Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC‐Derived Tensorial Constraints

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility

Production of isotope-labeled proteins in insect cells for NMR

Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host

Hyperpolarized Water to Study Protein-Ligand Interactions

The affinity between a chosen target protein and small molecules is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solutions of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concentrations below 20 M so that one can verify in a few seconds whether the proteins remain intact or have been denatured

Biosynthetic potential of the global ocean microbiome

8 pages, 4 figures, supplementary information https://doi.org/10.1038/s41586-022-04862-3.-- This Article is contribution number 130 of Tara OceansNatural 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 environmentsThis work was supported by funding from the ETH and the Helmut Horten Foundation; the Swiss National Science Foundation (SNSF) through project grants 205321_184955 to S.S., 205320_185077 to J.P. and the NCCR Microbiomes (51NF40_180575) to S.S.; by the Gordon and Betty Moore Foundation (https://doi.org/10.37807/GBMF9204) and the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 101000392 (MARBLES) to J.P.; by an ETH research grant ETH-21 18-2 to J.P.; and by the Peter and Traudl Engelhorn Foundation and by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 897571 to C.C.F. S.L.R. was supported by an ETH Zurich postdoctoral fellowship 20-1 FEL-07. M.L., L.M.C. and G.Z. were supported by EMBL Core Funding and the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft, project no. 395357507, SFB 1371 to G.Z.). M.B.S. was supported by the NSF grant OCE#1829831. C.B. was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement Diatomic, no. 835067). S.G.A. was supported by the Spanish Ministry of Economy and Competitiveness (PID2020-116489RB-I00). M.K. and H.M. were funded by the SNSF grant 407540_167331 as part of the Swiss National Research Programme 75 ‘Big Data’. M.K., H.M. and A.K. are also partially funded by ETH core funding (to G. Rätsch)With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe

Relaxation optimized double acquisition (RODA) as an alternative for virtual decoupling of NMR spectra

We introduce an alternative way for spin-state selection, RODA, which yields higher sensitivity for spin systems exhibiting a TROSY effect. With RODA, the TROSY component of a doublet is recorded twice using a double acquisition scheme. RODA works by simple addition of consecutive NMR signals, and does not require any special processing. Thus, this pulse sequence element can seamlessly be integrated into existing experiments. We demonstrate the broad applicability of RODA with several systems exhibiting a TROSY effect on 15N–1H, 19F–13C or 1H–13C moieties. Further, we show that virtual decoupling with increased sensitivity is possible in a single double acquisition experiment in situations as encountered with dissolution DNP.ISSN:1090-780

Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling

Current biological research increasingly focusses on large human proteins and their complexes. Such proteins are difficult to study by NMR spectroscopy because they often can only be produced in higher eukaryotic expression systems, where deuteration is hardly feasible. Here, we present the XL-ALSOFAST-[C-13,H-1]-HMQC experiment with much improved sensitivity for fully protonated high molecular weight proteins. For the tested systems ranging from 100 to 240 kDa in size, 3-fold higher sensitivity was obtained on average for fast relaxing signals compared to current state-of-the-art experiments. In the XL-ALSOFAST approach, non-observed magnetisation is optimally exploited and transverse relaxation is minimized by the newly introduced concept of delayed decoupling. The combination of high sensitivity and superior artefact suppression makes it ideal for studying inherently unstable membrane proteins or for analysing therapeutic antibodies at natural(13)C abundance. The XL-ALSOFAST and delayed decoupling will therefore expand the range of biomolecular systems accessible to NMR spectroscopy.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083

Automated NMR resonance assignment of large proteins for protein-ligand interaction studies

The detection and structural characterization of protein-ligand interactions by solution NMR is central to functional biology research as well as to drug discovery. Here we present a robust and highly automated procedure for obtaining the resonance assignments necessary for studies of such interactions. The procedure relies on a combination of three automated projection spectroscopy (APSY) experiments, including the new 4D APSY-HNCACB, and the use of fractionally deuterated protein samples. This labeling pattern increases the experimental sensitivity on the one hand, but it leads to peak multiplets on the other hand. The latter complications are however overcome by the geometric APSY analysis of the projection spectra. The three APSY experiments thus provide high precision chemical shift correlations of the backbone and side chain methyl groups, allowing a reliable and robust assignment of the protein by suitable algorithms. The present approach doubles the molecular size limit of APSY-based assignments to 25 kDa, thus providing the basis for efficient characterization of protein-ligand interactions at atomic resolution by NMR, such as structure-based drug design. We show the application to two human proteins with molecular weights of 15 and 22 kDa, respectively, at concentrations of 0.4 mM and discuss the general applicability to studies of protein-protein and protein-nucleic acid complexes

NMR assignment of the E. coli type 1 pilus protein FimF

ISSN:0925-2738ISSN:1573-500