Faculty of Biochemistry, Chemistry and Pharmacy: Research Data
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- Research DataNMR structure and dynamics of the stem loop II motif (s2m) from the Omicron variant of SARS-CoV-22025-04-30The stem-loop-II motif (s2m) is a conserved viral RNA element located in the 3’UTR of different viruses including SARS-CoV-2. High resolution 3D structural data for s2m is only available for the fundamentally different SCoV-1 version and difficult to access for SARS-CoV-2 due to the highly dynamic nature of the RNA element. With the omicron variant a large deletion occurred for s2m resulting in a relatively short hairpin with an apical pentaloop. We attempted to determine the NMR solution structure of s2m_omicron by including CCRs and J-coupling derived dihedral restraints in addition to NOE distance restraints. Surprisingly, relatively high 1H,13C heteronuclear NOE values, averaged ribose 3JHH-couplings (H1’H2’; H3’H4’) and dipole(H1’-C1’) dipole(H6/8-C6/8)-CCRs hinted towards significant dynamics for the small pentaloop making structure calculations solely relying on NMR data insufficient. To address this problem, we performed MD-simulations with the NMR structure bundle as a starting point and applied BME reweighting to refine the ensemble with the 3J-coupling data. Our results provide a detailed view of the conformational dynamics of the omicron variant of s2m characterized by different stacking patterns, ribose repuckering and overall heterogeneity of the torsion angles for the loop nucleotides. Strikingly, despite the deletion of the initial nonaloop, as present in the Wuhan and Delta variants of s2m, a dynamic UAC triplet is conserved at the tip of the pentaloop hinting towards a possible connection to the still unknown function of the RNA element.
18 7 - Research DataStructural heterogeneity and dynamics in the apical stem loop of s2m from SARS-CoV-2 Delta by an integrative NMR spectroscopy and MD simulation approach2025-02-28In structured RNAs, helical elements are often capped by apical loops that are integral structural elements, ranging from 3 to >20 nts of size on average, and display a highly heterogeneous energy landscape profile. This renders their structural characterization particularly challenging. We here provide an example for comprehensive characterization of the SARS-CoV-2 s2m element containing a highly dynamic nonaloop using an integrative approach of nuclear magnetic resonance spectroscopy (NMR), small angle X-ray scattering (SAXS) and molecular dynamics simulations (MD). As representation of the structure by a single conformation is unsuitable, we here present ensemble of NMR structures in agreement with SAXS data. We further explored the conformational space in the s2m nonaloop and its transient closing 5’-G-U-3’ base pair by MD simulations weighted by experimental NMR observables, leading to a comprehensive representation of the s2m nonaloop motif. Our deconvolution of the ensemble into conformations and dynamics provides a basis for future ensemble-functional characterisation of functional RNA structures featuring dynamic motifs.
34 3 - Research DataTargeting the SARS-CoV-2 RNA translation initiation element SL1 by molecules of low molecular weight2025-03-25We present the development of low molecular weight inhibitors that target the 5’-terminal RNA stem-loop 1 (SL1) of the SARS-CoV-2 genome. SL1 is crucial for allowing viral protein synthesis in the context of global translation repression in infected cells. We applied compound- and RNA-detected nuclear magnetic resonance spectroscopy (NMR) experiments to guide a fragment-growth strategy based on two primary NMR screening hits from a diverse fragment library poised for follow-up chemistry. These primary hits with molecular weights of around 200 Da were derivatized with the aim to improve solubility, binding affinity, and target specificity. We used NMR to monitor solubility changes, binding affinity, and specific binding to the SL1 binding pocket along the fragment derivatization campaign. The six compounds scoring best in all three categories were tested for their inhibitory effect on SL1 in a cell-free translation assay, where the best two compounds, A.2 and A.13, showed both significant and selective inhibition. Our results demonstrate that small molecules targeting translation initiation of SARS-CoV-2 can be rapidly obtained using NMR-guided medicinal chemistry, and that the correlation between affinity, selectivity, and in situ function of the derived compounds is still to be explored.
