Small-angle X-ray Scattering Studies Research Articles

Solvent selective gelation of cetyltrimethylammonium bromide: structure, phase evolution and thermal characteristics.

We report herein the gelation behaviour of cetyltrimethylammonium bromide (CTAB), a cationic surfactant, in a variety of solvent compositions. A turbid gel of CTAB in a binary solvent mixture at a critical composition was observed to be 1 : 3 v/v toluene : water. The molecular structure of the as-formed gel was investigated by X-ray diffraction and microscopic techniques, namely, optical and polarizing microscopy, scanning electron microscopy and small-angle X-ray scattering (SAXS). The phase evolution has been studied using UV-visible transmittance measurements and the thermal characteristics of the gel by differential scanning calorimetry measurements. SAXS studies, in conjunction with molecular modelling, revealed the gel to assemble as lamellae with high interdigitation of bilayer assembly of CTAB molecules with predominant non-covalent interactions, where the gel lamellae were inferred from the interplanar spacings. Rheological studies revealed the viscoelastic nature of the CTAB gels. The ability to form a gel has been evaluated in several polar solvents, such as methanol and chloroform, and non-polar solvents, such as toluene and carbon tetrachloride.

Synchrotron radiation SAXS study of the species structure evolution in the synthesis process of MCM-56 zeolite

In the process of synthesizing zeolite, the evolution behavior of aluminosilicate species is crucial for investigating the crystallization mechanism and controlling the structure of desired zeolite. Herein, the synthesis process of MCM-56 zeolite was investigated by using the synchrotron radiation Small Angle X-ray Scattering (SAXS) technique. Based on the distance distribution function curves and Porod analysis, the particle distribution of the sol in the synthesis system transformed from a monodisperse spherical particle to a polydisperse lamellar structure. The results indicated that the particle size distribution, fractal dimension, and the evolution behavior of particles in sol vary with the variation of crystallization time. Specifically, the initial step involves the depolymerization of particles forming small particles approximately 1 nm. Subsequently, these small particles polymerize to form structural units ranging in size from 1 to 5 nm, and finally grow to form MCM-56 zeolite crystal by consuming aluminosilicate species. The SAXS results in conjunction with the XRD, SEM, FT-IR, and N2 adsorption-desorption analysis elucidate the evolution of aluminosilicate species and the crystallization process of MCM-56 zeolite at three stages, including the induction/nucleation period (0–84 h), the crystal growth period (84–108 h) and the crystal stability period (108–168 h). By virtue of the understanding for the structural evolution of the sol species, this work is expected to highlight the controllable synthesis of MCM-56 zeolite.

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.

New formulation technology to boost sun protection.

As every skin type worldwide is concerned by photoprotection, with consumers preferring cosmetic elegant and efficient sunscreen products, we aim at developing the most performant and desirable sun care products. We selected an interesting polymer, abbreviated AAHCP and designed scanning electron cryomicroscopy (cryoSEM), small angle and wide angle X-ray scattering and confocal laser scanning microscopy studies to understand its behaviour in solution and in simplex sun care formulations. This allowed us to develop innovative sunscreen formulation technology that was demonstrated by in vitro and in vivo photoprotection methods. Comprehensive photoprotection evaluations were made on the fully developed sun protection product. We observed the polymer oil structuring properties as well as its ability to form small and stable droplets in simplex emulsions. In vitro and in vivo sun protection factor (SPF) measurements demonstrated the sun protection boosting efficacy of AAHCP polymer in several emulsions or as a stand-alone emulsifier. This formulation technology also allowed to filtering system concentration optimization. Use-test performed on a fully developed AAHCP-based sunscreen validated its optimal performances as well as its ideal cosmetic features, with non-sticky, non-greasy perception and invisible skin result. For the first time, thanks to a new specific polymer creating a new type of emulsion, we succeed in reconciliate in a single sun care product maximal SPF efficacy, resistance to numerous stresses and optimal sensoriality.

Assessment of structural behaviour of a new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form

The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, Km, Vmax, and kcat were 9.49 × 10−3 M, 25.13 IUmL−1 min−1, and 3.01 × 103 s−1, respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Differing from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37 °C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC50 of 7.4 μg.mL−1 and 5.6 μg.mL−1, respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.

