Faculty of Biochemistry, Chemistry and Pharmacy: Research Data
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- Research Data1H, 13C and 15N chemical shift assignment for stem-loop 5a from the 5‘UTR of HCoV-229E2025-06-18Due to the emergence of the SARS-CoV-2 virus, research on coronaviruses has been massively accelerated. In addition to SARS-CoV-2, there are other human infectious coronaviruses, including HCoV-229E. In all coronaviruses, secondary structure predictions indicate the presence of conserved structural elements in the 5'-untranslated region (5'-UTR). These conserved elements play crucial roles in RNA translation and replication. Stem-loop 5 (SL5), consisting of three substructures (5a, 5b, 5c), is highly conserved and harbours the start codon for viral translation. SL5 has repetitive structural motifs (RSMs), 5'-UUYYGU-3', which are conserved in many Alpha- and Betacoronaviruses. In the following, we present the 1H, 13C and 15N NMR resonance assignment of the SL5a RNA element from HCoV-229E and variations in the RSMs to show the effect of loop mutations on the structure of the hexaloop, revealing the different impact of each loop nucleotide on RNA dynamics.
46 11 - Research DataArchitectural principles of transporter-chaperone coupling within the native MHC I peptide-loading complex2025Adaptive immune responses are initiated by major histocompatibility class I (MHC I) presentation of antigenic peptides on the cell surface. This process relies on the peptide-loading complex (PLC), a dynamic transporter-multichaperone assembly in the endoplasmic reticulum (ER), to ensure high-fidelity selection, editing, and loading of peptides onto MHC I heterodimers. The PLC is the primary target for viral immune evasion, elicited in particular by human cytomegalovirus (HCMV), causing lifelong infections with severe risks for immunocompromised individuals. While the overall architecture of the PLC is known, how its activity is jeopardized by viral immune evasins remains unclear. Here, we present the 2.59–2.88 Å cryogenic electron microscopy structure of native human PLC associated with the HCMV immune evasin US6. US6 inhibits the heterodimeric transporter associated with antigen processing (TAP1/2) by latching its transmembrane helix laterally onto TAP2 and using its central disulfide-rich domain to mimic a translocating peptide. This effectively plugs the ER-lumenal exit and locks TAP in an outward-facing open conformation with closed nucleotide-binding domains and asymmetrically occluded ATP and ADP. In addition, the structure highlights the role of the unique N-terminal transmembrane domains of TAP as dynamic scaffolds that recruit the MHC I-specific chaperone tapasin by clamping its transmembrane helix to the core transmembrane domain of each transporter subunit. Our findings uncover how structural dynamics within human PLC are modulated by US6-mediated viral immune evasion and reveal potential targets for therapeutic modulation of antigen presentation in cancer and infectious diseases.
58 7 - Research DataArchitectural principles of transporter-chaperone coupling within the native MHC I peptide-loading complex2025-11-25Adaptive immunity depends on major histocompatibility complex class I (MHC I) presentation of peptides, a process orchestrated by the peptide-loading complex (PLC) in the endoplasmic reticulum. The PLC ensures precise peptide selection and loading and is a major target of viral immune evasion, notably by human cytomegalovirus (HCMV). Here, we report the 2.59–2.88 Å cryo-electron microscopy structure of native human PLC bound to the HCMV immune evasin US6. US6 inhibits the TAP1/2 transporter by laterally attaching its transmembrane helix to TAP2 using a disulfide-rich domain to mimic a translocating peptide. This domain blocks the ER-lumenal exit and locks TAP in an outward-facing conformation with closed nucleotide-binding domains and asymmetric ATP/ADP occlusion. The structure also reveals how TAP’s N-terminal transmembrane domains scaffold the MHC I chaperone tapasin. These findings elucidate the mechanism of US6-mediated immune evasion and highlight potential targets for therapeutic modulation of immune presentation in infection and cancer.
31 2 - 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.
47 7 - Research DataDissecting the stabilities of tetrads in G-quadruplexes by hydrogen exchange to derive a structure-activity relation for a thrombin-binding DNA G-quadruplex aptamer2025-09-18Guanosine- and deoxyguanosine-rich nucleic acids can form G-quadruplex structures (G4) that are stabilized by guanine tetrads (G4 tetrads). G4s find numerous application in biotechnology and we study here the so called TBA aptamer, developed by SELEX procedures, that adopts a G4 conformation and inhibits clotting of thrombin. We investigate the TBA G4 and its variants with either four adenosine desoxynucleotides or four abasic sites attached either to the 5’-terminus (A4-TBA and ab4-TBA) or the 3’-terminus (TBA-ab4 and TBA-A4). Biophysical characterization of the variants reveals that the structure of the aptamer remains unchanged but that their different thermal stabilities correlate with the anti-clotting activity of TBA. Hydrogen exchange quantified by nuclear magnetic resonance spectroscopy (NMR) reveals individual G4 tetrad thermodynamics. Our data indicate that while enthalpy, entropy and free energy of base pair opening shows surprisingly low variation, a hotspot for stabilization of the G4 is present at the 3’-terminal tetrad of TBA.