1 14 - Research DataCodon Wizard programe2019-04-04Optimization 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 optimization 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.
18 2 - Research DataStructural Insights into Spare-Tire DNA G-Quadruplex from the Human VEGF Promoter2025-03-10The vascular endothelial growth factor (VEGF) promoter region, which is involved in cancer progression, con-tains guanine-rich sequences capable of forming G-quadruplex (G4) structures. G4s play a critical role in transcriptional regulation and genomic stability and exhibit high structural polymorphism. The major VEGF G4 adopts a parallel topology involving the first four of five G-tracts (VEGF1234), while a potential "spare-tire" mechanism suggests the formation of VEGF1245 in response to oxidative damage. Here, we characterize this alternative G4 (VEGF1245), formed by excluding the third G-tract, using circular dichroism (CD) and nu-clear magnetic resonance (NMR) spectroscopy. Structural analysis reveals that VEGF1245 folds in a hybrid conformation. Different to other five tract containing G4s, for which various stand topologies can rapidly interconvert, VEGF1245 remains thermodynamically metastable and does not refold spontaneously into VEGF1234 at physiological temperatures. Further trapping of the VEGF1245 conformation by a photolabile protecting group and its in-situ release documents that the transition to VEGF1234 requires elevated tem-peratures, implicating kinetic barriers in the refolding process and the delineation of VEGF1245 as prominent metastable conformation. Our findings provide new insights into transcriptional regulation and DNA repair for the cancer-related VEGF-G4.
14 2 - Research DataNMR screening of low molecular weight inhibitors targeting the papain-like protease (PLPro) of SARS-CoV-22025-03-10The Papain-like protease (PLPro) from SARS-CoV-2 plays an important role in the cleavage of the polyproteins Pp1a and Pp1ab as well as in the suppression of the immune response by deISG15ylation. Considerable effort is therefore devoted to developing low molecular weight inhibitors as starting points for antiviral drugs. Here, we present the results of an NMR screening study of PLPro for binding to the DSI-poised fragment library containing 607 compounds. Based on Saturation-Transfer Difference (STD)- and WaterLOGSY-NMR experiments, we identified 86 binding compounds. We prioritized five candidates for further in-depth analysis. For three of those, we determined dissociation constants and two distinct binding sites on PLPro.
21 5 - Research DataNMR characterisation of the antibiotic resistance-mediating 32mer RNA from the 23S ribosomal RNA2025-02-26The increasing prevalence of antibiotic resistance represents a significant public health concern, underscoring the urgent need for the development of novel therapeutic strategies. Macrolides, the second most widely used class of antibiotics, are inhibited by Erm-proteins through the methylation of adenosine 2058 of the 23S ribosomal RNA (rRNA) (~2900 nucleotides). This methylation is the molecular basis for preventing macrolides from binding and bacteria (Staphylococcus, Streptococcus and Enterococcus) developing resistance. While Erm-proteins have received considerable attention, the role of the ribosomal RNA in acquiring antibiotic resistance is frequently undervalued, even though the ribosomal RNA is the chemical target for methylation. Here, we present the comprehensive resonance assignment for 1H, 13C and 15N for the part of the 23S RNA that serves as the Erm substrate in antimicrobial resistance by solution NMR spectroscopy. Furthermore, we compare the chemical shift signature of the methylated and dimethylated RNA construct and show that changes in the RNA upon methylation are locally restricted. The chemical shift assignments provide a starting point for investigating and targeting the molecular mechanism of the resistance-conferring Erm proteins.