In Situ Ultra-Small- and Small-Angle X-ray Scattering Study of ZnO Nanoparticle Formation and Growth through Chemical Bath Deposition in the Presence of Polyvinylpyrrolidone.

ZnO inverse opals combine the outstanding properties of the semiconductor ZnO with the high surface area of the open-porous framework, making them valuable photonic and catalysis support materials. One route to produce inverse opals is to mineralize the voids of close-packed polymer nanoparticle templates by chemical bath deposition (CBD) using a ZnO precursor solution, followed by template removal. To ensure synthesis control, the formation and growth of ZnO nanoparticles in a precursor solution containing the organic additive polyvinylpyrrolidone (PVP) was investigated by in situ ultra-small- and small-angle X-ray scattering (USAXS/SAXS). Before that, we studied the precursor solution by in-house SAXS at T = 25 °C, revealing the presence of a PVP network with semiflexible chain behavior. Heating the precursor solution to 58 °C or 63 °C initiates the formation of small ZnO nanoparticles that cluster together, as shown by complementary transmission electron microscopy images (TEM) taken after synthesis. The underlying kinetics of this process could be deciphered by quantitatively analyzing the USAXS/SAXS data considering the scattering contributions of particles, clusters, and the PVP network. A nearly quantitative description of both the nucleation and growth period could be achieved using the two-step Finke-Watzky model with slow, continuous nucleation followed by autocatalytic growth.

Solvent selectivity governed self-assembly of block copolymer in nanofabrication

Block copolymer-based nanoproducts including particles, fibers and films show promise in high-end applications including nanotemplating, nanolithography, and nanoporous membranes due to their advantageous self-assembled structure. However, few studies probe the nanofabrication process in detail and monitor the concomitant self-assembled structural transitions. Herein, time-resolved small-angle X-ray scattering (SAXS) is exploited to follow such nanostructural changes. Evaporation-induced casting, electrospraying or electrospinning, followed by a final solvent vapor annealing step, have been used to create a series of nanoproducts with reversibly tunable morphologies from a model block copolymer, polystyrene-b-poly(ethylene-co-butylene)-b-polystyrene (SEBS). During evaporation-induced casting, which is considered as an unconfined system, a selective (co)solvent system composed of tetrahydrofuran (THF) and dimethylformamide (DMF) can lead to the formation of various SEBS structures. For DMF-based unconfined systems, the final SEBS lamellar structures were found to be different from the structure formed from THF only, demonstrating the critical role of solvent selection in evaporation-induced casting. The comparison of SEBS electrospun fibers reveals the unique and complex self-assembled structural transition occurring in a confined system. Time-resolved SAXS studies of our model triblock copolymer provide guidelines for a more general approach to access various nanostructures from self-assembling block copolymers.

Thermal-/pH-triggered hollow mesoporous carbon nanocarrier for NIR-responsive drug release

Intelligent drug-delivery systems are considered one of the most important techniques for improving cancer treatment using existing over-the-counter medicines. However, metallic materials are always accompanied by metabolism problems, whereas chemotherapy produces several side effects in humans. Carbon-based materials exhibit exceptional features such as bio-affinity and bio-degradability. Herein, hollow mesoporous carbon nanoparticles (HMCs) are reported as effective nanocarriers of anti-cancer small drug molecules. Near IR (NIR) sources, which can penetrate most organs, induce thermal effects via non-invasive pathways. NIR radiation not only provides thermal therapy but also is compatible with temperature-sensitive coated responsive polymer shells. The template method was used to synthesize HMCs with size 200 ± 50 nm, under various conditions, to obtain suitably sized and hollow structures for liver-cancer treatment. Additional pH/thermal-bi-responsive poly(N-isopropylacrylamide) (PNIPAM) shells were further coated onto the HMCs to produce multiple shells that could trigger swelling motions in PNIPAM@HMCs, as confirmed via small-angle X-ray scattering (SAXS). NIR results demonstrated an extreme increase to the ∆T of 8.7 and 14.2 °C for HMC and PNIPAM@HMCs, respectively. The SAXS spectra analyzed using SasView simulations demonstrated the multi-shell structures of synthesized HMCs and the release mechanism of PNIPAM@HMCs. Based on the model simulation of SAXS, the different rates of polymer swelling indicated the core shrinkage (229.7 to 134.2 Å) and shell expansion (324.3 to 514.3 Å) at 37 °C and 42 °C, respectively. In addition, the first-order, Higuchi, Korsmeyer–Peppas, and Weibull mathematical models were used to verify the drug-release kinetics, and the model with the highest R2 value was considered most suitable for further application. This paper presents the first SAXS study on PNIPAM@HMCs release kinetics and related mechanisms. This phenomenon indicates NIR-induced PNIPAM@HMCs as an effective strategy for cancer treatment via doxorubicin release.

