@misc{21145, abstract = {Protein conformational energy landscapes are shaped not only by intramolecular interactions but also by their environment. In protein crystals and protein-protein complexes, intermolecular contacts alter this energy landscape, but the exact nature of this alteration is difficult to decipher. Understanding how the crystal lattice affects protein dynamics is crucial for crystallography-based studies of motion, yet its influence on collective motions remains unclear. Aromatic ring flips in the hydrophobic core represent sensitive probes of such dynamics. Here, we compare the kinetics of aromatic ring flips in the protein GB1 in crystals, in complex with its binding partner IgG, and in solution, combining advanced isotope labeling with quantitative NMR methods. We show that rings in the core flip nearly a thousand times less frequently in crystals than in solution. Enhanced-sampling molecular dynamics simulations, based on a new crystal structure, reproduce these elevated barriers and reveal how the crystal restrains motions. }, author = {Becker, Lea Marie and Schanda, Paul and Chipot, Christophe}, publisher = {Institute of Science and Technology Austria}, title = {{Additional Data for "Aromatic Ring Flips Reveal Reshaping of Protein Dynamics in Crystals and Complexes"}}, doi = {10.15479/AT-ISTA-21145}, year = {2026}, } @misc{21284, abstract = {The advantageous characteristics attributed to the 19F nucleus have made it a popular target for NMR once again in recent years. Aside from solution NMR, an increasing number of studies have been conducted applying solid-state magic-angle-spinning NMR to fluorine-labeled samples. Here, the high chemical shift anisotropy and strong dipolar couplings can be utilized to get structural insights into proteins and measure long distances. Despite increasing popularity and promising benefits, the sensitivity of biomolecular 19F MAS NMR often suffers from slow longitudinal T1 relaxation and therefore long recycle delays. In this work, we expand paramagnetic doping, an approach commonly used to reduce proton T1 relaxation times, to 19F-labeled biological samples. We study the effect of Gd(DTPA) and Gd(DTPA-BMA) on 19F and 13C T1 and T2 relaxation in a [5-19F13C]-tryptophan-labeled protein via 19F-detected MAS NMR experiments. The observed paramagnetic relaxation enhancement substantially reduces measurement times of 19F MAS NMR experiments without compromising resolution. Additionally, we report the chemical-shift assignments of all four fluorotryptophan signals in the 12 × 39 kDa large protein using a mutagenesis approach.}, author = {Becker, Lea Marie and Schanda, Paul}, publisher = {Institute of Science and Technology Austria}, title = {{Research data for "Accelerated 19F biomolecular magic-angle spinning NMR with paramagnetic dopants"}}, doi = {10.15479/AT-ISTA-21284}, year = {2026}, } @misc{19956, abstract = {The specific introduction of 1H-13C or 1H-15N moieties into otherwise deuterated proteins holds great potential for high-resolution solution and magic-angle spinning (MAS) NMR studies of protein structure and dynamics. Arginine residues play key roles for example at active sites of enzymes. Taking advantage of a chemically synthesized Arg with a 13C-1H2 group in an otherwise deuterated backbone, we demonstrate here the usefulness of proton-detected arginine MAS NMR approaches to probe arginine dynamics. In experiments on crystalline ubiquitin and the 134 kDa tetrameric enzyme malate dehydrogenase we detected a wide range of motions, from sites that are rigid on time scales of at least tens of milliseconds to residues undergoing predominantly nanosecond motions. Spin-relaxation and dipolar-coupling measurements enabled quantitative determination of these dynamics. We observed microsecond dynamics of residue Arg54 in crystalline ubiquitin, whose backbone is known to sample different β-turn conformations on this time scale. The labeling scheme and experiments presented here expand the toolkit for high-resolution proton-detected MAS NMR}, author = {Schanda, Paul}, publisher = {Institute of Science and Technology Austria}, title = {{Arginine Dynamics Probed by Magic-Angle Spinning NMR with a Specific