@article{13095, abstract = {Disulfide bond formation is fundamentally important for protein structure and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive μs time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfill other favorable contacts.}, author = {Troussicot, Laura and Vallet, Alicia and Molin, Mikael and Burmann, Björn M. and Schanda, Paul}, issn = {1520-5126}, journal = {Journal of the American Chemical Society}, number = {19}, pages = {10700–10711}, publisher = {American Chemical Society}, title = {{Disulfide-bond-induced structural frustration and dynamic disorder in a peroxiredoxin from MAS NMR}}, doi = {10.1021/jacs.3c01200}, volume = {145}, year = {2023}, } @misc{12820, abstract = {Disulfide bond formation is fundamentally important for protein structure, and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive microsecond time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a designed dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfil other favorable contacts. This data repository contains NMR data presented in the associated manuscript}, author = {Schanda, Paul}, publisher = {Institute of Science and Technology Austria}, title = {{Research data of the publication "Disulfide-bond-induced structural frustration and dynamic disorder in a peroxiredoxin from MAS NMR"}}, doi = {10.15479/AT:ISTA:12820}, year = {2023}, } @article{12114, abstract = {Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein’s hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10–20 ns time scale, and Phe45 are broadened in all crystals, presumably due to µs motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects.}, author = {Gauto, Diego F. and Lebedenko, Olga O. and Becker, Lea Marie and Ayala, Isabel and Lichtenecker, Roman and Skrynnikov, Nikolai R. and Schanda, Paul}, issn = {2590-1524}, journal = {Journal of Structural Biology: X}, keywords = {Structural Biology}, publisher = {Elsevier}, title = {{Aromatic ring flips in differently packed ubiquitin protein crystals from MAS NMR and MD}}, doi = {10.1016/j.yjsbx.2022.100079}, volume = {7}, year = {2023}, } @article{13096, abstract = {Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1,2,3,4,5,6,7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.}, author = {Degen, Morris and Santos, José Carlos and Pluhackova, Kristyna and Cebrero, Gonzalo and Ramos, Saray and Jankevicius, Gytis and Hartenian, Ella and Guillerm, Undina and Mari, Stefania A. and Kohl, Bastian and Müller, Daniel J. and Schanda, Paul and Maier, Timm and Perez, Camilo and Sieben, Christian and Broz, Petr and Hiller, Sebastian}, issn = {1476-4687}, journal = {Nature}, pages = {1065--1071}, publisher = {Springer Nature}, title = {{Structural basis of NINJ1-mediated plasma membrane rupture in cell death}}, doi = {10.1038/s41586-023-05991-z}, volume = {618}, year = {2023}, } @misc{14861, abstract = {Cover Page}, author = {Becker, Lea Marie and Berbon, Mélanie and Vallet, Alicia and Grelard, Axelle and Morvan, Estelle and Bardiaux, Benjamin and Lichtenecker, Roman and Ernst, Matthias and Loquet, Antoine and Schanda, Paul}, booktitle = {Angewandte Chemie International Edition}, issn = {1521-3773}, keywords = {General Chemistry, Catalysis}, number = {19}, publisher = {Wiley}, title = {{Cover Picture: The rigid core and flexible surface of amyloid fibrils probed by Magic‐Angle‐Spinning NMR spectroscopy of aromatic residues}}, doi = {10.1002/anie.202304138}, volume = {62}, year = {2023}, } @article{14835, abstract = {Aromatische Seitenketten sind wichtige Indikatoren für die Plastizität von Proteinen und bilden oft entscheidende Kontakte bei Protein‐Protein‐Wechselwirkungen. Wir untersuchten aromatische Reste in den beiden strukturell homologen cross‐β Amyloidfibrillen HET‐s und HELLF mit Hilfe eines spezifischen Ansatzes zur Isotopenmarkierung und Festkörper NMR mit Drehung am magischen Winkel. Das dynamische Verhalten der aromatischen Reste Phe und Tyr deutet darauf hin, dass der hydrophobe Amyloidkern starr ist und keine Anzeichen von “atmenden Bewegungen” auf einer Zeitskala von Hunderten von Millisekunden zeigt. Aromatische Reste, die exponiert an der Fibrillenoberfläche sitzen, haben zwar eine starre Ringachse, weisen aber Ringflips auf verschiedenen Zeitskalen von Nanosekunden bis Mikrosekunden auf. Unser Ansatz bietet einen direkten Einblick in die Bewegungen des hydrophoben Kerns und ermöglicht eine bessere Bewertung der Konformationsheterogenität, die aus einem NMR‐Strukturensemble einer solchen Cross‐β‐Amyloidstruktur hervorgeht.