CRYSTAF, DSC and SAXS Study of the Co-Crystallization, Phase Separation and Lamellar Packing of the Blends with Different Polyethylenes.

The crystallization of polyethylene (PE) blends is a highly complex process, owing to the significant differences in crystallizability of the various PE components and the varying PE sequence distributions resulting from short- or long-chain branching. In this study, we examined both the resins and their blends through crystallization analysis fractionation (CRYSTAF) to understand the PE sequence distribution and differential scanning calorimetry (DSC) to investigate the non-isothermal crystallization behavior of the bulk materials. Small-angle X-ray scattering (SAXS) was utilized to study the crystal packing structure. The results showed that the PE molecules in the blends crystallize at different rates during cooling, resulting in a complicated crystallization behavior characterized by nucleation, co-crystallization, and fractionation. We compared these behaviors to those of reference immiscible blends and found that the extent of the differences is related to the disparity in crystallizability between components. Furthermore, the lamellar packing of the blends is closely associated with their crystallization behaviors, and the crystalline structure varies significantly depending on the components' compositions. Specifically, the lamellar packing of the HDPE/LLDPE and HDPE/LDPE blends is similar to that of the HDPE component owing to its strong crystallizability, while the lamellar packing of the LLDPE/LDPE blend is approximately an average of the two neat components.

Crystallinity of side-chain fatty acid containing block copolymers with polyisobutylene segment

Block copolymers of polyisobutylene (PIB) with side-chain fatty acid-containing polymethacrylate segment with varying n-alkyl side-chains were synthesized by the combination of living cationic and reversible addition-fragmentation chain transfer (RAFT) polymerizations. Hydroxyl-terminated PIB (PIB-OH) was converted into macro-chain transfer agent (PIB-CDP macro-CTA) and used during the RAFT polymerization of fatty ester methacrylate (FEMA) monomers to obtain the block copolymers. Block copolymers were characterized by 1H NMR spectroscopy, size exclusion chromatography (SEC), and thermogravimetric analysis (TGA). The block copolymers showed varied crystalline behaviour depending on the chain length of the fatty acids in the polymethacrylate segment, confirmed by differential scanning calorimetry (DSC), small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), polarized optical microscopy (POM), and transmission electron microscopy (TEM). SAXS and POM analysis above the crystalline melting temperature (Tm) of the block copolymers showed crystalline to amorphous phase transformation, which upon cooling below their Tm regenerated crystalline morphology (lamella-like structure). Density functional theory (DFT) calculations were performed to support interlamellar distances determined from the SAXS study. Overall the modification in the orderliness of the lamellar orientation due to the amorphous PIB segment within the fatty acid-containing block copolymer leads to a new direction toward materials with modified physical properties.

Time-Resolved Small-Angle X-ray Scattering Studies during the Aqueous Emulsion Polymerization of Methyl Methacrylate.

