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.
59 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.
43 2 - Research DataATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter2026-01-16ATP-binding cassette (ABC) transporters are essential molecular machines whose conformational dynamics have largely been inferred from ensemble-averaged measurements. Resolving dynamic heterogeneity and transient intermediates, however, requires single-molecule approaches. Here, we use single-molecule Förster resonance energy transfer (smFRET) to directly monitor conformational changes of the heterodimeric type IV ABC transporter TmrAB, a functional homolog of the human antigen transporter TAP. Fluorophores positioned at the nucleotide-binding domains and the periplasmic gate were validated by accessible-volume simulations, fluorescence lifetimes, and ensemble FRET, demonstrating that these reporters reliably track conformational transitions. Single-molecule analysis distinguishes ATP-free and ATP-bound states and reveals ATP-dependent population shifts from micromolar to physiological ATP concentrations. Probing conformational dwell-times further uncovers an unexpectedly long ATP-bound dwell time of ~300 ms. Using complementary stabilization strategies–including a slow-turnover variant, Mg²⁺ depletion, or substrate trans-inhibition–we resolve a previously hidden outward-facing open state that rapidly interconverts with occluded intermediates under turnover conditions. These results provide the first single-molecule characterization of TmrAB and establish a general framework for dissecting ATP-coupled conformational dynamics in heterodimeric ABC transporters.
15 1 - 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.
48 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 DataDiverging pH dependence and photocycle dynamics across members of the CryoRhodopsin clade2026-01-15A new clade of microbial rhodopsins (MRs) called CryoRhodopsins (CryoRs) was recently discovered and characterized to possess a near-UV-absorbing intermediate populated for minutes at neutral to alkaline conditions. A detailed characterization of one member, CryoR1, showed a strong pH dependence, causing a hindered retinal isomerization and a significant acceleration of the photocycle dynamics at acidic conditions. Here, we present a spectroscopic study on the pH dependence of four other CryoRs (CryoR2-5), revealing strongly deviating photophysical properties within this subfamily under acidic conditions. While all investigated CryoRs possess a minute long photocycle duration at neutral to alkaline conditions, acidification shortens the photocycle to seconds and leads to variation in the observed photocycle schemes. Furthermore, this is not directly reflected in the absorption spectra and initial photoreactions, since CryoR4 and CryoR5 show very weak pH dependence, while CryoR2 and CryoR3 do show pH dependent shifts. In addition, CryoR3 is unique within both CryoRs and MRs in general, since a near-UV-absorbing species is clearly present throughout the whole pH range in the dark spectrum. The observed diversity suggests strong mechanistic divergence within this clade of MRs, a feature that was not observed for other clades of MRs. It also further supports functional relevance of the long-living near-UV-absorbing state of CryoRs under neutral and alkaline conditions.
15 2 - 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.
19 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.
7 78 - Research DataMANGO-“Fruitcage”: A conditional photocage utilizing a fluorescence light-up aptamer2026-02-10Photolabile protecting groups (PPGs) offer light-controlled molecular activation, but improving their photolysis quantum yield (QY) remains a challenge. Here, we present a proof of concept for an approach that we call "fruitcaging" which pertains to new conditionally activatable photocages. We show that a photocleavable thiazole orange 1 derivative (TO1-cage) alone has a QY below 0.1%, rendering it nearly inactive. However, in the presence of the fluorescence light-up aptamer (FLAP) Mango, the fluorescence emission increased 180-fold and the photocleavage reaction 14-fold. Femtosecond time resolved experiments revealed a drastically prolonged excited state lifetime of the TO1-cage upon binding to the Mango-aptamer, which facilitates the cleavage reaction and turned an inactive photocage to an active aptamer-photocage system - the “fruitcage”. This strategy offers detailed insights in the engineering of the excited state dynamics of a TO1-based PPG and therefore presents a new tool for efficient conditionally activated PPG for a variety of applications in the field of photochemistry.
1 8 - 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.
20 2 - 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.
38 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.
41 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.
57 6 - Research DataQuantifying protein homodimer affinities and the effect of molecular glues and interface residues using native mass spectrometry2025-12-19Biological processes rely on finely tuned homo- and heteromeric interactions between (biomacro)molecules. The strength of an interaction, typically given by the dissociation constant (KD), plays a crucial role in basic research and must be monitored throughout the development of drugs and agrochemicals. An ideal method for KD determination is applicable to various analytes with a large range of affinities, tolerates complex matrix compositions, does not require labeling, and simultaneously provides information on the structural integrity of the binding partners. Native mass spectrometry meets these criteria but typically struggles with homooligomeric complexes due to overlapping mass signals. To overcome this, we resolve monomer/dimer contributions to overlapping MS-peaks by separately analyzing the charge state distribution of each oligomeric species via sample dilution and covalent crosslinking. Following this approach, we show that quantitative Laser-Induced Liquid Bead Ion Desorption mass spectrometry (qLILBID-MS) accurately captures the affinities of Bovine Serum Albumin (BSA) and chemically induced dimers of Tryparedoxin (Tpx), an oxidoreductase from human pathogenic Trypanosoma brucei parasites, with various molecular glues and homodimer affinities. Conveniently, qLILBID-MS requires a fraction of sample used by other methods such as isothermal titration calorimetry (ITC) and yields previously inaccessible protein homodimer KDs in the high micromolar range, which allowed us to monitor the gradual decrease in homodimer affinity via mutation of crucial dimer interface contacts. Overall, qLILBID-MS is a sensitive, robust, fast, scalable, and cost-effective alternative to quantify protein/protein interactions, that can accelerate contemporary drug discovery workflows, e.g. the efficient screening for proximity inducing molecules like proteolysis targeting chimera (PROTACs) and molecular glues.
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