Combined In-Solution Fragment Screening and Crystallographic Binding-Mode Analysis with a Two-Domain Hsp70 Construct.

Heat shock protein 70 (Hsp70) isoforms are key players in the regulation of protein homeostasis and cell death pathways and are therefore attractive targets in cancer research. Developing nucleotide-competitive inhibitors or allosteric modulators, however, has turned out to be very challenging for this protein family, and no Hsp70-directed therapeutics have so far become available. As the field could profit from alternative starting points for inhibitor development, we present the results of a fragment-based screening approach on a two-domain Hsp70 construct using in-solution NMR methods, together with X-ray-crystallographic investigations and mixed-solvent molecular dynamics simulations. The screening protocol resulted in hits on both domains. In particular, fragment binding in a deeply buried pocket at the substrate-binding domain could be detected. The corresponding site is known to be important for communication between the nucleotide-binding and substrate-binding domains of Hsp70 proteins. The main fragment identified at this position also offers an interesting starting point for the development of a dual Hsp70/Hsp90 inhibitor.

Enhanced TROSY Effect in [2-19 F, 2-13 C] Adenosine and ATP Analogs Facilitates NMR Spectroscopy of Very Large Biological RNAs in Solution.

Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2-19 F, 2-13 C]-adenosine, which allow the incorporation of aromatic 19 F-13 C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2-19 F, 2-13 C]-adenosine-phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2-19 F, 2-13 C]-adenosine triphosphate, the 124 nt pre-miR-17-NPSL1-RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2-19 F, 2-13 C]-A-labeled RNA featured sharper resonances than the [2-1 H, 2-13 C]-A sample. The mutual cancelation of the chemical-shift-anisotropy and the dipole-dipole-components of TROSY-resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non-hydrolysable [2-19 F, 2-13 C]-adenosine-triphosphate, we facilitate the probing of co-factor binding in kinase complexes and NMR-based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide-and-conquer/mutational approach. The new [2-19 F, 2-13 C]-adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells.

Teaching under lockdown: the change in the social practice of teaching.

Due to the unprecedented situation caused by a global pandemic, the traditional way of teaching that is reliant on face-to-face interaction between teachers and students has been dismantled. This article looks into university teachers' experiences of teaching under lockdown, with an intention to understand what the change meant in terms of social practice. The research follows a qualitative design, in which ten university teachers were interviewed using a semi-structured interview guide. Three themes interwoven with a common thread were identified through teachers' reflections, including displacement, routine, and role. The common thread was identified as the interaction between teachers and students, and analysing the quality of this interaction led to understanding the social kernel of teaching as embedded in social practice, suggesting that physical dislocation demands teachers to recreate meaning in the new situation. This change has been seen as difficult, yet unpacking teachers' perceptions provided valuable lessons for the future.

Time-resolved structural analysis of an RNA-cleaving DNA catalyst.

The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.

Surviving but not thriving: Comparing primary, vocational and higher education teachers' experiences during the COVID-19 lockdown.

In this paper we examine the impacts of the global pandemic in 2020 on different levels of education system, particularly looking at the changes in teaching practice. The health emergency caused closure of schools, and online distance education became a temporary solution, creating discomfort for many teachers for whom this was the first time engaged with online education. In our research we investigated two important dimensions, namely, how technology was used and what the newfound distance meant in terms of the teacher-student relationship. The article offers insights into experiences of teaching from lockdown reported by 41 teachers at primary, vocational and higher education level in the region of Vaud, Switzerland. This comparative qualitative research has provided an opportunity for an in-depth analysis of the main similarities and differences at three distinctly different educational levels and a possibility to learn more about common coping practices in teaching. The study gives a contribution to a lack of comparative studies of teacher experiences at different educational levels. Results show two dimensions in handling the lockdown crisis: mastering the digital tools and the importance of student-teacher interaction. Whilst the interviewed teachers largely overcame the challenges of mastering digital tools, optimizing the quality interaction and ensuring the transactional presence online remained a problem. This indicates the importance of the social aspect in education at all levels, and implies that teacher support needs to expand beyond technical pedagogical knowledge of online distance education.