Recently, we reported time-resolved synchrotron small-angle X-ray scattering (TR-SAXS) studies during aqueous emulsion polymerization using a bespoke stirrable reaction cell (J. Am. Chem. Soc. 2021, 143, 1474-1484). This proof-of-concept study utilized a semifluorinated specialty monomer (2,2,2-trifluoroethyl methacrylate) to ensure high X-ray contrast relative to water. Herein, we extend this approach to emulsion polymerization of methyl methacrylate (MMA) in the presence or absence of sodium dodecyl sulfate (SDS) at 70 °C. Solution conductivity measurements for this anionic surfactant indicated a critical micelle concentration (CMC) of 10.9 mM at this temperature. Thus, SDS was employed at either 1.0 or 20.0 mM, which corresponds to well below or well above its CMC. Postmortem analysis by 1H NMR spectroscopy indicated MMA conversions of 93-95% for these three formulations. We demonstrate that the X-ray contrast between water and PMMA is sufficiently large to produce high-quality scattering patterns during TR-SAXS experiments. Such patterns were fitted using a hard-sphere scattering model to monitor the evolution in particle diameter. This enabled (i) determination of the time point for the onset of nucleation and (ii) the evolution in particle size to be monitored during the MMA polymerization. The final particle diameters obtained from such TR-SAXS studies were consistent with postmortem DLS analyses, while TEM studies confirmed that near-monodisperse latex particles were formed. Micellar nucleation occurs within just 2 min when the SDS concentration is well above its CMC, resulting in a high particle number concentration and relatively small latex particles. In contrast, when SDS is either absent or present below its CMC, particle nuclei are formed by homogeneous nucleation over significantly longer time scales (14-15 min). In the latter case, adsorption of SDS onto nascent particles reduces their coagulation, giving rise to a larger number of smaller particles compared to the surfactant-free polymerization. However, the characteristic time required for the onset of nucleation is barely affected because this is mainly controlled by the kinetics of homogeneous polymerization of the relatively water-soluble MMA monomer within the aqueous phase. These results suggest that the aqueous emulsion polymerization of several other (meth)acrylic monomers, and perhaps also vinyl acetate, may be amenable to TR-SAXS studies.

In situ SAXS study on the evolution of coal nanopore structures with uniaxial compressive stress

In this study, an in situ synchrotron radiation small-angle X-ray scattering (SAXS) experiment is used to characterize the evolution of coal nanopore structures under uniaxial compression. The variation in the coal nanopores is measured by analyzing the obtained scattering data. From the results, the coal scattering data show a positive Porod deviation in which the deviation slope decreases with increasing stress. Smaller nanopores are more sensitive to uniaxial compressive stress. There is a positive correlation between scattering intensity I and stress at less than 30 nm. The scattering intensity I of pores larger than 30 nm is negatively correlated with uniaxial compressive stress. The structure of smaller pores is more complex. The surface fractal dimension DS and pore fractal dimension DP increases and decreases with increasing uniaxial compressive stress, respectively. The specific surface area is positively correlated with DS. In the measuring range of 3–80 nm, the coal nanopores show a bimodal distribution. At the stage of below 0.6σp, the average diameter decreases by 1.98%, the porosity and specific surface area increases by 6.21% and 31.5%, respectively; At the stage of above 0.6σp, the average diameter decreases by 1.04%, the porosity and specific surface area increases by 1.18% and 5.57%, respectively. These results suggest that the variation of coal nanopores with stress have phased characteristics, and the evolution of the coal nanopores under uniaxial stress can be divided into two stages. In the nanopore fracture stage, the nanopores are deform, fracture and create new smaller pores with increasing stress, and the roughness of pore surface increase. In the nanopore closure stage, high stress intensifies the degree of pore fracture, and nanopores begin to close and disappear. This study reveals the evolution characteristics of the pore structures of coal under uniaxial compression at the nanoscale.

Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity.

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes.

A combined TEM and SAXS study of the growth and self-assembly of ultrathin Pt nanowires

Ultrathin Pt nanowires possess high activity for various electrocatalytic applications. However, little work has focused on understanding their growth mechanisms. Herein, we utilize a combination of time-dependent, ex situ transmission electron microscopy (TEM) and small angle x-ray scattering (SAXS) techniques to observe the growth process in addition to associated surfactant-based interactions. TEM images indicate that initially nanoparticles are formed within 30 s; these small ‘seed’ particles quickly elongate to form ultrathin nanowires after 2 min. These motifs remain relatively unchanged in size and shape up to 480 min of reaction. Complementary SAXS data suggests that the initial nanoparticles, which are coated by a surfactant bilayer, arrange into a bcc superlattice. With increasing reaction time, the bcc lattice disappears as the nanoparticles grow into nanowires, which then self-assemble into a columnar hexagonal structure in which the individual nanowires are covered by a CTAB monolayer. The hexagonal structure eventually degrades, thereby leading to the formation of lamellar stacking phases comprised of surfactant bilayers. To the best of our knowledge, this is the first time that SAXS has been used to monitor the growth and self-assembly of Pt nanowires. These insights can be used to better understand and rationally control the formation of anisotropic motifs of other metallic nanostructures.

