Simulated annealing-omit Fo-<i>D</i>Fc electron density contoured at 2 σ of PylRS in complex with (A) Kalk (orange), (B) Kbu (green), (C) Kcr (blue) and (D) Kpr (red).

<p>The protein is shown as cartoon, overlaid with its semi-transparent surface representation. Amino acids providing key interactions are drawn as sticks, hydrogen-bonds as dashed lines.</p

Comparison of the binding pockets of PylRS and LysRS.

<p>Two-dimensional plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096198#pone.0096198-Wallace1" target="_blank">[52]</a> of residues interacting with Lys (A), Pyl (B) and Kalk (C). Van-der-Waals contacts are shown as red half-spheres. Surface representation of the binding pockets of LysRS (D) and PylRS (E) with Lys, Pyl and Kalk, respectively. (F) Superposition of Pyl (grey), Kalk (orange), Kbu (green), Kcr (blue) and Kpr (red). The surface of the binding pocket is shown as mesh, with the charge distribution indicated by coloring (red  =  negative, blue  =  positive). The surrounding residues are drawn as sticks. (PDB codes PylRS: 2Q7H and LysRS: 3A74).</p

Data collection, processing and structure refinement statistics.

<p>Numbers in parentheses correspond to the high resolution shell.</p

Lysine, pyrrolysine and analogs used in this study.

<p>Lysine, pyrrolysine and analogs used in this study.</p

Cartoon representation of the overall structure of the catalytic domain of PylRS.

<p>(A) Type-II tRNA-synthetase folding topology of the tRNA synthetase domain from PylRS, shown as cartoon representation, overlaid with its semi-transparent surface. The adenylated Kbu is highlighted as green stick model in the active site. (B-F) Zoom in the active site: PylRS in complex with Kalk (orange), Kbu (green), Kcr (blue) and Kpr (red), drawn as stick models. (r. m. s. d ∼0.35 Å). All four non-natural amino acids bind in the same position.</p

A Subfamily of Bacterial Ribokinases Utilizes a Hemithioacetal for Pyridoxal Phosphate Salvage

Pyridoxal 5′-phosphate (PLP) is the active vitamer of vitamin B₆ and acts as an essential cofactor in many aspects of amino acid and sugar metabolism. The virulence and survival of pathogenic bacteria such as Mycobacterium tuberculosis depend on PLP, and deficiencies in humans have also been associated with neurological disorders and inflammation. While PLP can be synthesized by a de novo pathway in bacteria and plants, most higher organisms rely on a salvage pathway that phosphorylates either pyridoxal (PL) or its related vitamers, pyridoxine (PN) and pyridoxamine (PM). PL kinases (PLKs) are essential for this phosphorylation step and are thus of major importance for cellular viability. We recently identified a pyridoxal kinase (SaPLK) as a target of the natural product antibiotic rugulactone (Ru) in Staphylococcus aureus. Surprisingly, Ru selectively modified SaPLK not at the active site cysteine, but on a remote cysteine residue. Based on structural and biochemical studies, we now provide insight into an unprecedented dual Cys charge relay network that is mandatory for PL phosphorylation. The key component is the reactive Cys 110 residue in the lid region that forms a hemithioactetal intermediate with the 4′-aldehyde of PL. This hemithioacetal, in concert with the catalytic Cys 214, increases the nucleophilicity of the PL 5′-OH group for the inline displacement reaction with the γ-phosphate of ATP. A closer inspection of related enzymes reveals that Cys 110 is conserved and thus serves as a characteristic mechanistic feature for a dual-function ribokinase subfamily herein termed CC-PLKs

Unveiling the Dynamic Self-Assembly of a Recombinant Dragline-Silk-Mimicking Protein