Efficient affinity ranking of fluorinated ligands by 19F NMR: CSAR and FastCSAR.

Fluorine NMR has recently gained high popularity in drug discovery as it allows efficient and sensitive screening of large numbers of ligands. However, the positive hits found in screening must subsequently be ranked according to their affinity in order to prioritize them for follow-up chemistry. Unfortunately, the primary read-out from the screening experiments, namely the increased relaxation rate upon binding, is not proportional to the affinity of the ligand, as it is polluted by effects such as exchange broadening. Here we present the method CSAR (Chemical Shift-anisotropy-based Affinity Ranking) for reliable ranking of fluorinated ligands by NMR, without the need of isotope labeled protein, titrations or setting up a reporter format. Our strategy is to produce relaxation data that is directly proportional to the binding affinity. This is achieved by removing all other contributions to relaxation as follows: (i) exchange effects are efficiently suppressed by using high power spin lock pulses, (ii) dipolar relaxation effects are approximately subtracted by measuring at two different magnetic fields and (iii) differences in chemical shift anisotropy are normalized using calculated values. A similar ranking can be obtained with the simplified approach FastCSAR that relies on a measurement of a single relaxation experiment at high field (preferably > 600 MHz). An affinity ranking obtained in this simple way will enable prioritizing ligands and thus improve the efficiency of fragment-based drug design.

Aromatic 19F-13C TROSY: a background-free approach to probe biomolecular structure, function, and dynamics.

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.

Parallel NMR spectroscopy with simultaneous detection of (1) H and (19) F nuclei.

Recording NMR signals of several nuclear species simultaneously by using parallel receivers provides more information from a single measurement and at the same time increases the measurement sensitivity per unit time. Here we present a comprehensive series of the most frequently used NMR experiments modified for simultaneous direct detection of two of the most sensitive NMR nuclei - (1) H and (19) F. We hope that the presented material will stimulate interest in and further development of this technique.

Direct ¹³C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA.

In RNA secondary structure determination, it is essential to determine whether a nucleotide is base-paired and not. Base-pairing of nucleotides is mediated by hydrogen bonds. The NMR characterization of hydrogen bonds relies on experiments correlating the NMR resonances of exchangeable protons and can be best performed for structured parts of the RNA, where labile hydrogen atoms are protected from solvent exchange. Functionally important regions in RNA, however, frequently reveal increased dynamic disorder which often leads to NMR signals of exchangeable protons that are broadened beyond (1)H detection. Here, we develop (13)C direct detected experiments to observe all nucleotides in RNA irrespective of whether they are involved in hydrogen bonds or not. Exploiting the self-decoupling of scalar couplings due to the exchange process, the hydrogen bonding behavior of the hydrogen bond donor of each individual nucleotide can be determined. Furthermore, the adaption of HNN-COSY experiments for (13)C direct detection allows correlations of donor-acceptor pairs and the localization of hydrogen-bond acceptor nucleotides. The proposed (13)C direct detected experiments therefore provide information about molecular sites not amenable by conventional proton-detected methods. Such information makes the RNA secondary structure determination by NMR more accurate and helps to validate secondary structure predictions based on bioinformatics.

Fast experiments for structure elucidation of small molecules: Hadamard NMR with multiple receivers.

We propose several significant improvements to the PANSY (Parallel NMR SpectroscopY) experiments-PANSY COSY and PANSY-TOCSY. The improved versions of these experiments provide sufficient spectral information for structure elucidation of small organic molecules from just two 2D experiments. The PANSY-TOCSY-Q experiment has been modified to allow for simultaneous acquisition of three different types of NMR spectra-1D C-13 of non-protonated carbon sites, 2D TOCSY and multiplicity edited 2D HETCOR. In addition the J-filtered 2D PANSY-gCOSY experiment records a 2D HH gCOSY spectrum in parallel with a (1) J-filtered HC long-range HETCOR spectrum as well as offers a simplified data processing. In addition to parallel acquisition, further time savings are feasible because of significantly smaller F1 spectral windows as compared to the indirect detection experiments. Use of cryoprobes and multiple receivers can significantly alleviate the sensitivity issues that are usually associated with the so called direct detection experiments. In cases where experiments are sampling limited rather than sensitivity limited further reduction of experiment time is achieved by using Hadamard encoding. In favorable cases the total recording time for the two PANSY experiments can be reduced to just 40 s. The proposed PANSY experiments provide sufficient information to allow the CMCse software package (Bruker) to solve structures of small organic molecules.