15 3 - Research DataDNP-enhanced magic angle spinning solid-state NMR spectroscopy to determine RNA-ligand interactions2025-10-10Understanding the molecular recognition underlying RNA-ligand complex formation is of key importance to explain RNA regulatory function of riboswitches and to support the development of low molecular weight RNA binders as starting points for RNA-targeting drugs. Here, we report magic-angle spinning solid-state NMR spectroscopic studies enhanced by dynamic nuclear polarization (MAS-DNP) to determine the molecular recognition of a ligand-RNA riboswitch complex. We benchmarked different labeling strategies for four large RNAs (70-86 nt) of the aptamer domain of a 2’deoxyguanosine-sensing riboswitch from Mesoplasma florum. Samples were either prepared by chemo-enzymatic approaches or by solid-phase chemical RNA synthesis employing different labeling schemes of riboswitches of up to 86 nucleotides. RNA-ligand complexes were prepared by addition of their cognate metabolite. We show that nucleotide- and ligand-selective labeling are a prerequisite for the MAS-DNP studies to reduce the NMR signal overlap present in such large RNAs. We further extend site-specific labeling to atom-specific labeling that allowed us to derive the structure of the ligand binding pocket extending the application of 2D-13C,15N-TEDOR experiments. The work described here opens the avenue for the investigation of large RNA-ligand complexes by MAS-DNP.
14 3 - Research DataDNP-enhanced magic angle spinning solid-state NMR spectroscopy to determine RNA-ligand interactions2025-10-10Understanding the molecular recognition underlying RNA-ligand complex formation is of key importance to explain RNA regulatory function of riboswitches and to support the development of low molecular weight RNA binders as starting points for RNA-targeting drugs. Here, we report magic-angle spinning solid-state NMR spectroscopic studies enhanced by dynamic nuclear polarization (MAS-DNP) to determine the molecular recognition of a ligand-RNA riboswitch complex. We benchmarked different labeling strategies for four large RNAs (70-86 nt) of the aptamer domain of a 2’deoxyguanosine-sensing riboswitch from Mesoplasma florum. Samples were either prepared by chemo-enzymatic approaches or by solid-phase chemical RNA synthesis employing different labeling schemes of riboswitches of up to 86 nucleotides. RNA-ligand complexes were prepared by addition of their cognate metabolite. We show that nucleotide- and ligand-selective labeling are a prerequisite for the MAS-DNP studies to reduce the NMR signal overlap present in such large RNAs. We further extend site-specific labeling to atom-specific labeling that allowed us to derive the structure of the ligand binding pocket extending the application of 2D-13C,15N-TEDOR experiments. The work described here opens the avenue for the investigation of large RNA-ligand complexes by MAS-DNP.
62 1 - 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 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.
6 74 - Research DataMechanism of the glycan-driven MHC I quality control cycle mediated by a dedicated chaperone network2025-04-24Protein folding in the endoplasmic reticulum (ER) is crucial for about one third of the mammalian proteome. N-linked glycosylation and subsequent restructuring of glycans barcodes glycoproteins during their maturation. UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) and the chaperones calnexin and calreticulin together with glucosidase I play a vital role in this process. MHC I molecules, key for adaptive immunity, additionally rely on the specialized chaperones tapasin and TAPBPR (TAP-binding protein-related) for their maturation and loading of antigenic peptides. Here, we delineate the functional interplay between tapasin, TAPBPR, UGGT1, and calreticulin, during recycling of MHC I molecules via purified components. The transfer of peptide-receptive MHC I from TAPBPR back to tapasin relies on the recognition of the mono-glucosylated glycan by calreticulin. Our findings unveil a finetuned dynamic network of glycan-dependent and MHC I-specific chaperones that guarantee maturation of MHC I molecules and highlight the fundamental processes driving ER protein quality control.
48 6 - 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.
117 21 - Research DataMultistate Dihydroazulene-Spiropyran Dyads: Path-Dependent Switchings and Refinement of the “Meta-rule” of Photoactivity2025-04-08Multistate switches are interesting systems for a plethora of potential applications, such as for data storage involving many different states or for logic operations characterized by specific outputs. The main challenge is to achieve a precise control of accessibility to a specific state via a given sequence of multiple stimuli. Here, we have connected dihydroazulene (DHA) and spiropyran (SP) photoswitches in dyads to elucidate differences in optical and switching properties between ortho-, meta-, and para-phenylene-bridged dyads. Dyads were prepared by Suzuki and Sonogashira coupling reactions and photoisomerizations studied in detail by stationary and ultrafast spectroscopies. Moreover, the kinetics of thermal back-reactions of meta-stable states were studied. The results show path-dependent switchings of the dyads using light in combination with other stimuli (acid/base/heat), allowing access to eight distinct states. The accessibility to some specific states via only one sequence of external stimuli provides an additional degree of data storage—information is not only stored as the state itself but also as the unique sequence of stimuli required to reach this state. By changing the bridging unit between the photoswitches, various properties (outputs) were finely tuned such as absorption and fluorescence behaviors, lifetime of meta-stable state, and photoisomerization dynamics.