Isotope-Labeling Scheme}}, doi = {10.15479/AT-ISTA-19956}, year = {2025}, } @article{20184, abstract = {Specialized DNA polymerases facilitate various cellular processes. Despite extensive research, the mutagenic effects of these error-prone enzymes on genomes are not fully understood. Here we show that Pol IV promotes genomic instability in Pseudomonas aeruginosa by misincorporating oxidized guanine nucleotides. This activity led to a distinctive mutational signature, characterized by A-to-C transversions occurring preferentially at AT sites flanked by a 5’G and/or 3’C. Furthermore, Pol IV preferentially targeted pathogenicity genes located at specific chromosomal locations near the replication termination region and rRNA-encoding operons. Half of the mutation events catalyzed by Pol IV impaired gene function. This can be attributed to the bias of Pol IV for mutating codons with its preferred sequence contexts, leading to substitutions to unreactive alanine and glycine residues. Remarkably, mutation signatures identified for Pol IV were found in clinical isolate genomes of P. aeruginosa, providing compelling evidence for its role in genetic diversification during pathogen adaptation.}, author = {Castell, Sofía D. and Fernandez, Consuelo M. and Tumas, Ignacio N. and Margara, Lucía M. and Miserendino, Maria C and Ceschin, Danilo G. and Pezza, Roberto J. and Monti, Mariela R.}, issn = {2399-3642}, journal = {Communications Biology}, publisher = {Springer Nature}, title = {{The low-fidelity DNA Pol IV accelerates evolution of pathogenicity genes in Pseudomonas aeruginosa}}, doi = {10.1038/s42003-025-08589-5}, volume = {8}, year = {2025}, } @article{20258, abstract = {The specific introduction of ^1H-^13C or ^1H-^15N moieties into otherwise deuterated proteins holds great potential for high-resolution solution and magic-angle spinning (MAS) NMR studies of protein structure and dynamics. Arginine residues play key roles for example at active sites of enzymes. Taking advantage of a chemically synthesized Arg with a ^13C-^1H2 group in an otherwise deuterated backbone, we demonstrate here the usefulness of proton-detected MAS NMR approaches to probe arginine dynamics. In experiments with crystalline ubiquitin and the 134 kDa tetrameric enzyme malate dehydrogenase we detected a wide range of motions, from sites that are rigid on time scales of at least tens of milliseconds to residues undergoing predominantly nanosecond motions. Spin-relaxation and dipolar-coupling measurements enabled quantitative determination of these dynamics. We observed microsecond dynamics of residue Arg54 in crystalline ubiquitin, whose backbone is known to sample different β-turn conformations on this time scale. The labeling scheme and experiments presented here expand the toolkit for high-resolution proton-detected MAS NMR.}, author = {Rohden, Darja and Napoli, Federico and Kapitonova, Anna and Tatman, Benjamin and Lichtenecker, Roman J. and Schanda, Paul}, issn = {1089-8638}, journal = {Journal of Molecular Biology}, number = {23}, publisher = {Elsevier}, title = {{Arginine dynamics probed by magic-angle spinning NMR with a specific isotope-labeling scheme}}, doi = {10.1016/j.jmb.2025.169379}, volume = {437}, year = {2025}, } @article{20321, abstract = {Microsecond-to-millisecond motions are instrumental for many biomolecular functions, including enzymatic activity and ligand binding. Bloch-McConnell Relaxation Dispersion (BMRD) Nuclear Magnetic Resonance (NMR) spectroscopy is a key technique for studying these dynamic processes. While BMRD experiments are routinely used to probe protein motions in solution, the experiment is more demanding in the solid state, where dipolar couplings complicate the spin dynamics. It is believed that high deuteration levels are required and sufficient to obtain accurate and quantitative data. Here we show that even under fast magic-angle spinning and high levels of deuteration artifactual “bumps” in 15N R1ρ BMRD profiles are common. The origin of these artifacts is identified as a second-order three-spin Mixed Rotational and Rotary Resonance (MIRROR) recoupling condition. These artifacts are found to be a significant confounding factor for the accurate quantification of microsecond protein dynamics using BMRD in the solid state. We show that the application of low-power continuous wave (CW) decoupling simultaneously with the 15N spin-lock leads to the suppression of these conditions and enables quantitative measurements of microsecond exchange in the solid state. Remarkably, the application of decoupling allows the measurement of accurate BMRD even in fully protonated proteins at 100 kHz MAS, thus extending the scope of μs dynamics measurements in MAS NMR.