}, author = {Becker, Lea Marie and Berbon, Mélanie and Vallet, Alicia and Grelard, Axelle and Morvan, Estelle and Bardiaux, Benjamin and Lichtenecker, Roman and Ernst, Matthias and Loquet, Antoine and Schanda, Paul}, issn = {1521-3757}, journal = {Angewandte Chemie}, keywords = {General Medicine}, number = {19}, publisher = {Wiley}, title = {{Der starre Kern und die flexible Oberfläche von Amyloidfibrillen – Magic‐Angle‐Spinning NMR Spektroskopie von aromatischen Resten}}, doi = {10.1002/ange.202219314}, volume = {135}, year = {2023}, } @inbook{14847, abstract = {Understanding the mechanisms of chaperones at the atomic level generally requires producing chaperone–client complexes in vitro. This task comes with significant challenges, because one needs to find conditions in which the client protein is presented to the chaperone in a state that binds and at the same time avoid the pitfalls of protein aggregation that are often inherent to such states. The strategy differs significantly for different client proteins and chaperones, but there are common underlying principles. Here, we discuss these principles and deduce the strategies that can be successfully applied for different chaperone–client complexes. We review successful biochemical strategies applied to making the client protein “binding competent” and illustrate the different strategies with examples of recent biophysical and biochemical studies.}, author = {Sučec, I. and Schanda, Paul}, booktitle = {Biophysics of Molecular Chaperones}, editor = {Hiller, Sebastian and Liu, Maili and He, Lichun}, isbn = {9781839162824}, pages = {136--161}, publisher = {Royal Society of Chemistry}, title = {{Preparing Chaperone–Client Protein Complexes for Biophysical and Structural Studies}}, doi = {10.1039/bk9781839165986-00136}, volume = {29}, year = {2023}, } @article{14036, abstract = {Magic-angle spinning (MAS) nuclear magnetic resonance (NMR) is establishing itself as a powerful method for the characterization of protein dynamics at the atomic scale. We discuss here how R1ρ MAS relaxation dispersion NMR can explore microsecond-to-millisecond motions. Progress in instrumentation, isotope labeling, and pulse sequence design has paved the way for quantitative analyses of even rare structural fluctuations. In addition to isotropic chemical-shift fluctuations exploited in solution-state NMR relaxation dispersion experiments, MAS NMR has a wider arsenal of observables, allowing to see motions even if the exchanging states do not differ in their chemical shifts. We demonstrate the potential of the technique for probing motions in challenging large enzymes, membrane proteins, and protein assemblies.}, author = {Napoli, Federico and Becker, Lea Marie and Schanda, Paul}, issn = {1879-033X}, journal = {Current Opinion in Structural Biology}, number = {10}, publisher = {Elsevier}, title = {{Protein dynamics detected by magic-angle spinning relaxation dispersion NMR}}, doi = {10.1016/j.sbi.2023.102660}, volume = {82}, year = {2023}, } @article{12675, abstract = {Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein--protein interactions. By studying a pair of structurally homologous cross-β amyloid fibrils, HET-s and HELLF, with a specific isotope-labeling approach and magic-angle-spinning (MAS) NMR, we have characterized the dynamic behavior of Phe and Tyr aromatic rings to show that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips, on a variety of time scales from ns to µs. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from a NMR structural ensemble of such amyloid cross-β architecture.}, author = {Becker, Lea Marie and Berbon, Mélanie and Vallet, Alicia and Grelard, Axelle and Morvan, Estelle and Bardiaux, Benjamin and Lichtenecker, Roman and Ernst, Matthias and Loquet, Antoine and Schanda, Paul}, issn = {1521-3773}, journal = {Angewandte Chemie International Edition}, keywords = {General Chemistry, Catalysis}, number = {19}, publisher = {Wiley}, title = {{The rigid core and flexible surface of amyloid fibrils probed by Magic‐Angle Spinning NMR of aromatic residues}}, doi = {10.1002/anie.202219314}, volume = {62}, year = {2023}, } @misc{12497, abstract = {Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein–protein interactions. We studied aromatic residues in the two structurally homologous cross-β amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-β architecture.