NMR structure analysis of uniformly 13C-labeled carbohydrates.

In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of (13)C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly (13)C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-(13)C)-sucrose, 342 Da] and one compound of medium molecular weight ((13)C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The (13)C resonances are traced using (13)C-(13)C correlations from homonuclear experiments, such as (H)CC-CT-COSY, (H)CC-NOESY, CC-CT-TOCSY and/or virtually decoupled (H)CC-TOCSY. Based on the assignment of the (13)C resonances, the (1)H chemical shifts are derived in a straightforward manner using one-bond (1)H-(13)C correlations from heteronuclear experiments (HC-CT-HSQC). In order to avoid the (1) J CC splitting of the (13)C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either (13)C or (1)H detected experiments, namely CC-CT-COSY, band-selective (H)CC-TOCSY, HC-CT-HSQC-NOESY or long-range HC-CT-HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the (1)H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the (13)C-enriched polysaccharide were assigned by using HC-H2BC spectra. The assignment of the N-acetyl groups with (15)N at natural abundance was completed by using HN-SOFAST-HMQC, HNCA, HNCO and (13)C-detected (H)CACO spectra.

Improved NMR experiments with ¹³C-isotropic mixing for assignment of aromatic and aliphatic side chains in labeled proteins.

Three improved ¹³C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(C(ali)C(aro))H-TOCSY, is an HCCH-TOCSY in which all CHn moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the ¹³C-detected C(all)-TOCSY, magnetization transfer comprises all carbons--aliphatic, aromatic as well as the carbonyl carbons--making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct ¹³C-¹³C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa.

Probing water-protein contacts in a MMP-12/CGS27023A complex by nuclear magnetic resonance spectroscopy.

Using the case of the catalytic domain of MMP-12 in complex with the known inhibitor CGS27023A, a recently assembled 3D (15)N-edited/(14)N,(12)C-filtered ROESY experiment is used to monitor and distinguish protein amide protons in fast exchange with bulk water from amide protons close to water molecules with longer residence times, the latter possibly reflecting water molecules of structural or functional importance. The (15)N-edited/(14)N,(12)C-filtered ROESY spectra were compared to the original (15)N-edited/(14)N,(12)C-filtered NOESY and the conventional amide-water exchange experiment, CLEANEX. Three protein backbone amide protons experiencing direct dipolar cross relaxation with water in the (15)N-edited/(14)N,(12)C-filtered ROESY spectrum were assigned. In an ensemble of six crystal structures, two conserved water molecules within 3 Å of the three amide protons were identified. These two water molecules are buried into cavities in the protein surface and thus sufficiently slowed down by the protein topology to account for the observed dipolar interaction. Structural analysis of an ensemble of six crystal structures ruled out any exchange-relayed contributions for the amide-water interactions of interest.

Synovial fluid metabolomics in different forms of arthritis assessed by nuclear magnetic resonance spectroscopy.

