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Transcription and translation obey to the genetic code of four nucleobases and 21 amino acids evolved over billions of years. Both these processes have been engineered to facilitate the use of non-natural building blocks in both nucleic acids and proteins, enabling researchers with a decent toolbox for structural and functional analyses. Here, we review the most common approaches for how labeling of both nucleic acids as well as proteins in a site-selective fashion with either modifiable building blocks or spectroscopic probes can be facilitated by genetic code expansion. We emphasize methodological approaches and how these can be adapted for specific modifications, both during as well as after biomolecule synthesis. These modifications can facilitate, for example, a number of different spectroscopic analysis techniques and can under specific circumstances even be used in combination., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Post-translational modifications of proteins are widespread in eukaryotes. To elucidate the functional role of these modifications, detection methods need to be developed that provide information at atomic resolution. Here, we report on the development of a novel Arg-specific NMR experiment that detects the methylation status and symmetry of each arginine side chain even in highly repetitive RGG amino acid sequence motifs found in numerous proteins within intrinsically disordered regions. The experiment relies on the excellent resolution of the backbone H,N correlation spectra even in these low complexity sequences. It requires 13 C, 15 N labeled samples.

Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure-function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The adenine-sensing riboswitch from the Gram-negative bacterium Vibrio vulnificus is an RNA-based gene regulatory element that acts in response to both its cognate low-molecular weight ligand and temperature. The combined sensitivity to environmental temperature and ligand concentration is maintained by an equilibrium of three distinct conformations involving two ligand-free states and one ligand-bound state. The key structural element that undergoes refolding in the ligand-free states comprises a 35-nucleotide temperature response module. Here, we present the structural characterization of this temperature response module. We employ high-resolution NMR spectroscopy and photocaged RNAs as molecular probes to decipher the kinetic and thermodynamic framework of the secondary structure transition in the apo state of the riboswitch. We propose a model for the transition state adopted during the thermal refolding of the temperature response module that connects two mutually exclusive long-lived and stable conformational states. This transition state is characterized by a comparatively low free activation enthalpy. A pseudoknot conformation in the transition state, as commonly seen in RNA refolding, is therefore unlikely. More likely, the transition state of the adenine-sensing riboswitch temperature response module features a linear conformation.

Non-canonical DNA i-motifs and G-quadruplexes are postulated as genetic switches for the transcriptional regulation of proto-oncogenes. However, in comparison to G-quadruplexes, the therapeutic potential of i-motifs is less explored. The development of i-motif selective ligands by conventional approaches is challenging due to the structural complexity of i-motifs. The target guided synthetic (TGS) approach involving in situ cycloaddition could provide specific ligands for these dynamic DNA structures. Herein, we have used i-motif forming C-rich DNA and their complementary G-quadruplex forming DNA sequences of c-MYC and BCL2 promoter regions as well as a control self-complementary duplex DNA sequence as the templates to generate selective ligands from a pool of reactive azide-alkyne building blocks. In our approach, thiolated DNA targets are immobilized on the surface of gold-coated iron nanoparticles to enable efficient isolation of the newly generated ligands from the solution mixture by simple magnetic decantation. The combinatorial in situ cycloaddition generated cell-membrane permeable triazole leads for respective DNA targets ( c-MYC and BCL2 i-motifs and G-quadruplexes) that selectively promote their formation. In vitro cellular studies reveal that the c-MYC i-motif and G-quadruplex leads downregulate c-MYC gene expression whereas the BCL2 i-motif lead upregulates and the BCL2 G-quadruplex lead represses BCL2 gene expression. The TGS strategy using i-motif DNA nanotemplates represents a promising platform for the direct in situ formation of i-motif specific ligands for therapeutic intervention., (This journal is © The Royal Society of Chemistry 2020.)

Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A-T and G-C base-pairs are therefore uniquely suitable to facilitate position-specific labeling of nucleic acids. Here, we employ the orthogonal kappa-xanthosine-base-pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy-version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7-deaza-xanthosine, which allows to site-specifically modify transcribed RNA molecules with fluorophores or other labels., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1 H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW's enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15 N- 1 H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSR apt ) of the xpt-pbuX operon in Bacillus subtilis Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1 H NMR spectra allowed us to monitor in real time the changes that GSR apt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1 H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSR apt , is briefly discussed., Competing Interests: The authors declare no competing interest.

Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in-cell NMR spectroscopy experiments. We are able to monitor real-time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer-based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Past sequencing campaigns overlooked small proteins as they seemed to be irrelevant due to their small size. However, their occurrence is widespread, and there is growing evidence that these small proteins are in fact functionally very important in organisms found in all kingdoms of life. Within a global proteome analysis for small proteins of the archaeal model organism Haloferax volcanii, the HVO_2922 protein has been identified. It is differentially expressed in response to changes in iron and salt concentrations, thus suggesting that its expression is stress-regulated. The protein is conserved among Haloarchaea and contains an uncharacterized domain of unknown function (DUF1508, UPF0339 family protein). We elucidated the NMR solution structure, which shows that the isolated protein forms a symmetrical dimer. The dimerization is found to be concentration-dependent and essential for protein stability and most likely for its functionality, as mutagenesis at the dimer interface leads to a decrease in stability and protein aggregation., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The complex folding energy landscape of DNA G-quadruplexes leads to numerous conformations for this functionally important class of noncanonical DNA structures. A new layer of conformational heterogeneity comes from sequences with different numbers of G-nucleotides in each of the DNA G-strands that form the four-stranded G-quartet core. The mechanisms by which G-quadruplexes transition from one folded conformation to another are currently unknown. To address this question, we studied two different G-quadruplexes, selecting a single conformation by blocking hydrogen bonding with photolabile protection groups. Upon irradiation, the block can be released and the kinetics of re-equilibration to the native conformational equilibrium can be determined by time-resolved NMR. We compared the NMR-derived refolding kinetics with data derived from thermal hysteresis folding kinetic experiments and found excellent agreement. The outlined methodological approach allows separation of K + -induced G-quadruplex formation and subsequent refolding and provides key insight into rate-limiting steps of G-quadruplex conformational dynamics.

Ribonucleic acid oligonucleotides (RNAs) play pivotal roles in cellular function (riboswitches), chemical biology applications (SELEX-derived aptamers), cell biology and biomedical applications (transcriptomics). Furthermore, a growing number of RNA forms (long non-coding RNAs, circular RNAs) but also RNA modifications are identified, showing the ever increasing functional diversity of RNAs. To describe and understand this functional diversity, structural studies of RNA are increasingly important. However, they are often more challenging than protein structural studies as RNAs are substantially more dynamic and their function is often linked to their structural transitions between alternative conformations. NMR is a prime technique to characterize these structural dynamics with atomic resolution. To extend the NMR size limitation and to characterize large RNAs and their complexes above 200 nucleotides, new NMR techniques have been developed. This Minireview reports on the development of NMR methods that utilize detection on low-γ nuclei (heteronuclei like 13 C or 15 N with lower gyromagnetic ratio than 1 H) to obtain unique structural and dynamic information for large RNA molecules in solution. Experiments involve through-bond correlations of nucleobases and the phosphodiester backbone of RNA for chemical shift assignment and make information on hydrogen bonding uniquely accessible. Previously unobservable NMR resonances of amino groups in RNA nucleobases are now detected in experiments involving conformational exchange-resistant double-quantum 1 H coherences, detected by 13 C NMR spectroscopy. Furthermore, 13 C and 15 N chemical shifts provide valuable information on conformations. All the covered aspects point to the advantages of low-γ nuclei detection experiments in RNA., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