}, author = {Becker, Lea Marie and Schanda, Paul}, keywords = {aromatic side chains, isotopic labeling, protein dynamics, ring flips, spin relaxation}, publisher = {Institute of Science and Technology Austria}, title = {{Research data to: The rigid core and flexible surface of amyloid fibrils probed by magic-angle-spinning NMR spectroscopy of aromatic residues}}, doi = {10.15479/AT:ISTA:12497}, year = {2023}, } @article{11179, abstract = {Large oligomeric enzymes control a myriad of cellular processes, from protein synthesis and degradation to metabolism. The 0.5 MDa large TET2 aminopeptidase, a prototypical protease important for cellular homeostasis, degrades peptides within a ca. 60 Å wide tetrahedral chamber with four lateral openings. The mechanisms of substrate trafficking and processing remain debated. Here, we integrate magic-angle spinning (MAS) NMR, mutagenesis, co-evolution analysis and molecular dynamics simulations and reveal that a loop in the catalytic chamber is a key element for enzymatic function. The loop is able to stabilize ligands in the active site and may additionally have a direct role in activating the catalytic water molecule whereby a conserved histidine plays a key role. Our data provide a strong case for the functional importance of highly dynamic - and often overlooked - parts of an enzyme, and the potential of MAS NMR to investigate their dynamics at atomic resolution.}, author = {Gauto, Diego F. and Macek, Pavel and Malinverni, Duccio and Fraga, Hugo and Paloni, Matteo and Sučec, Iva and Hessel, Audrey and Bustamante, Juan Pablo and Barducci, Alessandro and Schanda, Paul}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR}}, doi = {10.1038/s41467-022-29423-0}, volume = {13}, year = {2022}, } @article{10323, abstract = {Molecular chaperones are central to cellular protein homeostasis. Dynamic disorder is a key feature of the complexes of molecular chaperones and their client proteins, and it facilitates the client release towards a folded state or the handover to downstream components. The dynamic nature also implies that a given chaperone can interact with many different client proteins, based on physico-chemical sequence properties rather than on structural complementarity of their (folded) 3D structure. Yet, the balance between this promiscuity and some degree of client specificity is poorly understood. Here, we review recent atomic-level descriptions of chaperones with client proteins, including chaperones in complex with intrinsically disordered proteins, with membrane-protein precursors, or partially folded client proteins. We focus hereby on chaperone-client interactions that are independent of ATP. The picture emerging from these studies highlights the importance of dynamics in these complexes, whereby several interaction types, not only hydrophobic ones, contribute to the complex formation. We discuss these features of chaperone-client complexes and possible factors that may contribute to this balance of promiscuity and specificity.}, author = {Sučec, Iva and Bersch, Beate and Schanda, Paul}, issn = {2296-889X}, journal = {Frontiers in Molecular Biosciences}, publisher = {Frontiers}, title = {{How do chaperones bind (partly) unfolded client proteins?}}, doi = {10.3389/fmolb.2021.762005}, volume = {8}, year = {2021}, } @article{8402, abstract = {Background: The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results: Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions: The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.}, author = {Rampelt, Heike and Sucec, Iva and Bersch, Beate and Horten, Patrick and Perschil, Inge and Martinou, Jean-Claude and van der Laan, Martin and Wiedemann, Nils and Schanda, Paul and Pfanner, Nikolaus}, issn = {1741-7007}, journal = {BMC Biology}, keywords = {Biotechnology, Plant Science, General Biochemistry, Genetics and Molecular Biology, Developmental Biology, Cell Biology, Physiology, Ecology, Evolution, Behavior and Systematics, Structural Biology, General Agricultural and Biological Sciences}, publisher = {Springer Nature}, title = {{The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments}}, doi = {10.1186/s12915-019-0733-6}, volume = {18}, year = {2020}, } @unpublished{8404, abstract = {The mitochondrial Tim chaperones are responsible for the transport of membrane proteins across the inter-membrane space to the inner and outer mitochondrial membranes. TIM9·10, a hexameric 70 kDa protein complex formed by 3 copies of Tim9 and Tim10, guides its clients across the aqueous compartment. The TIM9·10·12 complex is the anchor point at the inner-membrane insertase complex TIM22. The mechanism of client transport by TIM9·10 has been resolved recently, but the structure