18 1 - 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.
37 6 - 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.
40 8 - 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.
47 11 - 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.
56 6 - Research DataReconstitution of glycan-driven MHC I recycling reveals calreticulin as mediator between TAPBPR and tapasin2025-04-24Protein folding in the endoplasmic reticulum (ER) is essential for about one-third of the mammalian proteome. N-linked glycosylation and subsequent glycan remodeling barcodes glycoproteins during their maturation in the ER. Major histocompatibility complex class I (MHC I) molecules, key for adaptive immunity, rely on a dedicated quality control cycle that involves specialized chaperones and glycan-modifying enzymes for their maturation and loading of immunogenic peptides. However, the functional interplay of the MHC I editors tapasin as part of the peptide-loading complex (PLC), TAP-binding protein-related (TAPBPR), the UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), and calreticulin in glycan-dependent transfer of MHC I clients has not been determined in a reconstituted system. With isolated components, we show that transfer of peptide-receptive MHC I from the downstream quality control factor TAPBPR back to tapasin depends on the recognition of the monoglucosylated glycan of MHC I by calreticulin. While calreticulin’s C-terminal acidic helix is dispensable for disengaging reglucosylated MHC I from TAPBPR, it is essential for docking MHC I onto tapasin. Our data provide a mechanistic basis for glycan-surveillance by calreticulin necessary for retrograde trafficking of misfolded or suboptimally loaded MHC I that escaped the first quality control at the PLC and were trapped by TAPBPR. Such finetuned dynamic network of glycan-dependent and MHC I-specific chaperones guarantees maturation of MHC I molecules and highlight the fundamental processes driving ER protein quality control.
57 7 - Research DataRNA•DNA:DNA Triplex Formation Modulates Individual Base Pair Stabilities in the DNA Target Duplex Revealed by NMR Observed Solvent Exchange2025-09-30Long non-coding RNAs (lncRNAs) play a key role in numerous biological processes, including gene regulation. One potential mechanism involves the formation of RNA•DNA:DNA triplex structures, where the lncRNA binds in the major groove of a target DNA via Hoogsteen base pairs. Here, we investigated the impact of the lncRNA binding on the stability of the DNA duplex target to gain more insights into the triplex formation process. Quantification of the temperature-dependent imino proton solvent exchange allows the determination of the changes of individual DNA duplex base pairs upon triplex formation. The data shown here demonstrate that triplex formation alters DNA structure and stability by affecting both hydrogen bonding and base-stacking interactions. These thermodynamic insights support the prediction of triplex stability and enhance the understanding of RNA•DNA:DNA triplex formation.
23 3 - Research DataSingle-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC transporter2025-07-25ATP-binding cassette (ABC) transporters are molecular machines involved in diverse physiological processes, including antigen processing by TAP, a key component of adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes, yet the precise mechanism linking ATP binding to substrate movement remains unclear. Here, we employ a single-molecule FRET sensor to visualize single translocation events by individual ABC transporters, overcoming the limitations of ensemble averaging. This approach reveals that substrate transport is driven by a conformational switch from the inward- to the outward-facing state. Using a slow-turnover TmrAB variant, we demonstrate that ATP binding alone, even in the absence of Mg2+, is sufficient to drive a single round of peptide translocation. Cryo-EM structures of wild-type and slow-turnover TmrAB show that ATP binding induces the outward-facing conformation without Mg2+. In wild-type TmrAB, this conformational transition supports a single translocation event, whereas Mg2+-dependent ATP hydrolysis is required to reset the transporter. These findings establish a direct mechanistic link between ATP binding and substrate translocation at single-molecule resolution, providing new insights into the catalytic cycle of ABC transporters.
10 2 - Research DataSingle-molecule dynamics reveals ATP binding alone powers substrate translocation by an ABC transporter2025-07-25ATP-binding cassette (ABC) transporters are molecular machines that are involved in a wide range of physiological processes, including antigen presentation by TAP, a key player in adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes. To elucidate the mechanism of substrate transport, we employed a single-molecule FRET sensor to visualize single-translocation events by individual ABC transporters, overcoming the limitations of ensemble averaging. Using this approach, we tracked the membrane translocation of single peptides driven by a conformational switch from the inward- to the outward-facing state. In a slow-turnover TmrAB variant, even in the absence of Mg2+, we show that ATP binding alone is sufficient to drive peptide translocation. Structural analyses via cryogenic electron microscopy reveal that ATP binding induces an outward-facing conformation in both wild-type and mutant TmrAB, even without Mg2+. In wild-type TmrAB, this was sufficient for a single peptide translocation. However, Mg2+ was essential for the later stages of ATP hydrolysis and transporter resetting. Together, these results reveal a direct mechanistic link between ATP binding and substrate translocation at single-molecule resolution, providing new insights into the catalytic cycle of ABC transporters.
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