We herein report a cell-membrane-permeable molecular probe ADG , prepared by conjugating guanosine with anthracene, selectively interacts with c-MYC G-quadruplex over other promoter and telomeric quadruplexes as well as duplex DNA. NMR spectroscopy suggests that ADG interacts with terminal G-quartets as well as with the nearby G-rich tract (G13-G14-G15 and G8-G9-G10) of c-MYC quadruplex. In vitro cellular studies indicate that ADG represses c-MYC expression by stabilizing its promoter G-quadruplex and alters c-MYC -related cellular events. ADG suppresses hTERT and BCL2 gene expressions in a promoter-independent manner, inhibits elongation of telomere length, and activates apoptotic cascades in cancer cells.

Paramagnetic relaxation enhancement (PRE) can be used to determine long-range distance restraints in biomolecules. The PREs are typically determined by analysis of intensity differences in HSQC experiments of paramagnetic and diamagnetic spin labels. However, this approach requires both isotope- and spin-labelling. Herein, we report a novel method to evaluate NOESY intensities in the presence of a paramagnetic moiety to determine PRE restraints. The advantage of our approach over HSQC-based approaches is the increased number of available signals without the need for isotope labelling. NOESY intensities affected by a paramagnetic center were evaluated during a structure calculation within the paramagnetic iterative relaxation matrix approach (P-IRMA). We applied P-IRMA to a 14-mer RNA with a known NMR solution structure, which allowed us to assess the quality of the PRE restraints. To this end, three different spin labels have been attached at different positions of the 14-mer to test the influence of flexibility on the structure calculation. Structural disturbances introduced by the spin label have been evaluated by chemical shift analysis. Furthermore, the impact of P-IRMA on the quality of the structure bundles were tested by intentionally leaving out available diamagnetic restraints. Our analyses show that P-IRMA is a powerful tool to refine RNA structures for systems that are insufficiently described by using only diamagnetic restraints.

Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

We describe the design and construction of a modular, triple-resonance, fully balanced, DNP-MAS probe based on transmission line technology and its integration into a 500 MHz/330 GHz DNP-NMR spectrometer. A novel quantitative probe design and characterization strategy is developed and employed to achieve optimal sensitivity, RF homogeneity and excellent isolation between channels. The resulting three channel HCN probe has a modular design with each individual, swappable module being equipped with connectorized, transmission line ports. This strategy permits attachment of a mating connector that facilitates accurate impedance measurements at these ports and allows characterization and adjustment (e.g. for balancing or tuning/matching) of each component individually. The RF performance of the probe is excellent; for example, the 13 C channel attains a Rabi frequency of 280 kHz for a 3.2 mm rotor. In addition, a frequency tunable 330 GHz gyrotron operating at the second harmonic of the electron cyclotron frequency was developed for DNP applications. Careful alignment of the corrugated waveguide led to minimal loss of the microwave power, and an enhancement factor ε = 180 was achieved for U- 13 C urea in the glassy matrix at 80 K. We demonstrated the operation of the system with acquisition of multidimensional spectra of cross-linked lysozyme crystals which are insoluble in glycerol-water mixtures used for DNP and samples of RNA., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two new dimeric compounds of the alternariol class, (±)-alternarlactones A ( 1 ) and B ( 2 ), were isolated along with 11 known compounds from the fungus Alternaria alternata P1210. Their structures were elucidated with the assistance of long-range HSQMBC to address inadequate cross-peaks in HMBC that result from the highly dense quaternary carbons, as well as theoretical calculations. All isolated altenuisol derivatives were screened for their antiparasitic activities, which provide a preliminary structure-activity relationship of this class of compounds against neglected tropical diseases.