17 - Research DataPELDOR on multi-nitroxide model compounds2025-01-31We investigated the accuracy and limitation of using the modulation depth of pulsed electron-electron double resonance experiments to count the number of coupled spins. For this purpose synthesized multi-nitroxide molecules with two to six spins were used. We could show that the main limitation on accurately counting larger number of coupled spins at Q-band frequencies is determined by the reproducibility of adjusting and calibrating the pump pulse excitation efficiency. Contrariwise, with broadband sech/tanh or short 10 ns rectangular pump pulses modulation depth suppression effects arising from non-ideal coverage of the dipolar-split signals can be avoided for molecules with intra-molecular spin distances larger than 2nm. The transverse relaxation times for our model compounds with one to six spins did not depend on the spin number and were all the same. Nevertheless, the signal intensity of the primary Hahn echo signal in a 4-pulse PELDOR sequence decreased strongly with the number of coupled spins. This is due to the dipolar defocusing if more than one spin is excited by the first two pulses at the detection frequency, resulting in a loss of refocused echo intensity of the PELDOR experiments. This effect further reduces the accuracy of using the PELDOR modulation depth for spin counting. Altogether, our results demonstrate, that this method can potentially be applied for application up to hexameric complexes.
33 4 - Research DataExploring the modulation of the complex folding landscape of Human Telomeric DNA by low molecular weight ligands2025-02-04Telomeric DNA forms G-quadruplex (G4) structures. G4s are crucial for genomic stability and therapeutic targeting. Using time-resolved NMR and CD spectroscopies, we investigated how the ligand Phen-DC3 modulates the folding of the human telomeric repeat 23TAG DNA sequence into G4. The kinetics are modulated by the ligand and by the presence of potassium cations (K+). Ligand binding to G4 occurs via a triphasic process with fast and slow phases. Notably, for the G4 structure in the presence of K+, the slow rate is ten times slower than without K+. These findings offer key insights into the modulation of the complex folding landscape of G4s by ligands, advancing our understanding of G4-ligand interactions for potential therapeutic applications.
5 61 - Research DataEnergy Transfer Booster: How a Leaving group controls the excited state pathway within a caging BASHY-BODIPY dyad - All spectrocopic and theoretical data2024-12-19All spectroscopic and theoretical data from the manuscript are available.
45 4 - Research DataStructural response of G protein binding to the cyclodepsipeptide inhibitor FR900359 probed by NMR spectroscopy2024-07-04The cyclodepsipeptide FR900359 (FR) and its analogs are able to selectively inhibit the class of Gq proteins by blocking GDP/GTP exchange. The inhibitor binding site of Gq has been characterized by X-ray crystallography, and various binding and functional studies have determined binding kinetics and mode of inhibition. Here we investigate isotope-labeled FR bound to the membrane-anchored G protein heterotrimer by solid-state nuclear magnetic resonance (ssNMR) and in solution by liquid-state NMR. The resulting data allowed us to identify regions of the inhibitor which show especially pronounced effects upon binding and revealed a generally rigid binding mode in the cis conformation under native-like conditions. The inclusion of the membrane environment allowed us to show a deep penetration of FR into the lipid bilayer illustrating a possible access mode of FR into the cell. Dynamic nuclear polarization (DNP)-enhanced ssNMR was used to observe the structural response of specific segments of the Gα subunit to inhibitor binding. This revealed rigidification of the switch I binding site and an allosteric response in the α5 helix as well as suppression of structural changes induced by nucleotide exchange due to inhibition by FR. Our NMR studies of the FR-G protein complex conducted directly within a native membrane environment provide important insights into the inhibitors access via the lipid membrane, binding mode, and structural allosteric effects.
29 4 - Research DataMolecular Mechanisms and Evolutionary Robustness of a Color Switch in Proteorhodopsins2023-10-17The data set is associated with manuscript: Jiafei Mao, Xinsheng Jin, Man Shi, David Heidenreich, Lynda J. Brown, Richard C.D. Brown, Moreno Lelli, Xiao He, Clemens Glaubitz: Molecular Mechanisms and Evolutionary Robustness of a Color Switch in Proteorhodopsins; submitted to Science Advances (adj0384). It contains the NMR data from Figures 2, 3, 4, S2-15 as well as data for the bioinformatics analysis in Figues 1d, 7, S22-S24. An overview of all samples and spectra is provided in Table S1 of the manuscript.
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