(Digital Presentation) Small Angle X-Ray Scattering Studies Investigating the Degradation of Electrocatalysts

In the presentation, the application of small angle X-ray scattering (SAXS) in the investigation of degradation phenomena for electrocatalysts is discussed based on recent examples from my work group. In contrast to local methods such as identical location (IL) microscopy1, SAXS probes a microscopic volume of a catalyst film providing representative data for the catalyst. Furthermore, the method can be coupled to operando measurements or to ex situ measurements in gas diffusion electrode setups2. It is demonstrated that the analysis of SAXS data leads to reliable information of change in mean particle size of the catalysts as function of the applied degradation protocol, however, that a proper measurement and control of a background is essential due to the concomitant degradation of the support material. Keeping this in mind SAXS can play a crucial role for not only elucidating and rationalizing degradation phenomena, but also for the testing of concepts for new electrocatalysts with improved stability. Arenz, M. & Zana, A. Fuel cell catalyst degradation: Identical location electron microscopy and related methods. Nano Energy 29, 299–313 (2016).Schröder, J. et al. A New Approach to Probe the Degradation of Fuel Cell Catalysts under Realistic Conditions: Combining Tests in a Gas Diffusion Electrode Setup with Small Angle X-ray Scattering. J. Electrochem. Soc. 167, 134515 (2020).

Dynamics of Human Serum Transferrin in Varying Physicochemical Conditions Explored by Using Molecular Dynamics Simulations.

Conformational stability of human serum transferrin (Tf) at varying pH values and salt and excipient concentrations were investigated using molecular dynamics (MD) simulations, and the results are compared with previously published small-angle X-ray scattering (SAXS) experiments. SAXS study showed that at pH 5, Tf is predominantly present in a partially open (PO) form, and the factions of PO differ based on the physicochemical condition and drift toward the closed form (HO) as the pH increases. Tf is a bilobal glycoprotein that is composed of homologous halves termed the N- and C-lobes. The current study shows that the protonation of Y188 and K206 at pH 5 is the primary conformational drive into PO, which shifts toward the closed (HO) conformer as the pH increases. Furthermore, at pH 6.5, PO is unfavorable due to negative charge-charge repulsion at the N/C-lobe interface linker region causing increased hinge distance when compared to HO, which has favorable attractive electrostatic interactions in this region. Subsequently, the effect of salt concentration was studied at 70 and 140 mM NaCl. At 70 mM NaCl and pH 5, chloride ions bind strongly in the N-lobe iron-binding site, whereas these interactions are weak at pH 6.5. With increasing salt concentration at pH 5, the regions surrounding the N-lobe iron-binding site are saturated, and as a consequence, sodium and chloride ions accumulate into the bulk. Additionally, protein-excipient interactions were investigated. At pH 5, the excipients interact in similar loop regions, E89-T93, and D416-D420, located in the N- and C-lobes of the HO conformer, respectively. It is anticipated that interactions of additives in these two loop regions cause conformational changes that lead to iron-coordinating residues in the N-lobe to drift away from iron and thus drive HO to PO conversion. Furthermore, at pH 6.5 and 140 mM histidine, these interactions are negligible leading to the stabilization of HO.

Time-Resolved SAXS Study of Polarity- and Surfactant-Controlled Superlattice Transformations of Oleate-Capped Nanocubes During Solvent Removal.

Structural transformations and lattice expansion of oleate-capped iron oxide nanocube superlattices are studied by time-resolved small-angle X-ray scattering (SAXS) during solvent removal. The combination of conductor-like screening model for real solvents (COSMO-RS) theory with computational fluid dynamics (CFD) modeling provides information on the solvent composition and polarity during droplet evaporation. Evaporation-driven poor-solvent enrichment in the presence of free oleic acid results in the formation of superlattices with a tilted face-centered cubic (fcc) structure when the polarity reaches its maximum. The tilted fcc lattice expands subsequently during the removal of the poor solvent and eventually transforms to a regular simple cubic (sc) lattice during the final evaporation stage when only free oleic acid remains. Comparative studies show that both the increase in polarity as the poor solvent is enriched and the presence of a sufficient amount of added oleic acid is required to promote the formation of structurally diverse superlattices with large domain sizes.