Optimization of coding sequences to maximize protein expression yield is often outsourced to external service providers during commercial gene synthesis and thus unfortunately remains a black box for many researchers. The presented software program "CodonWizard" offers scientists a powerful but easy-to-use tool for customizable codon optimization: The intuitive graphical user interface empowers even scientists inexperienced in the art to straightforward design, modify, test and save complex codon optimization strategies and to publicly share successful otimization strategies among the scientific community. "Codon Wizard" provides highly flexible features for sequence analysis and completely customizable modification/optimization of codon usage of any given input sequence data (DNA/RNA/peptide) using freely combinable algorithms, allowing for implementation of contemporary, well-established optimization strategies as well as novel, proprietary ones alike. Contrary to comparable tools, "Codon Wizard" thus finally opens up ways for an empirical approach to codon optimization and may also >be used completely offline to protect resulting intellectual property. As a benchmark, the reliability, intuitiveness and utility of the application could be demonstrated by increasing the yield of recombinant TEV-protease expressed in E. coli by several orders of magnitude after codon optimization using "CodonWizard" - Permanently available for download on the web at http://schwalbe.org.chemie.uni-frankfurt.de/node/3324., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Acute myeloid leukemia (AML) is one of the most common and life-threatening leukemias. A highly diverse and flexible metabolism contributes to the aggressiveness of the disease that is still difficult to treat. By using different sources of nutrients for energy and biomass supply, AML cells gain metabolic plasticity and rapidly outcompete normal hematopoietic cells. This review aims to decipher the diverse metabolic strategies and the underlying oncogenic and environmental changes that sustain continuous growth, mediate redox homeostasis and induce drug resistance in AML. We revisit Warburg's hypothesis and illustrate the role of glucose as a provider of cellular building blocks rather than as a supplier of the tricarboxylic acid (TCA) cycle for energy production. We discuss how the diversity of fuels for the TCA cycle, including glutamine and fatty acids, contributes to the metabolic plasticity of the disease and highlight the roles of amino acids and lipids in AML metabolism. Furthermore, we point out the potential of the different metabolic effectors to be used as novel therapeutic targets., Competing Interests: The authors declare no conflict of interest.

The bile acid-sensing transcription factor farnesoid X receptor (FXR) regulates multiple metabolic processes. Modulation of FXR is desired to overcome several metabolic pathologies but pharmacological administration of full FXR agonists has been plagued by mechanism-based side effects. We have developed a modulator that partially activates FXR in vitro and in mice. Here we report the elucidation of the molecular mechanism that drives partial FXR activation by crystallography- and NMR-based structural biology. Natural and synthetic FXR agonists stabilize formation of an extended helix α11 and the α11-α12 loop upon binding. This strengthens a network of hydrogen bonds, repositions helix α12 and enables co-activator recruitment. Partial agonism in contrast is conferred by a kink in helix α11 that destabilizes the α11-α12 loop, a critical determinant for helix α12 orientation. Thereby, the synthetic partial agonist induces conformational states, capable of recruiting both co-repressors and co-activators leading to an equilibrium of co-activator and co-repressor binding.

Up to now, NMR spectroscopic investigations of RNA have utilized imino proton resonances as reporters for base pairing and RNA structure. The nucleobase amino groups are often neglected, since most of their resonances are broadened beyond detection due to rotational motion around the C-NH 2 bond. Here, we present 13 C-detected NMR experiments for the characterization of all RNA amino groups irrespective of their motional behavior. We have developed a C(N)H-HDQC experiment that enables the observation of a complete set of sharp amino resonances through the detection of proton-NH 2 double quantum coherences. Further, we present an "amino"-NOESY experiment to detect NOEs to amino protons, which are undetectable by any other conventional NOESY experiment. Together, these experiments allow the exploration of additional chemical shift information and inter-residual proton distances important for high-resolution RNA secondary and tertiary structure determination., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Osteoarthritis (OA) of the knee is a widespread disease, often resulting in pain, restricted mobility and a reduction of activities and participation. Initial studies gave hints that Acoustic Emission Analysis (AEA) is capable of detecting early changes in cartilage structure. However, up to date no in vivo validation studies have been conducted. A prospective pilot study was conducted to investigate this diagnostic capability and the accuracy of the AEA, using magnetic resonance imaging (MRI) as a reference standard. Additionally, potential factors influencing false positive or negative results were studied. Twenty-eight patients, receiving MRI due to discomfort of the knee, were examined with AEA. Sensitivity was 0.92 for the whole knee and 0.86 to 1 for different parts of the knee. The specificity was 0.7 and 0.59 to 0.78, respectively. Confidence intervals varied between 0 and 0.33 for sensitivity and 0.1 and 0.24 for specificity. The diagnostic accuracy of the AEA was shown to be good to very good. However, because of the relatively small number of patients involved, interpretation of the data should be handled with care. Future studies with greater sample sizes have to be conducted to confirm the results of this investigation., (Copyright © 2019. Published by Elsevier Ltd.)

Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Photolabile protecting groups are widely used to trigger oligonucleotide activity. The ON/OFF-amplitude is a critical parameter. An experimental setup has been developed to identify protecting group derivatives with superior caging properties. Bulky rests are attached to the cage moiety via Cu-catalyzed azide-alkyne cycloaddition post-synthetically on DNA. Interestingly, the decrease in melting temperature upon introducing o-nitrobenzyl-caged (NPBY-) and diethylaminocoumarin-cages (DEACM-) in DNA duplexes reaches a limiting value. NMR spectroscopy was used to characterize individual base-pair stabilities and determine experimental structures of a selected number of photocaged DNA molecules. The experimental structures agree well with structures predicted by MD simulations. Combined, the structural data indicate that once a sterically demanding group is added to generate a tri-substituted carbon, the sterically less demanding cage moiety points towards the neighboring nucleoside and the bulkier substituents remain in the major groove., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Initiation of bacterial translation requires that the ribosome-binding site in mRNAs adopts single-stranded conformations. In Gram-negative bacteria the ribosomal protein S1 (rS1) is a key player in resolving of structured elements in mRNAs. However, the exact mechanism of how rS1 unfolds persistent secondary structures in the translation initiation region (TIR) is still unknown. Here, we show by NMR spectroscopy that Vibrio vulnificus rS1 displays a unique architecture of its mRNA-binding domains, where domains D3 and D4 provide the mRNA-binding platform and cover the nucleotide binding length of the full-length rS1. D5 significantly increases rS1's chaperone activity, although it displays structural heterogeneity both in isolation and in presence of the other domains, albeit to varying degrees. The heterogeneity is induced by the switch between the two equilibrium conformations and is triggered by an order-to-order transition of two mutually exclusive secondary structures (β-strand-to-α-helix) of the 'AERERI' sequence. The conformational switching is exploited for melting of structured 5'-UTR's, as the conformational heterogeneity of D5 can compensate the entropic penalty of complex formation. Our data thus provides a detailed understanding of the intricate coupling of protein and RNA folding dynamics enabling translation initiation of structured mRNAs.

NMR and chromatography methods combined with mass spectrometry are the most important analytical techniques employed for plant metabolomics screening. Metabolomic analysis integrated to transcriptome screening add an important extra dimension to the information flow from DNA to RNA to protein. The most useful NMR experiment in metabolomics analysis is the proton spectra due the high receptivity of 1 H and important structural information, through proton-proton scalar coupling. Routinely, databases have been used in identification of primary metabolites, however, there is currently no comparable data for identification of secondary metabolites, mainly, due to signal overlap in normal 1 H NMR spectra and natural variation of plant. Related to spectra overlap, alternatively, better resolution can be find using 1 H pure shift and 2D NMR pulse sequence in complex samples due to spreading the resonances in a second dimension. Thus, in data brief we provide a catalogue of metabolites and expression levels of genes identified in soy leaves and roots under flooding stress.

