time can be fitted well by this model. The effect of initial Ag+ concentration on the final particle size and the growth mechanism of Ag nanoparticles are discussed in this paper.

Mechanism of the Rpn13-induced activation of Uch37.

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other’s ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37ΔHb,Hc,KEKE, a truncation removal of the C-terminal extension region (residues 256–329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270–407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-014-0046-z) contains supplementary material, which is available to authorized users.

Resolving Early Stages of Homogeneous Iron(III) Oxyhydroxide Formation from Iron(III) Nitrate Solutions at pH 3 Using Time-Resolved SAXS

Small angle X-ray scattering (SAXS) measurements coupled to a stopped-flow device has permitted the observation of the kinetics of Fe(III) oxyhydroxide (FeOx) formation and transformation from around 1 s to 30 min after initiation under environmentally relevant conditions at pH 3. The Unified Model approach was used to determine the evolution of multiple key parameters (particle scattering mass, mean particle volume, particle concentration, particle dimensionality, and particle size) for two separate structural levels as a function of time, with the results obtained enabling clarification of the mechanisms underlying FeOx formation and transformation under these conditions. Colloidal primary particles (radius of gyration 2–10 nm) that were observable by SAXS formed within 1 s of stopping the flow and subsequently grew over several minutes, first by cluster–cluster addition and then by a monomer-addition mechanism. Aggregation of these primary particles via a secondary cluster–cluster addition mechanism simultaneously resulted in a distinct population of larger (25–40 nm radius of gyration) secondary particles. The primary particles evolved into compact spheroidal forms with fractally rough surfaces, while the secondary particles were relatively open mass fractal structures. Comparison of the observed rates of these processes with those predicted for Fe polymerization indicates that kinetics of primary particle formation were likely controlled initially by rates of exchange between water molecules coordinated with Fe and those in the bulk solution. These findings provide new insights into the mechanisms underlying FeOx formation and transformation, and the kinetics of these mechanisms, at pH 3.

Probing Anisotropic Structure Changes in Proteins with Picosecond Time-Resolved Small-Angle X-ray Scattering

We have exploited the principle of photoselection and the method of time-resolved small-angle X-ray scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ∼150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly contorted, short-lived, red-shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.3% along the direction defined by the electronic transition dipole moment of the chromophore. Contraction along this dimension is accompanied by expansion along the orthogonal directions, with the net protein volume change being approximately -0.25%. More than half the strain arising from formation of pR0 is relieved by the pR0 to pR1 structure transition (1.8 ± 0.2 ns), with the persistent strain presumably contributing to the driving force needed to generate the spectroscopically blue-shifted pB signaling state. The results reported here are consistent with the near-atomic resolution structural dynamics reported in a recent time-resolved Laue crystallography study of PYP crystals and suggest that the early time structural dynamics in the crystalline state carry over to proteins in solution.

Evolution of microstructure in mixed niobia-hybrid silica thin films from sol–gel precursors

The evolution of structure in sol–gel derived mixed bridged silsesquioxane-niobium alkoxide sols and drying thin films was monitored in situ by small-angle X-ray scattering. Since sol–gel condensation of metal alkoxides proceeds much faster than that of silicon alkoxides, the incorporation of d-block metal dopants into silica typically leads to formation of densely packed nano-sized metal oxide clusters that we refer as metal oxide building blocks in a silica-based matrix. SAXS was used to study the process of niobia building block formation while drying the sol as a thin film at 40–80°C. The SAXS curves of mixed niobia-hybrid silica sols were dominated by the electron density contrast between sol particles and surrounding solvent. As the solvent evaporated and the sol particles approached each other, a correlation peak emerged. Since TEM microscopy revealed the absence of mesopores, the correlation peak was caused by a heterogeneous system of electron-rich regions and electron poor regions. The regions were assigned to small clusters that are rich in niobium and which are dispersed in a matrix that mainly consisted of hybrid silica. The correlation peak was associated with the typical distances between the electron dense clusters and corresponded with distances in real space of 1–3nm. A relationship between the prehydrolysis time of the silica precursor and the size of the niobia building blocks was observed. When 1,2-bis(triethoxysilyl)ethane was first hydrolyzed for 30min before adding niobium penta-ethoxide, the niobia building blocks reached a radius of 0.4nm. Simultaneous hydrolysis of the two precursors resulted in somewhat larger average building block radii of 0.5–0.6nm. This study shows that acid-catalyzed sol–gel polymerization of mixed hybrid silica niobium alkoxides can be rationalized and optimized by monitoring the structural evolution using time-resolved SAXS.