and subunit composition of the TIM9·10·12 complex remains largely unresolved. Furthermore, the assembly process of the hexameric TIM chaperones from its subunits remained elusive. We investigate the structural and dynamical properties of the Tim subunits, and show that they are highly dynamic. In their non-assembled form, the subunits behave as intrinsically disordered proteins; when the conserved cysteines of the CX3C-Xn-CX3C motifs are formed, short marginally stable α-helices are formed, which are only fully stabilized upon hexamer formation to the mature chaperone. Subunits are in equilibrium between their hexamer-embedded and a free form, with exchange kinetics on a minutes time scale. Joint NMR, small-angle X-ray scattering and MD simulation data allow us to derive a structural model of the TIM9·10·12 assembly, which has a 2:3:1 stoichiometry (Tim9:Tim10:Tim12) with a conserved hydrophobic client-binding groove and flexible N- and C-terminal tentacles.}, author = {Weinhäupl, Katharina and Wang, Yong and Hessel, Audrey and Brennich, Martha and Lindorff-Larsen, Kresten and Schanda, Paul}, booktitle = {bioRxiv}, publisher = {Cold Spring Harbor Laboratory}, title = {{Architecture and subunit dynamics of the mitochondrial TIM9·10·12 chaperone}}, doi = {10.1101/2020.03.13.990150}, year = {2020}, } @unpublished{8403, abstract = {Chaperones are essential for assisting protein folding, and for transferring poorly soluble proteins to their functional locations within cells. Hydrophobic interactions drive promiscuous chaperone–client binding, but our understanding of how additional interactions enable client specificity is sparse. Here we decipher what determines binding of two chaperones (TIM8·13, TIM9·10) to different integral membrane proteins, the all-transmembrane mitochondrial carrier Ggc1, and Tim23 which has an additional disordered hydrophilic domain. Combining NMR, SAXS and molecular dynamics simulations, we determine the structures of Tim23/TIM8·13 and Tim23/TIM9·10 complexes. TIM8·13 uses transient salt bridges to interact with the hydrophilic part of its client, but its interactions to the transmembrane part are weaker than in TIM9·10. Consequently, TIM9·10 outcompetes TIM8·13 in binding hydrophobic clients, while TIM8·13 is tuned to few clients with both hydrophilic and hydrophobic parts. Our study exemplifies how chaperones fine-tune the balance of promiscuity vs. specificity.}, author = {Sučec, Iva and Wang, Yong and Dakhlaoui, Ons and Weinhäupl, Katharina and Jores, Tobias and Costa, Doriane and Hessel, Audrey and Brennich, Martha and Rapaport, Doron and Lindorff-Larsen, Kresten and Bersch, Beate and Schanda, Paul}, booktitle = {bioRxiv}, publisher = {Cold Spring Harbor Laboratory}, title = {{Structural basis of client specificity in mitochondrial membrane-protein chaperones}}, doi = {10.1101/2020.06.08.140772}, year = {2020}, } @article{8405, abstract = {Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.}, author = {Gauto, Diego F. and Estrozi, Leandro F. and Schwieters, Charles D. and Effantin, Gregory and Macek, Pavel and Sounier, Remy and Sivertsen, Astrid C. and Schmidt, Elena and Kerfah, Rime and Mas, Guillaume and Colletier, Jacques-Philippe and Güntert, Peter and Favier, Adrien and Schoehn, Guy and Schanda, Paul and Boisbouvier, Jerome}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Biochemistry, Genetics and Molecular Biology, General Physics and Astronomy, General Chemistry}, publisher = {Springer Nature}, title = {{Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex}}, doi = {10.1038/s41467-019-10490-9}, volume = {10}, year = {2019}, } @article{8406, abstract = {Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.}, author = {Felix, Jan and Weinhäupl, Katharina and Chipot, Christophe and Dehez, François and Hessel, Audrey and Gauto, Diego F. and Morlot, Cecile and Abian, Olga and Gutsche, Irina and Velazquez-Campoy, Adrian and Schanda, Paul and Fraga, Hugo}, issn = {2375-2548}, journal = {Science Advances}, number = {9}, publisher = {American Association for the Advancement of Science}, title = {{Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors}}, doi = {10.1126/sciadv.aaw3818}, volume = {5}, year = {2019}, } @article{8413, abstract = {NMR relaxation dispersion methods provide a holistic way to observe microsecond time-scale protein backbone motion both in solution and in the solid state. Different nuclei (1H and 15N) and different relaxation dispersion techniques (Bloch–McConnell and near-rotary-resonance) give complementary information