Production of recombinant prion proteins is of crucial relevance in food technology (analytical standards, assay development) but also in basic research, most importantly structural biology (NMR, X-ray diffraction). Structural approaches conveniently allow for sophisticated investigation of prion disease pathogenesis, but usually require large amounts of sample material. Recently, working with recombinant prion proteins has been recategorized to biosafety levels > S1 as infectious prions may readily be generated de novo and become airborne via aerosols. Heterologous expression should therefore be established with appropriately adjusted safety precautions. We have developed a protocol for high-yield expression, purification and refolding of recombinant mammalian prion proteins at elevated biological safety levels by introducing means of abolishing aerosol formation and propagation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A ligand-binding study is presented focusing on thermodynamics of fragment expansion. The binding of four compounds with increasing molecular weight to protein kinase A (PKA) was analyzed. The ligands display affinities between low-micromolar to nanomolar potency despite their low molecular weight. Binding free energies were measured by isothermal titration calorimetry, revealing a trend toward more entropic and less enthalpic binding with increase in molecular weight. All protein-ligand complexes were analyzed by crystallography and solution NMR spectroscopy. Crystal structures and solution NMR data are highly consistent, and no major differences in complex dynamics across the series are observed that would explain the differences in the thermodynamic profiles. Instead, the thermodynamic trends result either from differences in the solvation patterns of the conformationally more flexible ligand in aqueous solution prior to protein binding as molecular dynamics simulations suggest, or from local shifts of the water structure in the ligand-bound state. Our data thus provide evidence that changes in the solvation pattern constitute an important parameter for the understanding of thermodynamic data in protein-ligand complex formation., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The investigation of non-coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo-enzymatic RNA synthesis containing photoactivatable or 13 C, 15 N-labelled nucleosides. All four ribonucleotides containing ortho-nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C-nucleotides and 13 C, 15 N-labelled nucleotides were incorporated position-specifically in high yields. We applied this approach for the synthesis of light-inducible 2'dG-sensing riboswitch variants and detected ligand-induced structural reorganization upon irradiation by NMR spectroscopy. This chemo-enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid-phase synthesis., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Erythropoietin-producing hepatocellular (EPH) receptors are transmembrane receptor tyrosine kinases. Their extracellular domains bind specifically to ephrin A/B ligands, and this binding modulates intracellular kinase activity. EPHs are key players in bidirectional intercellular signaling, controlling cell morphology, adhesion, and migration. They are increasingly recognized as cancer drug targets. We analyzed the binding of NVP-BHG712 (NVP) to EPHA2 and EPHB4. Unexpectedly, all tested commercially available NVP samples turned out to be a regioisomer (NVPiso) of the inhibitor, initially described in a Novartis patent application. They only differ by the localization of a single methyl group on either one of two adjacent nitrogen atoms. The two compounds of identical mass revealed different binding modes. Furthermore, both in vitro and in vivo experiments showed that the isomers differ in their kinase affinity and selectivity., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate., Competing Interests: The authors have declared that no competing interests exist.

The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis ( Mtb ) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr 128 and Tyr 129 Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDD PtkA ) and the C-terminal rigid kinase core domain (KCD PtkA ). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDD PtkA is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCD PtkA This interaction modulates the accessibility of the KCD PtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in Mtb virulence., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Biophysical parameters can accelerate drug development; e.g., rigid ligands may reduce entropic penalty and improve binding affinity. We studied systematically the impact of ligand rigidification on thermodynamics using a series of fasudil derivatives inhibiting protein kinase A by crystallography, isothermal titration calorimetry, nuclear magnetic resonance, and molecular dynamics simulations. The ligands varied in their internal degrees of freedom but conserve the number of heteroatoms. Counterintuitively, the most flexible ligand displays the entropically most favored binding. As experiment shows, this cannot be explained by higher residual flexibility of ligand, protein, or formed complex nor by a deviating or increased release of water molecules upon complex formation. NMR and crystal structures show no differences in flexibility and water release, although strong ligand-induced adaptations are observed. Instead, the flexible ligand entraps more efficiently water molecules in solution prior to protein binding, and by release of these waters, the favored entropic binding is observed.