}, author = {Tatman, Benjamin and Sridharan, Vidhyalakshmi and Uttarkabat, Motilal and Jaroniec, Christopher P. and Ernst, Matthias and Rovo, Petra and Schanda, Paul}, issn = {1520-5126}, journal = {Journal of the American Chemical Society}, number = {32}, pages = {29315--29326}, publisher = {American Chemical Society}, title = {{Bumps on the road: The way to clean relaxation dispersion magic-angle spinning NMR}}, doi = {10.1021/jacs.5c09057}, volume = {147}, year = {2025}, } @article{20538, abstract = {In this study, we describe an integrated approach for methyl group assignment comprising precursor-based selective methyl group labeling, a novel pulse sequence for methyl to backbone coherence transfer and chemical shift predictions using UCBShift 2.0. The utility of this novel α-ketoacid isotopologue is shown by the adaptation of an HMBC-HMQC pulse sequence that simultaneously connects geminal methyl groups of leucine and valine residues to each other and to the protein backbone. By additional 13C,2H-labeling of residues other than valine and leucine residues of the protein, important chemical shift information about neighboring residues (following valine and leucine residues) can be achieved. Thus, different valine and leucine residues in a protein can be characterized as a specific chemical shift vector. Frequency matching with predicted chemical shifts via UCBShift 2.0 using experimental data taken from a subset of the BMRB database revealed a correct assignment performance of about 90%. With applications to proteins of 60.2 kDa and 134 kDa (4 × 33.5 kDa) in size, we demonstrate that the approach provides valuable information even for very large proteins.}, author = {Knödlstorfer, Sonja and Toscano, Giorgia and Ptaszek, Aleksandra L. and Kontaxis, Georg and Napoli, Federico and Schneider, Jakob and Maier, Katharina and Kapitonova, Anna and Lichtenecker, Roman J. and Schanda, Paul and Konrat, Robert}, issn = {1089-8638}, journal = {Journal of Molecular Biology}, number = {23}, publisher = {Elsevier}, title = {{A novel HMBC-CC-HMQC NMR strategy for methyl assignment using triple-13C-labeled α-ketoisovalerate integrated with UCBShift 2.0}}, doi = {10.1016/j.jmb.2025.169465}, volume = {437}, year = {2025}, } @misc{20641, abstract = {Protein conformational energy landscapes are shaped not only by intramolecular interactions but also by their environment. In protein crystals and protein-protein complexes, intermolecular contacts alter this energy landscape, but the exact nature of this alteration is difficult to decipher. Understanding how the crystal lattice affects protein dynamics is crucial for crystallography-based studies of motion, yet its influence on collective motions remains unclear. Aromatic ring flips in the hydrophobic core represent sensitive probes of such dynamics. Here, we compare the kinetics of aromatic ring flips in the protein GB1 in crystals, in complex with its binding partner IgG, and in solution, combining advanced isotope labeling with quantitative NMR methods. We show that rings in the core flip nearly a thousand times less frequently in crystals than in solution. Enhanced-sampling molecular dynamics simulations, based on a new crystal structure, reproduce these elevated barriers and reveal how the crystal restrains motions. }, author = {Becker, Lea Marie and Schanda, Paul}, publisher = {Institute of Science and Technology Austria}, title = {{Data for "Aromatic Ring Flips Reveal Reshaping of Protein Dynamics in Crystals and Complexes"}}, doi = {10.15479/AT-ISTA-20641}, year = {2025}, } @article{19072, abstract = {Pathogenic fungal and bacterial cells are enveloped within a cell wall, a molecular barrier at their cell surface, and a critical architecture