Despite the considerable interest in the recombinant production of synthetic spider silk fibers that possess mechanical properties similar to those of native spider silks, such as the cost-effectiveness, tunability, and scalability realization, is still lacking. To address this long-standing challenge, we have constructed an artificial spider silk gene using Golden Gate assembly for the recombinant bacterial production of dragline-mimicking silk, incorporating all the essential components: the N-terminal domain, a 33-residue-long major-ampullate-spidroin-inspired segment repeated 16 times, and the C-terminal domain (N16C). This designed silk-like protein was successfully expressed in Escherichia coli, purified, and cast into films from formic acid. We produced uniformly 13C–15N-labeled N16C films and employed solid-state magic-angle spinning nuclear magnetic resonance (NMR) for characterization. Thus, we could demonstrate that our bioengineered silk-like protein self-assembles into a film where, when hydrated, the solvent-exposed layer of the rigid, β-nanocrystalline polyalanine core undergoes a transition to an α-helical structure, gaining mobility to the extent that it fully dissolves in water and transforms into a highly dynamic random coil. This hydration-induced behavior induces chain dynamics in the glycine-rich amorphous soft segments on the microsecond time scale, contributing to the elasticity of the solid material. Our findings not only reveal the presence of structurally and dynamically distinct segments within the film’s superstructure but also highlight the complexity of the self-organization responsible for the exceptional mechanical properties observed in proteins that mimic dragline silk

Additional file 1 of SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency

Additional file 1: Supplemental Figure 1. Screening of healthy donors 1-93 for WT allele. Supplemental Figure 2. Screening of healthy donors 1-93 for mutant allele. Supplemental Figure 3. Screening of healthy donors 94-186 for WT allele. Supplemental Figure 4. Screening of healthy donors 94-186 for mutant allele. Supplemental Figure 5. Screening of healthy donors 187-279 for WT allele. Supplemental Figure 6. Screening of healthy donors 187-279 for mutant allele. Supplemental Figure 7. Screening of healthy donors 280-360 for WT allele. Supplemental Figure 8. Screening of healthy donors 280-360 for mutant allele. Supplemental Figure 9. Repetition of PCRs with no or positive results in first PCR

Data_Sheet_1_Protoplast-Esculin Assay as a New Method to Assay Plant Sucrose Transporters: Characterization of AtSUC6 and AtSUC7 Sucrose Uptake Activity in Arabidopsis Col-0 Ecotype.pdf

<p>The best characterized function of sucrose transporters of the SUC family in plants is the uptake of sucrose into the phloem for long-distance transport of photoassimilates. This important step is usually performed by one specific SUC in every species. However, plants possess small families of several different SUCs which are less well understood. Here, we report on the characterization of AtSUC6 and AtSUC7, two members of the SUC family in Arabidopsis thaliana. Heterologous expression in yeast (Saccharomyces cerevisiae) revealed that AtSUC6_Col-0 is a high-affinity H⁺-symporter that mediates the uptake of sucrose and maltose across the plasma membrane at exceptionally low pH values. Reporter gene analyses revealed a strong expression of AtSUC6_Col-0 in reproductive tissues, where the protein product might contribute to sugar uptake into pollen tubes and synergid cells. A knockout of AtSUC6 did not interfere with vegetative development or reproduction, which points toward physiological redundancy of AtSUC6_Col-0 with other sugar transporters. Reporter gene analyses showed that AtSUC7_Col-0 is expressed in roots and pollen tubes and that this sink specific expression of AtSUC7_Col-0 is regulated by intragenic regions. Transport activity of AtSUC7_Col-0 could not be analyzed in baker’s yeast or Xenopus oocytes because the protein was not correctly targeted to the plasma membrane in both heterologous expression systems. Therefore, a novel approach to analyze sucrose transporters in planta was developed. Plasma membrane localized SUCs including AtSUC6_Col-0 and also sucrose specific SWEETs were able to mediate transport of the fluorescent sucrose analog esculin in transformed mesophyll protoplasts. In contrast, AtSUC7_Col-0 is not able to mediate esculin transport across the plasma membrane which implicates that AtSUC7_Col-0 might be a non-functional pseudogene. The novel protoplast assay provides a useful tool for the quick and quantitative analysis of sucrose transporters in an in planta expression system.</p

Summary of the kinematic parameter for the patients' group (PG) and control group (CG).

<p>Bars represent the mean of frequency (FREQ), number of inversions of velocity (NIV) and the average maximum velocity (Max Vel) for finger tapping (FT), hand tapping (HT) and forearm pronation-supination (PS). Error bars represent the standard deviation of the mean (SEM). Significant differences after training in the MG are indicated (* p<.05, ** p<.01).</p