The NMR structure of the recombinant elk prion protein (ePrP), which represents the cellular isoform (ePrP C ) in the healthy organism, is described here. As anticipated from the highly conserved amino acid sequence, ePrP C has the same global fold as other mammalian prion proteins (PrPs), with a flexibly disordered ''tail'' of residues 23-124 and a globular domain 125-226 with three ␣-helices and a short antiparallel -sheet. However, ePrP C shows a striking local structure variation when compared with most other mammalian PrPs, in particular human, bovine, and mouse PrP C . A loop of residues 166 -175, which links the -sheet with the ␣2-helix and is part of a hypothetical ''protein X'' epitope, is outstandingly well defined, whereas this loop is disordered in the other species. Based on NMR structure determinations of two mouse PrP variants, mPrP[N174T] and mPrP[S170N,N174T], this study shows that the structured loop in ePrP C relates to these two local amino acid exchanges, so that mPrP[S170N,N174T] exactly mimics ePrP C . These results are evaluated in the context of recent reports on chronic wasting disease (CWD) in captive and free-ranging deer and elk in the U.S. and Canada, and an animal model is proposed for support of future research on CWD.transmissible spongiform encephalopathy ͉ chronic wasting disease C hronic wasting disease (CWD) is a neurological disorder in cervids that has been shown to be a transmissible spongiform encephalopathy (TSE) or ''prion disease''; other prion diseases include scrapie in sheep, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease in humans (1-3). CWD was diagnosed in 1978 as a TSE in a captive mule deer (Odocoileus hemionus) (4, 5) and was diagnosed in 1981 in a free-ranging elk (Cervus elaphus nelsoni) (6). Today, CWD is known to affect captive and free-ranging elk, mule deer, and white-tailed deer (O. virginianus), and the disease seems to be endemic in areas of the western United States and Canada.CWD is unique among TSEs by the fact that is has been studied in free-ranging species (7). The natural route of exposure appears to be oral, possibly through direct interaction between animals or through environmental contamination (8, 9). Although there is evidence for transmission to different mammalian species by intracerebral inoculation (7, 10, 11), domestic animals such as cattle, sheep, and goats are not known to be naturally susceptible to CWD (12). Compared with bovine spongiform encephalopathy (BSE), CWD transmission to cattle, goats, and laboratory animals has been reported to be inefficient, suggesting that there is a rather stringent species barrier (13-15). Cell-free conversion experiments (15) led to the prediction that TSE transmission efficiency from cervids to humans might be similar to that from cattle to humans, which is clearly of serious concern. Overall, the potential threat to livestock and the human population from the recent spread of CWD in free-ranging cervids in the U.S. and Canada is still difficult to assess, and further r...
Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds2,3. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters (‘Candidatus Eudoremicrobiaceae’) that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments.
Fluorine chemistry has taken a pivotal role in chemical reaction discovery, drug development, and chemical biology. NMR spectroscopy, arguably the most important technique for the characterization of fluorinated compounds, is rife with highly inconsistent referencing of fluorine NMR chemical shifts, producing deviations larger than 1 ppm. Herein, we provide unprecedented evidence that both spectrometer design and the current unified scale system underpinning the calibration of heteronuclear NMR spectra have unintentionally led to widespread variation in the standardization of F NMR spectral data. We demonstrate that internal referencing provides the most robust, practical, and reproducible method whereby chemical shifts can be consistently measured and confirmed between institutions to less than 30 ppb deviation. Finally, we provide a comprehensive table of appropriately calibrated chemical shifts of reference compounds that will serve to calibrate F spectra correctly.
Fluorine NMR spectroscopy is widely used for detection of protein-ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug molecule to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiologically high protein concentrations and large ligand:protein ratios. An enhancement in the (19)F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concentrations of fluorinated small molecules. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR analysis, dissociation constants are determined. However, the ability to use a low ligand concentration permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and additional information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery.
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