The structural differences among different G-quadruplexes provide an opportunity for site-specific targeting of a particular G-quadruplex structure. However, majority of G-quadruplex ligands described thus far show little selectivity among different G-quadruplexes. In this work, we delineate the design and synthesis of a crescent-shaped thiazole peptide that preferentially stabilizes c-MYC quadruplex over other promoter G-quadruplexes and inhibits c-MYC oncogene expression. Biophysical analysis such as Förster resonance energy transfer (FRET) melting and fluorescence spectroscopy show that the thiazole peptide TH3 can selectively interact with the c-MYC G-quadruplex over other investigated G-quadruplexes and duplex DNA. NMR spectroscopy reveals that peptide TH3 binds to the terminal G-quartets and capping regions present in the 5'- and 3'-ends of c-MYC G-quadruplex with a 2:1 stoichiometry; whereas structurally related distamycin A is reported to interact with quadruplex structures via groove binding and end stacking modes with 4:1 stoichiometry. Importantly, qRT-PCR, western blot and dual luciferase reporter assay show that TH3 downregulates c-MYC expression by stabilizing the c-MYC G-quadruplex in cancer cells. Moreover, TH3 localizes within the nucleus of cancer cells and exhibits antiproliferative activities by inducing S phase cell cycle arrest and apoptosis.

The interaction of fibroblast growth factors (FGFs) with their fibroblast growth factor receptors (FGFRs) are important in the signaling network of cell growth and development. SSR128129E (SSR), a ligand of small molecular weight with potential anti-cancer properties, acts allosterically on the extracellular domains of FGFRs. Up to now, the structural basis of SSR binding to the D3 domain of FGFR remained elusive. This work reports the structural characterization of the interaction of SSR with one specific receptor, FGFR3, by NMR spectroscopy. This information provides a basis for rational drug design for allosteric FGFR inhibitors., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Small molecules that stabilize G-quadruplex structures in telomeres can prevent telomerase enzyme mediated telomere lengthening and subsequently lead to cell death. We herein report two fluoro-isoquinoline derivatives IQ1 and IQ2 as selective ligands for human telomeric G-quadruplex DNA. IQ1 and IQ2 containing different triazolyl side chains have been synthesized by Cu (I) catalyzed azide-alkyne cycloaddition. Fluorescence Resonance Energy Transfer (FRET) melting assay and fluorescence binding titrations indicate that both these ligands exhibit binding preference for telomeric G-quadruplex DNA ( h-TELO) over other promoter DNA quadruplexes and duplex DNA. However, ligand IQ1, containing pyrrolidine side chains, is capable of discriminating among quadruplexes by showing higher affinity toward h-TELO quadruplex DNA. On the contrary, IQ2, containing benzamide side chains, interacts with all the investigated quadruplexes. NMR analysis suggests that IQ1 interacts strongly with the external G-quartets of h-TELO. Biological studies reveal that IQ1 is more potent than IQ2 in inhibiting telomerase activity by selectively interacting with telomeric DNA G-quadruplex. Moreover, a dual luciferase reporter assay indicates that IQ1 is unable to reduce the cellular expression of c-MYC and BCL2 at transcriptional level. Significantly, IQ1 mostly stains the nucleus, induces cell cycle arrest in G0/G1 phase, triggers apoptotic response in cancer cells, and activates caspases 3/7.

Georatusin (1), featuring a highly reduced, methylated polyketide moiety fused to a tryptophan by an amide and ester bond forming a 13-membered ring, was produced by the soil fungus Geomyces auratus. An HMQC-COSY spectrum was measured to build up the connectivities despite the overlapping proton signals. DQF-COSY, HETLOC, J-HMBC, and ROESY were implemented to determine the relative configuration of the flexible moiety. Georatusin (1) shows specific antiparasitic activities against Leishmania donovani and Plasmodium falciparum without obvious cytotoxicity. The biosynthesis of 1 was also proposed.

Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription. RNA folding pathways leading to functional ON and OFF regulation involve the formation of metastable states within well-defined sequence intervals during transcription. The kinetic requirements for the formation and preservation of these metastable states in the context of transcription remain unresolved. Here, we reversibly trap the previously defined regulatory relevant metastable intermediate of the Mesoplasma florum 2'-deoxyguanosine (2'dG)-sensing riboswitch using a photocaging-ligation approach, and monitor folding to its native state by real-time NMR in both presence and absence of ligand. We further determine transcription rates for two different bacterial RNA polymerases. Our results reveal that the riboswitch functions only at transcription rates typical for bacterial polymerases (10-50 nt s -1 ) and that gene expression is modulated by 40-50% only, while subtle differences in folding rates guide population ratios within the structural ensemble to a specific regulatory outcome.

G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.

The impact of the incorporation of a non-natural amino acid (NNAA) on protein structure, dynamics, and ligand binding has not been studied rigorously so far. NNAAs are regularly used to modify proteins post-translationally in vivo and in vitro through click chemistry. Herein, structural characterisation of the impact of the incorporation of azidohomoalanine (AZH) into the model protein domain PDZ3 is examined by means of NMR spectroscopy and X-ray crystallography. The structure and dynamics of the apo state of AZH-modified PDZ3 remain mostly unperturbed. Furthermore, the binding of two PDZ3 binding peptides are unchanged upon incorporation of AZH. The interface of the AZH-modified PDZ3 and an azulene-linked peptide for vibrational energy transfer studies has been mapped by means of chemical shift perturbations and NOEs between the unlabelled azulene-linked peptide and the isotopically labelled protein. Co-crystallisation and soaking failed for the peptide-bound holo complex. NMR spectroscopy, however, allowed determination of the protein-ligand interface. Although the incorporation of AZH was minimally invasive for PDZ3, structural analysis of NNAA-modified proteins through the methodology presented herein should be performed to ensure structural integrity of the studied target., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The human genome contains GC-rich sequences able to form tetraplex secondary structures known as the G-quadruplex and i-motif. Such sequences are notably present in the promoter region of oncogenes and are proposed to function as regulatory elements of gene expression. The P1 promoter of BCL2 contains a 39-mer C-rich sequence (Py39wt) that can fold into a hairpin or an i-motif in a pH-dependent manner in vitro. The protein hnRNP LL was identified to recognise the i-motif over the hairpin conformation and act as an activating transcription factor. Thus, the Py39wt sequence would act as an ON/OFF switch, according to the secondary structure adopted. Herein, a structural study of the interaction between hnRNP LL and Py39wt is reported. Both N-terminal RNA recognition motifs (RRM12) cooperatively recognise one Py39wt DNA sequence and engage their β-sheet to form a large binding platform. In contrast, the C-terminal RRMs show no binding capacity. It is observed that RRM12 binds to Py39wt regardless of the DNA conformation. We propose that RRM12 recognises a single-stranded CTCCC element present in loop 1 of the i-motif and in the apical loop of the hairpin conformation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3'-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.

Recently, 15 N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15 N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three 15 N-detected H-N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15 N-detected BEST-TROSY experiment, which proves to be the most sensitive 15 N-detected H-N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the 15 N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1 H- than in the 15 N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15 N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.

We shall overcome: As a result of efforts to overcome the sensitivity challenge of liquid-state NMR spectroscopy, a thousand-fold signal enhancement was achieved by dynamic nuclear polarization (DNP) for 13 C signals at high magnetic field (3.4 T) and room temperature, thereby exceeding the predicted limitations of high-field liquid-state in situ DNP., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)