that constantly evolves during pathogenesis. Understanding the molecular composition, structural organization, and mobility of polysaccharides constituting this cell envelope is crucial to correlate cell wall organization with its role in pathogenicity and to identify potential antifungal targets. For the fungal pathogen Cryptococcus neoformans, the characterization of the cell envelope has been complexified by the presence of an additional external polysaccharide capsular shell. Here, we investigate how magic-angle spinning (MAS) solid-state NMR techniques increase the analytical capabilities to characterize the structure and dynamics of this encapsulated pathogen. The versatility of proton detection experiments, dynamic-based filters, and relaxation measurements facilitate the discrimination of the highly mobile external capsular structure from the internal rigid cell wall of C. neoformans. In addition, we report the in situ detection of triglyceride molecules from lipid droplets based on NMR dynamic filters. Together, we demonstrate a nondestructive technique to study the cell wall architecture of encapsulated microbes using C. neoformans as a model, an airborne opportunistic fungal pathogen that infects mainly immunocompromised but also competent hosts.}, author = {Lends, Alons and Lamon, Gaelle and Delcourte, Loic and Sturny-Leclere, Aude and Grélard, Axelle and Morvan, Estelle and Abdul-Shukkoor, Muhammed Bilal and Berbon, Mélanie and Vallet, Alicia and Habenstein, Birgit and Dufourc, Erick J. and Schanda, Paul and Aimanianda, Vishukumar and Loquet, Antoine}, issn = {1520-5126}, journal = {Journal of the American Chemical Society}, number = {8}, pages = {6813--6824}, publisher = {American Chemical Society}, title = {{Molecular distinction of cell wall and capsular polysaccharides in encapsulated pathogens by in situ magic-angle spinning NMR techniques}}, doi = {10.1021/jacs.4c16975}, volume = {147}, year = {2025}, } @article{19555, abstract = {The charged arginine side chain is unique in determining many innate properties of proteins, contributing to stability and interaction surfaces, and directing allosteric regulation and enzymatic catalysis. NMR experiments can be used to reveal these processes at the molecular level, but it often requires selective insertion of carbon-13, nitrogen-15, and deuterium at defined atomic positions. We introduce a method to endow arginine residues with defined isotope patterns, combining synthetic organic chemistry and cell-based protein overexpression. The resulting proteins feature NMR active spin systems with optimized relaxation pathways leading to simplified NMR spectra with a sensitive response to changes in the chemical environment of the nuclei observed.}, author = {Rohden, Darja and Toscano, Giorgia and Schanda, Paul and Lichtenecker, Roman J.}, issn = {1521-3765}, journal = {Chemistry - A European Journal}, number = {24}, publisher = {Wiley}, title = {{Synthesis of selectively 13C/2H/15N- labeled arginine to probe protein conformation and interaction by NMR spectroscopy}}, doi = {10.1002/chem.202500408}, volume = {31}, year = {2025}, } @misc{19696, author = {Tatman, Benjamin}, publisher = {Institute of Science and Technology Austria}, title = {{Dataset for "Bumps on the Road: The Way to Clean Relaxation Dispersion in the Solid State"}}, doi = {10.15479/AT-ISTA-19696}, year = {2025}, } @inproceedings{21327, abstract = {Proteins exist as a dynamic ensemble of multiple conformations, and these motions are often crucial for their functions. However, current structure prediction methods predominantly yield a single conformation, overlooking the conformational heterogeneity revealed by diverse experimental modalities. Here, we present a framework for building experiment-grounded protein structure generative models that infer conformational ensembles consistent with measured experimental data. The key idea is to treat stateof-the-art protein structure predictors (e.g., AlphaFold3) as sequence-conditioned structural priors, and cast ensemble modeling as posterior inference of protein structures given experimental measurements. Through extensive real-data experiments, we demonstrate the generality of our method to incorporate a variety of experimental measurements. In particular, our framework uncovers previously unmodeled conformational heterogeneity from crystallographic densities, and generates high-accuracy NMR ensembles orders of magnitude faster than the status quo. Notably, we demonstrate that our ensembles outperform AlphaFold3 (Abramson et al., 2024) and sometimes better fit experimental data than publicly deposited structures to the Protein Data Bank (PDB, Burley et al. (2017)). We believe that this approach will unlock building predictive models that fully embrace experimentally observed conformational diversity.