Preparation and characterization of inactivated tick-borne encephalitis virus samples for single-particle imaging at the European XFEL.

X-ray imaging of virus particles at the European XFEL could eventually allow their complete structures to be solved, potentially approaching the resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, about 1 ml of purified virus suspension containing at least 10 particles per millilitre is required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses.

The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces.

Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains.

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase.

X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion.

Hydrophilization of Magnetic Nanoparticles with Modified Alternating Copolymers. Part 2: Behavior in solution.

Aqueous solutions of iron oxide nanoparticles (NPs) stabilized by poly(maleic acid-alt-1-octadecene) (PMAcOD) modified with the 5,000 Da poly(ethylene glycol) (PEG) or the short ethylene glycol (EG) tails were analyzed by small-angle X-ray scattering (SAXS). Advanced SAXS data analysis methods were employed to systematically characterize the structure and interactions between the NPs. Depending on the type of the grafted tail and the grafting density all NPs can be separated into three groups.

Hydrophilization of Magnetic Nanoparticles with Modified Alternating Copolymers. Part 1: The Influence of the Grafting.

Iron oxide nanoparticles (NPs) with a diameter 21.6 nm were coated with poly(maleic acid-alt-1-octadecene) (PMAcOD) modified with grafted 5,000 Da poly(ethyelene glycol) (PEG) or short ethylene glycol (EG) tails. The coating procedure utilizes hydrophobic interactions of octadecene and oleic acid tails, while the hydrolysis of maleic anhydride moieties as well as the presence of hydrophilic PEG (EG) tails allows the NP hydrophilicity.

Human serum albumin binding ibuprofen: a 3D description of the unfolding pathway in urea.

Small angle X-ray scattering (SAXS) technique, supported by light scattering measurements and spectroscopic data (circular dichroism and fluorescence) allowed us to restore the 3D structure at low resolution of defatted human serum albumin (HSA) in interaction with ibuprofen. The data were carried out on a set of HSA solutions with urea concentrations between 0.00 and 9.

Urea-induced denaturation process on defatted human serum albumin and in the presence of palmitic acid.

We report a study on the unfolding behavior of the most abundant protein contained in plasma, human serum albumin. The unfolding mechanisms in denaturing conditions induced by urea are studied for the defatted form (HSA) and for the palmitic acid:albumin (HSAPalm) complex. We employed the singular value decomposition method to determine the minimum number of structural states present in the unfolding processes.

Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin.

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton.

Hydrophilic Monodisperse Magnetic Nanoparticles Protected by an Amphiphilic Alternating Copolymer.

Iron oxide nanoparticles (NPs) with diameters of 16.1, 20.5, and 20.

Kinetic and mechanistic characterization of Mycobacterium tuberculosis glutamyl-tRNA synthetase and determination of its oligomeric structure in solution.

Mycobacterium tuberculosis glutamyl-tRNA synthetase (Mt-GluRS), encoded by Rv2992c, was overproduced in Escherichia coli cells, and purified to homogeneity. It was found to be similar to the other well-characterized GluRS, especially the E. coli enzyme, with respect to the requirement for bound tRNA(Glu) to produce the glutamyl-AMP intermediate, and the steady-state kinetic parameters k(cat) (130 min(-1)) and K(M) for tRNA (0.

Intrinsic structural disorder of mouse proNGF.

The unprocessed precursor of the Nerve Growth Factor (NGF), proNGF, has additional functions, besides its initially described role as a chaperone for NGF folding. The precursor protein endows apoptotic and/or neurotrophic properties, in contrast to the mature part. The structural and molecular basis for such distinct activities are presently unknown.

Structural basis for sugar recognition, including the Tn carcinoma antigen, by the lectin SNA-II from Sambucus nigra.

Bark of elderberry (Sambucus nigra) contains a galactose (Gal)/N-acetylgalactosamine (GalNAc)-specific lectin (SNA-II) corresponding to slightly truncated B-chains of a genuine Type-II ribosome-inactivating protein (Type-II RIPs, SNA-V), found in the same species. The three-dimensional X-ray structure of SNA-II has been determined in two distinct crystal forms, hexagonal and tetragonal, at 1.90 A and 1.

Communication between the nucleotide site and the main molecular hinge of 3-phosphoglycerate kinase.

3-Phosphoglycerate kinase is a hinge-bending enzyme with substrate-assisted domain closure. However, the closure mechanism has not been described in terms of structural details. Here we present experimental evidence of the participation of individual substrate binding side chains in the operation of the main hinge which is distant from the substrate binding sites.

Dynamics in a pure encounter complex of two proteins studied by solution scattering and paramagnetic NMR spectroscopy.

