During storage in the silk gland, the n-terminal domain (nt) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that nts from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer's disease. A designed variant of nt from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.Orb-weaving spiders manufacture up to seven different silks, e.g. dragline silk derived from major ampullate silk proteins (spidroins, MaSp) and flagelliform silk derived from flagelliform spidroins (FlSp). The various spidroins share a common architecture -a large core repetitive region capped by globular N-and C-terminal domains (NT and CT) 1 . The divergent and large aggregation-prone repetitive regions of the spidroins determine the mechanical properties of the respective spider silks, while the terminal domains regulate silk fiber formation 2,3 . Despite their high aggregation propensity the spidroins can be stored at extremely high concentrations (30-50% w/v) in the spider silk gland, solubilized by the NT domain 1,4 .The NT dimerizes upon a drop in pH, which is crucial for silk fiber formation 1,5 . To ensure solubility also at low pH and widen the applicability of NT as a solubility enhancing fusion partner, a charged-reversed mutant has been designed (referred to as NT* MaSp ) 6 . The previously reported NT* MaSp tag is derived from the NT domain of Euprosthenops australis MaSp1 and folds as a five-helix bundle 6,7 . NT* MaSp is a pH insensitive constitutive monomer, highly stable and extremely soluble, and has been successfully applied for efficient production and purification of, among others, lung surfactant protein analogs, cholecystokinin-58, human antimicrobial cathelicidin and a designed β-sheet protein 6,8 .Aggregation-prone proteins and peptides are associated with several neurodegenerative disorders, e.g. Alzheimer's disease (AD), the most prevalent form of dementia 9,10 . These proteins/peptides often exhibit high β-sheet propensity, which make them prone to aggregate and form insoluble amyloid fibrils 11 . These intrinsic properties of amyloid-forming proteins make high-yield biochemical production challenging, yet the availability of pure protein samples is crucial for studying protein self-assembly and its associated neurotoxicity in vitro and in vivo. This is probably one important reason behind the fact that, despite immense efforts, the exact mechanisms of Aβ self-assembly are still unknown 9-11 . Recent advances have howe...
Spherical mesoporous carbon (with a particle size in the range of 40–75 μm) was synthesized by nanoreplication of a hard silica template using sucrose as the carbon precursor. The mesoporous carbon with BET surface areas higher than 1200 m2/g was doped with N by a treatment in an aqueous solution of nitric acid and/or in a flow of gaseous ammonia. The highest N content (3.2 wt % of N in bulk) was obtained when both modification methods were combined. Complementary physicochemical characterization techniques, including scanning electron microscopy (SEM), low-temperature N2 adsorption, powder X-ray diffraction (XRD), and Raman spectroscopy revealed the morphology, structure, and textural properties of the synthesized N-loaded carbon materials. For the identification of the detailed chemical structure on the surface of the carbons, 1H, 13C, and 15N solid-state nuclear magnetic resonance (NMR) measurements were performed, and the data were supported by chemical shift calculations with accurate quantum chemistry methods and X-ray photoelectron spectroscopic (XPS) analyses. All NMR experiments were performed at natural isotope abundance. The verified experimental data clearly showed that after the introduction of the N-containing moieties by the combined methods of treatment, a high concentration of pyridinic N at the edge, and pyrrolic N being external to the edge, was achieved for the mesoporous carbon. The distributed N surface species promoted the catalytic activity in the oxidative dehydrogenation of ethylbenzene to styrene but did not significantly influence the efficiency of the carbon materials in the electrochemical reduction of nitrate ions.
Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide β17. The fusion protein NT*-β17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that β17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, β17 adopts a β-sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.
-Leucinyl benzenesulfonamides have been discovered as a novel class of potent inhibitors of leucyl-tRNA synthetase. The binding of inhibitors to the enzyme was measured by using isothermal titration calorimetry. This provided information on enthalpy and entropy contributions to binding, which, together with docking studies, were used for structure-activity relationship analysis. Enzymatic assays revealed that-leucinyl benzenesulfonamides display remarkable selectivity for leucyl-tRNA synthetase compared to and human orthologues. The simplest analogue of the series, -leucinyl benzenesulfonamide (R = H), showed the highest affinity against leucyl-tRNA synthetase and also exhibited antibacterial activity against Gram-negative pathogens (the best MIC = 8 μg/mL, ATCC 25922), which renders it as a promising template for antibacterial drug discovery.
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