OBJECTIVES: Currently there are no reliable biomarkers in the synovial fluid available to differentiate between septic and non-septic arthritis or to predict the prognosis of osteoarthritis, respectively. Nuclear magnetic resonance (NMR) spectroscopy is an analytical technique that allows a rapid, high throughput metabolic profiling of biological fluids or tissues. METHODS: Proton (1H)-nuclear magnetic resonance (NMR) spectroscopy was performed in synovial fluid samples from patients with septic arthritis, crystal arthropathy, different forms of inflammatory arthritis or osteoarthritis (OA). The metabolic environment based on the low molecular weight components was compared in disease subsets and principal component analysis (PCA) was performed. RESULTS: Fifty-nine samples from patients with OA, gout, calcium pyrophosphate disease, spondylarthritis, septic arthritis and rheumatoid arthritis (RA) were analysed. NMR yielded stable and reproducible metabolites over time. Thirty-five different metabolites as well as paracetamol and ibuprofen were identified in synovial fluid. The metabolic profile of septic arthritis assessed by PCA was distinguishable from the other samples whereas no differences were seen in OA compared to crystal-associated arthritis, RA or spondylarthritis. CONCLUSIONS: 1H-NMR is a fast analytic tool with possible implications in synovial fluid diagnostics. A distinctive metabolism is observed in septic arthritis whereas metabolites in OA are similar to those in inflammatory arthritis.

13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides.

We present here a set of (13)C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose (13)C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4' nuclei of adjacent nucleotides via J(C,P) couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1',H1' ribose signals. The experiments were applied to two RNA hairpin structures. The current set of (13)C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification of the individual ribose moieties following their sequential assignment. Thus, (13)C-direct detected NMR methods constitute useful complements to the conventional (1)H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift dispersion. The developed methods can also be applied to large deuterated RNAs.

Metal ion-N7 coordination in a ribozyme branch domain by NMR.

The N7 of purine nucleotides presents one of the most dominant metal ion binding sites in nucleic acids. However, the interactions between kinetically labile metal ions like Mg(2+) and these nitrogen atoms are inherently difficult to observe in large RNAs. Rather than using the insensitive direct (15)N detection, here we have used (2)J-[(1)H,(15)N]-HSQC (Heteronuclear Single Quantum Coherence) NMR experiments as a fast and efficient method to specifically observe and characterize such interactions within larger RNA constructs. Using the 27 nucleotides long branch domain of the yeast-mitochondrial group II intron ribozyme Sc.ai5gamma as an example, we show that direct N7 coordination of a Mg(2+) ion takes place in a tetraloop nucleotide. A second Mg(2+) ion, located in the major groove at the catalytic branch site, coordinates mainly in an outer-sphere fashion to the highly conserved flanking GU wobble pairs but not to N7 of the sandwiched branch adenosine.

Does a fast nuclear magnetic resonance spectroscopy- and X-ray crystallography hybrid approach provide reliable structural information of ligand-protein complexes? A case study of metalloproteinases.

A human matrix metalloproteinase (MMP) hydroxamic acid inhibitor (CGS27023A) was cross-docked into 15 MMP-12, MMP-13, MMP-9, and MMP-1 cocrystal structures. The aim was to validate a fast protocol for ligand binding conformation elucidation and to probe the feasibility of using inhibitor-protein NMR contacts to dock an inhibitor into related MMP crystal structures. Such an approach avoids full NMR structure elucidation, saving both spectrometer- and analysis time. We report here that for the studied MMPs, one can obtain docking results well within 1 A compared to the corresponding reference X-ray structure, using backbone amide contacts only. From the perspective of the pharmaceutical industry, these results are relevant for the binding studies of inhibitor series to a common target and have the potential advantage of obtaining information on protein-inhibitor complexes that are difficult to crystallize.

Three-dimensional 13C-detected CH3-TOCSY using selectively protonated proteins: facile methyl resonance assignment and protein structure determination.

Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100 kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR.

Simultaneous 1H- or 2H-, 15N- and multiple-band-selective 13C-decoupling during acquisition in 13C-detected experiments with proteins and oligonucleotides.

Significant resolution improvement in 13C,13C-TOCSY spectra of uniformly deuterated and 13C, 15N-labeled protein and 13C,15N-labeled RNA samples is achieved by introduction of multiple-band-selective 13C-homodecoupling applied simultaneously with 1H- or 2H- and 15N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective 13C-decoupling in 2D-[13C,13C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear 13C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any 13C-detected experiment.

Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR.

Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin-alpha(v)beta3 docking model suggests that echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.