In the general view of protein-complex formation, a transient and dynamic encounter complex proceeds to form a more stable, well-defined, and active form. In weak protein complexes, however, the encounter state can represent a significant population of the complex. The redox proteins adrenodoxin (Adx) and cytochrome c (C c) associate to form such a weak and short-lived complex, which is nevertheless active in electron transfer.

Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding.

Heterotrimeric AMP-activated protein kinase (AMPK) is crucial for energy homeostasis of eukaryotic cells and organisms. Here we report on (i) bacterial expression of untagged mammalian AMPK isoform combinations, all containing gamma(1), (ii) an automated four-dimensional purification protocol, and (iii) biophysical characterization of AMPK heterotrimers by small angle x-ray scattering in solution (SAXS), transmission and scanning transmission electron microscopy (TEM, STEM), and mass spectrometry (MS). AMPK in solution at low concentrations (~1 mg/ml) largely consisted of individual heterotrimers in TEM analysis, revealed a precise 1:1:1 stoichiometry of the three subunits in MS, and behaved as an ideal solution in SAXS.

Role of side-chains in the operation of the main molecular hinge of 3-phosphoglycerate kinase.

The single mutants (F165A, E192A, F196A, S392A, T393A) at and near the main hinge (beta-strand L) of human 3-phosphoglycerate kinase (hPGK) exhibit variously reduced enzyme activity, indicating the cumulative effects of these residues in regulating domain movements. The residues F165 and E192 are also essential in maintaining the conformational integrity of the whole molecule, including the hinge-region. Shortening of betaL by deleting T393 has led to a dramatic activity loss and the concomitant absence of domain closure (as detected by small angle X-ray scattering), demonstrating the role of betaL in functioning of hPGK.

Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP.

l-Nucleoside-analogues, mirror images of the natural d-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound l-MgADP with hPGK.

Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein.

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR).

Fine-tuning of intrinsic N-Oct-3 POU domain allostery by regulatory DNA targets.

The 'POU' (acronym of Pit-1, Oct-1, Unc-86) family of transcription factors share a common DNA-binding domain of approximately 160 residues, comprising so-called 'POUs' and 'POUh' sub-domains connected by a flexible linker. The importance of POU proteins as developmental regulators and tumor-promoting agents is due to linker flexibility, which allows them to adapt to a considerable variety of DNA targets. However, because of this flexibility, it has not been possible to determine the Oct-1/Pit-1 linker structure in crystallographic POU/DNA complexes.

Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23-24 dimer.

Filamin C is a dimeric, actin-binding protein involved in organization of cortical cytoskeleton and of the sarcomere. We performed crystallographic, small-angle X-ray scattering and analytical ultracentrifugation experiments on the constructs containing carboxy-terminal domains of the protein (domains 23-24 and 19-21). The crystal structure of domain 23 of filamin C showed that the protein adopts the expected immunoglobulin (Ig)-like fold.

Release factors 2 from Escherichia coli and Thermus thermophilus: structural, spectroscopic and microcalorimetric studies.

Prokaryotic class I release factors (RFs) respond to mRNA stop codons and terminate protein synthesis. They interact with the ribosomal decoding site and the peptidyl-transferase centre bridging these 75 A distant ribosomal centres. For this an elongated RF conformation, with partially unfolded core domains II.

Structural characterization of beta-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro.

The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPSc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90-231). A novel in vitro conversion assay based on aerobic incubation of PrP in the presence of elemental copper pellets at pH 5 was established, resulting in aggregation of highly beta-sheeted prion proteins.

Substrate-induced double sided H-bond network as a means of domain closure in 3-phosphoglycerate kinase.

Closure of the two domains of 3-phosphoglycerate kinase, upon substrate binding, is essential for the enzyme function. The available crystal structures cannot provide sufficient information about the mechanism of substrate assisted domain closure and about the requirement of only one or both substrates, since lattice forces may hinder the large scale domain movements. In this study the known X-ray data, obtained for the open and closed conformations, were probed by solution small-angle X-ray scattering experiments.

The C-terminal domain of the transcriptional corepressor CtBP is intrinsically unstructured.

C-terminal binding proteins (CtBPs) are moonlighting proteins involved in nuclear transcriptional corepression and in Golgi membrane tubule fission. Structural information on CtBPs is available for their substrate-binding domain, responsible for transcriptional repressor recognition/binding, and for the nucleotide-binding domain, involved in NAD(H)-binding and dimerization. On the contrary, little is known about the structure of CtBP C-terminal region ( approximately 90 residues), hosting sites for post-translational modifications.