}, author = {Maddipatla, Sai A and Sellam, Nadav E and Bojan, Meital I and Vedula, Sanketh and Schanda, Paul and Marx, Ailie and Bronstein, Alexander}, booktitle = {Proceedings of the 42nd International Conference on Machine Learning}, issn = {2640-3498}, location = {Vancouver, Canada}, pages = {42366 -- 42393}, publisher = {ML Research Press}, title = {{Inverse problems with experiment-guided AlphaFold}}, volume = {267}, year = {2025}, } @article{20664, abstract = {Conference travel contributes to the climate footprint of academic research. Here, we provide a quantitative estimate of the carbon emissions associated with conference attendance by analyzing travel data from participants of 10 international conferences in the field of magnetic resonance, namely EUROMAR, ENC and ICMRBS. We find that attending a EUROMAR conference produces, on average, more than 1 t CO2 eq.. For the analyzed conferences outside Europe, the corresponding value is about 2–3 times higher, on average, with intercontinental trips amounting to up to 5 t. We compare these conference-related emissions to other activities associated with research and show that conference travel is a substantial portion of the total climate footprint of a researcher in magnetic resonance. We explore several strategies to reduce these emissions, including the impact of selecting conference venues more strategically and the possibility of decentralized conferences. Through a detailed comparison of train versus air travel – accounting for both direct and infrastructure-related emissions – we demonstrate that train travel offers considerable carbon savings. These data may provide a basis for strategic choices of future conferences in the field and for individuals deciding on their conference attendance.}, author = {Kapoor, Lucky and Ruzickova, Natalia and Zivadinovic, Predrag and Leitner, Valentin and Sisak, Maria A and Mweka, Cecelia N and Dobbelaere, Jeroen A and Katsaros, Georgios and Schanda, Paul}, issn = {2699-0016}, journal = {Magnetic Resonance}, number = {2}, pages = {243--256}, publisher = {Copernicus Publications}, title = {{Quantifying the carbon footprint of conference travel: The case of NMR meetings}}, doi = {10.5194/mr-6-243-2025}, volume = {6}, year = {2025}, } @misc{20242, abstract = {This repository contains calculations of carbon footprints of NMR conferences, as described in the article by Lucky N. Kapoor, Natalia Ruzickova, Predrag Živadinović, Valentin Leitner, Maria Anna Sisak, Cecelia Mweka, Jeroen Dobbelaere, Georgios Katsaros, and Paul Schanda Published in Magnetic Resonance, 2025.}, author = {Schanda, Paul}, keywords = {sustainability, conference travel}, publisher = {Institute of Science and Technology Austria}, title = {{Data of: "Quantifying the carbon footprint of conference travel: the case of NMR meetings"}}, doi = {10.15479/AT-ISTA-20242}, year = {2025}, } @inbook{18167, abstract = {Holdase chaperones are essential in the mitochondrial membrane-protein biogenesis as they stabilize preproteins and keep them in an import-competent state as they travel through the aqueous cytosol and intermembrane space. The small TIM chaperones of the mitochondrial intermembrane space function within a fine balance of client promiscuity and high affinity binding, while being also able to release their client proteins without significant energy barrier to the downstream insertases/translocases. The tendency of the preproteins to aggregate and the dynamic nature of the preprotein—chaperone complexes makes the preparation of these complexes challenging. Here we present two optimized methods for complex formation of highly hydrophobic precursor proteins and chaperones: a pull-down approach and an in-vitro translation strategy. In the former, attaching the client protein to an affinity resin keeps the individual client protein copies apart from each other and decreases the client self-aggregation probability, thereby favouring complex formation. In the latter approach, a purified chaperone, added to the cell-free protein synthesis, captures the nascent precursor protein. The choice of method will depend on the desired client-chaperone complex amount, or the need for specific labeling scheme.