Size and structure of hexanuclear plutonium oxo-hydroxo clusters in aqueous solution from synchrotron analysis

The size and shape of a water-soluble hexanuclear plutonium cluster were probed by combining synchrotron small-angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS). A specific setup coupling both techniques and dedicated to radioactive samples on the MARS beamline endstation at Synchrotron SOLEIL is described. The plutonium hexanuclear cores are well stabilized by the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ligands and this allows a good evaluation of the setup to probe the very small plutonium core. The results show that, in spite of the constrained conditions required to avoid any risk of sample dispersion, the flux and the sample environment are optimized to obtain a very good signal-to-noise ratio, allowing the detection of small plutonium aggregates in an aqueous phase. The structure of the well defined hexanuclear cluster has been confirmed by EXAFS measurements in solution and correlated with SAXS data processing and modelling. An iterative comparison of classical fit models (Guinier or sphere form factor) with the experimental results allowed a better interpretation of the SAXS signal that will be relevant for future work under environmentally relevant conditions.

High-affinity nanobodies as tools for structural and functional studies on mammalian Arc

Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro. This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo.

Eruca sativa seed napin structural insights and thorough functional characterization

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC–MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

Printable Alginate Hydrogels with Embedded Network of Halloysite Nanotubes: Effect of Polymer Cross-Linking on Rheological Properties and Microstructure

Rapidly growing 3D printing of hydrogels requires network materials which combine enhanced mechanical properties and printability. One of the most promising approaches to strengthen the hydrogels consists of the incorporation of inorganic fillers. In this paper, the rheological properties important for 3D printability were studied for nanocomposite hydrogels based on a rigid network of percolating halloysite nanotubes embedded in a soft alginate network cross-linked by calcium ions. Particular attention was paid to the effect of polymer cross-linking on these properties. It was revealed that the system possessed a pronounced shear-thinning behavior accompanied by a viscosity drop of 4–5 orders of magnitude. The polymer cross-links enhanced the shear-thinning properties and accelerated the viscosity recovery at rest so that the system could regain 96% of viscosity in only 18 s. Increasing the cross-linking of the soft network also enhanced the storage modulus of the nanocomposite system by up to 2 kPa. Through SAXS data, it was shown that at cross-linking, the junction zones consisting of fragments of two laterally aligned polymer chains were formed, which should have provided additional strength to the hydrogel. At the same time, the cross-linking of the soft network only slightly affected the yield stress, which seemed to be mainly determined by the rigid percolation network of nanotubes and reached 327 Pa. These properties make the alginate/halloysite hydrogels very promising for 3D printing, in particular, for biomedical purposes taking into account the natural origin, low toxicity, and good biocompatibility of both components.

Interpreting SAXS data recorded on cellulose rich pulps

A simulation method was developed for modelling SAXS data recorded on cellulose rich pulps. The modelling method is independent of the establishment of separate form factors and structure factors and was used to model SAXS data recorded on dense samples. An advantage of the modelling method is that it made it possible to connect experimental SAXS data to apparent average sizes of particles and cavities at different sample solid contents. Experimental SAXS data could be modelled as a superposition of a limited number of simulated intensity components and gave results in qualitative agreement with CP/MAS 13C-NMR data recorded on the same samples. For the water swollen samples, results obtained by the SAXS modelling method and results obtained from CP/MAS 13C-NMR measurements, agreed on the ranking of particle sizes in the different samples. The SAXS modelling method is dependent on simulations of autocorrelation functions and the time needed for simulations could be reduced by rescaling of simulated correlation functions due to their independence of the choice of step size in real space. In this way an autocorrelation function simulated for a specific sample could be used to generate SAXS intensity profiles corresponding to all length scales for that sample and used for efficient modelling of the experimental data recorded on that sample. Graphical abstract

Small-angle X-ray (SAXS) and Raman spectroscopy studies of biot-CMG(2)-DOPE quasicrystalline phases