Structural and Functional Characterization of PLCβ3

Phospholipase C β (PLCβ) plays an important role in cardiovascular diseases and opioid analgesia. PLCβ catalyzes the hydrolysis of the inner membrane lipid phosphatidylinositol‐4,5‐bisphosphate (PIP2) to inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG are crucial secondary messengers that activate multiple signaling pathways and modulate gene expression to control responses to extracellular signals. PLCβ is a downstream effector of G‐protein coupled receptors (GPCRs) and is activated by both the Gαq and Gβγ subunits. Activation by Gα q requires a unique 400 amino acid C‐terminal region of the lipase, which is subdivided into the proximal and distal C‐terminal domains (CTDs). These domains are required for allosteric activation by Gα q and for membrane binding. Our lab and others have reported that PLCβ is more flexible in solution, as compared to crystal structures of the protein. In small angle X‐ray scattering (SAXS) experiments, the solution structure of PLCβ had additional density that cannot be accounted by the crystal structure. Additionally, crosslinking the PH and EF hand domains decreased this density and the maximum particle diameter. We propose that this additional density corresponds to an open conformation, wherein the PH and EF hand domains are extended from the core of the lipase, and that this conformation of PLCβ represents an autoinhibited state. To test this hypothesis, we mutated residues in PLCβ3 at the interface between the PH and EF hand domains to disrupt their interaction, and then measured changes in PLCβ basal activity and Gβγ‐stimulated activity. These mutations had no significant impact on basal activity, however in many of these mutants, there was a decrease in apparent Gβγ activation. Since these mutations should stabilize an open conformation of PLCβ, this suggests the open conformation has less ability to bind to or be activated by Gβγ. We are currently working to solve the structure of PLCβ3 in solution using single particle cryo‐electron microscopy (cryo‐EM), which will allow us to determine a solution structure of PLCβ to a higher resolution than SAXS studies. We hypothesize that PLCβ3 will exist in the open, autoinhibited conformation, and will also shed light on possible allosteric interactions between the distal CTD and the core.

In situ SAXS study of non-fat milk model systems during heat treatment and acidification

Small-angle X-ray scattering (SAXS) was used to monitor structural changes induced by heat treatment and acid gelation in milk matrices with added whey protein concentrates (WPCs) and nano-particulated whey protein (NWP). In general, heat treatment was found to mainly affect whey protein components while pure casein micelles remained largely unaffected. Conversely, acidification mainly affected caseins while leaving pure whey protein components intact. In mixed systems, the overall behaviour could be understood as a combination of the above effects, however, the type of the added whey protein components influenced the resulting structure formation and dynamics. NWP led to formation of larger structures compared to WPC components during heat treatment, although the latter showed faster aggregation dynamics. During acidification the NWP containing samples exhibited structural changes at slightly higher pH values than the WPC samples. The modeling of pure liquid whey protein (LWPC) samples showed that the heat induced denaturation and resulting aggregation of individual whey proteins is mainly a surface effect leaving the overall protein shape and dimensions unaffected. Schematic diagrams based on the current SAXS data and previous studies were constructed to illustrate the suggested interaction mechanisms between casein and whey proteins during both heating and acidification.

Synergistic Role of Model‐Independent and Model‐Dependent Approaches for Determining Size and Shape of Au Nanostructures Using SAXS

AbstractThe main objective of this study is to showcase the use of synergistic utilization of model‐independent and model‐dependent approaches for analyzing small angle X‐ray scattering (SAXS) data to determine the shape and size of different nanostructures viz. spherical, nanorod, and core‐shell. Herein, different gold nanostructures viz. spherical, nanorod, and core‐shell are synthesized and analyzed using SAXS. First, the SAXS data are analyzed using Guinier law, PDDF (pair–distance distribution function) plots, double logarithmic plots, and electron density curves to get primary information about the size and shape of nanostructures. This information is utilized for choosing an appropriate model and initial input parameters for fitting the SAXS data. The observations obtained from SAXS studies are compared with transmission electron microscopy and UV–vis spectroscopy. It is believed that this study will help in understanding the analysis of SAXS data using a systematic and synergistic approach of utilizing the two methods. It is believed that the study will further add to the understanding of the technique as an alternative approach for transmission electron microscopy.