}, author = {Guillerm, Undina and Sučec, Iva and Schanda, Paul}, booktitle = {Methods in Enzymology}, issn = {0076-6879}, pages = {391--422}, publisher = {Elsevier}, title = {{Generation of TIM chaperone substrate complexes}}, doi = {10.1016/bs.mie.2024.07.051}, volume = {707}, year = {2024}, } @article{18910, abstract = {Proteins often undergo large-scale conformational transitions, in which secondary and tertiary structure elements (loops, helices, and domains) change their structures or their positions with respect to each other. Simple considerations suggest that such dynamics should be relatively fast, but the functional cycles of many proteins are often relatively slow. Sophisticated experimental methods are starting to tackle this dichotomy and shed light on the contribution of large-scale conformational dynamics to protein function. In this review, we focus on the contribution of single-molecule Förster resonance energy transfer and nuclear magnetic resonance (NMR) spectroscopies to the study of conformational dynamics. We briefly describe the state of the art in each of these techniques and then point out their similarities and differences, as well as the relative strengths and weaknesses of each. Several case studies, in which the connection between fast conformational dynamics and slower function has been demonstrated, are then introduced and discussed. These examples include both enzymes and large protein machines, some of which have been studied by both NMR and fluorescence spectroscopies.}, author = {Schanda, Paul and Haran, Gilad}, issn = {1936-1238}, journal = {Annual Review of Biophysics}, pages = {247--273}, publisher = {Annual Reviews}, title = {{NMR and single-molecule FRET insights into fast protein motions and their relation to function}}, doi = {10.1146/annurev-biophys-070323-022428}, volume = {53}, year = {2024}, } @misc{17042, abstract = {Bacterial cell walls are gigadalton-large cross-linked polymers with a wide range of motional amplitudes, including rather rigid as well as highly flexible parts. Magic-angle spinning NMR is a powerful method to obtain atomic-level information about intact cell walls. Here we investigate sensitivity and information content of different homonuclear 13C-13C and heteronuclear H-N, H-C and N-C correlation experiments. We demonstrate that a CPMAS CryoProbe yields ca. 8-fold increased signal-to-noise over a room-temperature probe, or a ca. 3-4-fold larger per-mass sensitivity. The increased sensitivity allowed to obtain high-resolution spectra even on intact bacteria. Moreover, we compare resolution and sensitivity of 1H MAS experiments obtained at 100 kHz vs. 55 kHz. Our study provides useful hints for choosing experiments to extract atomic-level details on cell-wall samples. }, author = {Schanda, Paul}, keywords = {nuclear magnetic resonance, NMR, cellwall, structural biology, spectroscopy}, publisher = {Institute of Science and Technology Austria}, title = {{Raw data to "MAS NMR experiments of corynebacterial cell walls: complementary 1H- and CPMAS CryoProbe-enhanced 13C-detected experiments"}}, doi = {10.15479/AT:ISTA:17042}, year = {2024}, } @article{17161, abstract = {Dynamic processes in molecules can occur on a wide range of timescales, and it is important to understand which timescales of motion contribute to different parameters used in dynamics measurements. For spin relaxation, this can easily be understood from the sampling frequencies of the spectral-density function by different relaxation-rate constants. In addition to data from relaxation measurements, determining dynamically averaged anisotropic interactions