Neoglycolipids due to their amphiphilic properties exhibit self-assembly in aqueous phases. In high concentrations the liquid crystalline or gel phases may form. So-called soft-material are a subject of interest of many scientists especially as biosensors and wound healing materials. In this study we examine the structure of a quasicrystalline phase of biot-CMG(2)-DOPE obtained at the concentration of 150 mg/ml (13wt.%) in PBS. The structural data such as interplanar spacing, order parameter and long-range order were obtained by SAXS, while the changes in chemical structure were studied by Raman spectroscopy. It was also in our interest to examine a correlation between the ionic strength and the self-assembly, so we also studied a similar quasicrystalline phase of the same compound but in a buffer containing CaCl2 at the concentration of 4wt.%. According to SAXS data, FSL-biotin construct formed a complex ordered phase consisting of overlapping latices of different kind. The addition of CaCl2 into PBS resulted in obtaining a more structured system demonstrating cubic-like crystal lattice. Change in peak intensities on Raman spectrums of -C-H- and -C-C- bonds vibrations explained the change in phase properties.

Improving the global dimensions of intrinsically disordered proteins in Martini 3

Coarse-grained molecular dynamics simulations are a useful tool to determine conformational ensembles of intrinsically disordered proteins (IDPs). Here, we show that the coarse-grained force field Martini 3 underestimates the global dimensions of IDPs when compared with small angle X-ray scattering (SAXS) data. Increasing the strength of protein-water interactions favors more expanded conformations, improving agreement with SAXS data and alleviating problems with overestimated IDP-IDP interactions.

Structural and functional characterization of Rv0792c from Mycobacterium tuberculosis: identifying small molecule inhibitors against GntR protein

ABSTRACTIn order to adapt in host tissues, microbial pathogens regulate their gene expression through an array of transcription factors. Here, we have functionally characterized Rv0792c, a GntR homolog from M. tuberculosis. In comparison to the parental strain, ΔRv0792c mutant strain of M. tuberculosis was compromised for survival upon exposure to oxidative stress, cell wall agents and infection in guinea pigs. RNA-seq analysis revealed that Rv0792c regulates the expression of genes that are involved in stress adaptation and virulence of M. tuberculosis. Solution small angle X-ray scattering (SAXS) data steered model building confirmed that the C-terminal region plays a pivotal role in dimer formation. Systematic evolution of ligands by exponential enrichment resulted in identification of ssDNA aptamers that can be used as a tool to identify small molecule inhibitors targeting Rv0792c. Using SELEX and SAXS data based modelling, we identified residues essential for the DNA binding activity of Rv0792c and I-OMe-Tyrphostin as an inhibitor of Rv0792c aptamer binding activity. Taken together, we provide a detailed shape-function characterization of GntR family of transcription factors from M. tuberculosis. To the best of our knowledge, this is the first study that has resulted in the identification of small molecule inhibitors against GntR family of transcription factors from bacterial pathogens.

Tandem domain structure determination based on a systematic enumeration of conformations

Protein structure determination is undergoing a change of perspective due to the larger importance taken in biology by the disordered regions of biomolecules. In such cases, the convergence criterion is more difficult to set up and the size of the conformational space is a obstacle to exhaustive exploration. A pipeline is proposed here to exhaustively sample protein conformations using backbone angle limits obtained by nuclear magnetic resonance (NMR), and then to determine the populations of conformations. The pipeline is applied to a tandem domain of the protein whirlin. An original approach, derived from a reformulation of the Distance Geometry Problem is used to enumerate the conformations of the linker connecting the two domains. Specifically designed procedure then permit to assemble the domains to the linker conformations and to optimize the tandem domain conformations with respect to two sets of NMR measurements: residual dipolar couplings and paramagnetic resonance enhancements. The relative populations of optimized conformations are finally determined by fitting small angle X-ray scattering (SAXS) data. The most populated conformation of the tandem domain is a semi-closed one, fully closed and more extended conformations being in minority, in agreement with previous observations. The SAXS and NMR data show different influences on the determination of populations.