in magic-angle spinning (MAS) solid-state NMR allows for better quantification of the amplitude of molecular motion. For partially averaged anisotropic interactions, the relevant timescales of motion are not so clearly defined. Whether the averaging depends on the experimental methods (e.g., pulse sequences) or conditions (e.g., MAS frequency, magnitude of anisotropic interaction, radio-frequency field amplitudes) is not fully understood. To investigate these questions, we performed numerical simulations of dynamic systems based on the stochastic Liouville equation using several experiments for recoupling the dipolar coupling, chemical-shift anisotropy or quadrupolar coupling. As described in the literature, the transition between slow motion, where parameters characterizing the anisotropic interaction are not averaged, and fast motion, where the tensors are averaged leading to a scaled anisotropic quantity, occurs over a window of motional rate constants that depends mainly on the strength of the interaction. This transition region can span 2 orders of magnitude in exchange-rate constants (typically in the microsecond range) but depends only marginally on the employed recoupling scheme or sample spinning frequency. The transition region often coincides with a fast relaxation of coherences, making precise quantitative measurements difficult. Residual couplings in off-magic-angle experiments, however, average over longer timescales of motion. While in principle one may gain information on the timescales of motion from the transition area, extracting such information is hampered by low signal-to-noise ratio in experimental spectra due to fast relaxation that occurs in the same region.}, author = {Aebischer, Kathrin and Becker, Lea Marie and Schanda, Paul and Ernst, Matthias}, issn = {2699-0016}, journal = {Magnetic Resonance}, number = {1}, pages = {69--86}, publisher = {Copernicus Publications}, title = {{Evaluating the motional timescales contributing to averaged anisotropic interactions in MAS solid-state NMR}}, doi = {10.5194/mr-5-69-2024}, volume = {5}, year = {2024}, } @article{17291, abstract = {Bacterial cell walls are gigadalton-large cross-linked polymers with a wide range of motional amplitudes, including rather rigid as well as highly flexible parts. Magic-angle spinning NMR is a powerful method to obtain atomic-level information about intact cell walls. Here we investigate sensitivity and information content of different homonuclear 13Csingle bond13C and heteronuclear 1Hsingle bond15N, 1Hsingle bond13C and 15Nsingle bond13C correlation experiments. We demonstrate that a CPMAS CryoProbe yields ca. 8-fold increased signal-to-noise over a room-temperature probe, or a ca. 3–4-fold larger per-mass sensitivity. The increased sensitivity allowed to obtain high-resolution spectra even on intact bacteria. Moreover, we compare resolution and sensitivity of 1H MAS experiments obtained at 100 kHz vs. 55 kHz. Our study provides useful hints for choosing experiments to extract atomic-level details on cell-wall samples.}, author = {Vallet, Alicia and Ayala, Isabel and Perrone, Barbara and Hassan, Alia and Simorre, Jean-Pierre and Bougault, Catherine and Schanda, Paul}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, publisher = {Elsevier}, title = {{MAS NMR experiments of corynebacterial cell walls: Complementary 1H- and CPMAS CryoProbe-enhanced 13C-detected experiments}}, doi = {10.1016/j.jmr.2024.107708}, volume = {364}, year = {2024}, } @article{15401, abstract = {Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H2O-based culture medium doped with deuterated cell lysate allow scientists to overcome this “reprotonation bottleneck” while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), the amide 1H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments.}, author = {Napoli, Federico and Guan, Jia-Ying and Arnaud, Charles-Adrien and Macek, Pavel and Fraga, Hugo and Breyton, Cécile and Schanda, Paul}, issn = {2699-0016}, journal = {Magnetic Resonance}, number = {1}, pages = {33--49}, publisher = {Copernicus Publications}, title = {{Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck}}, doi = {10.5194/mr-5-33-2024}, volume = {5}, year = {2024}, }