about the amplitudes and time scales of the conformational dynamics and provide comprehensive insights into the mechanistic details of the structural rearrangements. In this paper, we exemplify the benefits of the combination of various solution- and solid-state relaxation dispersion methods on a microcrystalline protein (α-spectrin SH3 domain), for which we are able to identify and model the functionally relevant conformational rearrangements around the ligand recognition loop occurring on multiple microsecond time scales. The observed loop motions suggest that the SH3 domain exists in a binding-competent conformation in dynamic equilibrium with a sterically impaired ground-state conformation both in solution and in crystalline form. This inherent plasticity between the interconverting macrostates is compatible with a conformational-preselection model and provides new insights into the recognition mechanisms of SH3 domains.}, author = {Rovó, Petra and Smith, Colin A. and Gauto, Diego and de Groot, Bert L. and Schanda, Paul and Linser, Rasmus}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, keywords = {Colloid and Surface Chemistry, Biochemistry, General Chemistry, Catalysis}, number = {2}, pages = {858--869}, publisher = {American Chemical Society}, title = {{Mechanistic insights into microsecond time-scale motion of solid proteins using complementary 15N and 1H relaxation dispersion techniques}}, doi = {10.1021/jacs.8b09258}, volume = {141}, year = {2019}, } @article{8412, abstract = {Microsecond to millisecond timescale backbone dynamics of the amyloid core residues in Y145Stop human prion protein (PrP) fibrils were investigated by using 15N rotating frame (R1ρ) relaxation dispersion solid‐state nuclear magnetic resonance spectroscopy over a wide range of spin‐lock fields. Numerical simulations enabled the experimental relaxation dispersion profiles for most of the fibril core residues to be modelled by using a two‐state exchange process with a common exchange rate of 1000 s−1, corresponding to protein backbone motion on the timescale of 1 ms, and an excited‐state population of 2 %. We also found that the relaxation dispersion profiles for several amino acids positioned near the edges of the most structured regions of the amyloid core were better modelled by assuming somewhat higher excited‐state populations (∼5–15 %) and faster exchange rate constants, corresponding to protein backbone motions on the timescale of ∼100–300 μs. The slow backbone dynamics of the core residues were evaluated in the context of the structural model of human Y145Stop PrP amyloid.}, author = {Shannon, Matthew D. and Theint, Theint and Mukhopadhyay, Dwaipayan and Surewicz, Krystyna and Surewicz, Witold K. and Marion, Dominique and Schanda, Paul and Jaroniec, Christopher P.}, issn = {1439-4235}, journal = {ChemPhysChem}, keywords = {Physical and Theoretical Chemistry, Atomic and Molecular Physics, and Optics}, number = {2}, pages = {311--317}, publisher = {Wiley}, title = {{Conformational dynamics in the core of human Y145Stop prion protein amyloid probed by relaxation dispersion NMR}}, doi = {10.1002/cphc.201800779}, volume = {20}, year = {2019}, } @article{8411, abstract = {Studying protein dynamics on microsecond‐to‐millisecond (μs‐ms) time scales can provide important insight into protein function. In magic‐angle‐spinning (MAS) NMR, μs dynamics can be visualized by R1p rotating‐frame relaxation dispersion experiments in different regimes of radio‐frequency field strengths: at low RF field strength, isotropic‐chemical‐shift fluctuation leads to “Bloch‐McConnell‐type” relaxation dispersion, while when the RF field approaches rotary resonance conditions bond angle fluctuations manifest as increased R1p rate constants (“Near‐Rotary‐Resonance Relaxation Dispersion”, NERRD). Here we explore the joint analysis of both regimes to gain comprehensive insight into motion in terms of geometric amplitudes, chemical‐shift changes, populations and exchange kinetics. We use a numerical simulation procedure to illustrate these effects and the potential of extracting exchange parameters, and apply the methodology to the study of a previously described conformational exchange process in microcrystalline ubiquitin.}, author = {Marion, Dominique and Gauto, Diego F. and Ayala, Isabel and Giandoreggio-Barranco, Karine and Schanda, Paul}, issn = {1439-4235}, journal = {ChemPhysChem}, keywords = {Physical and Theoretical Chemistry, Atomic and Molecular Physics, and Optics}, number = {2}, pages = {276--284}, publisher = {Wiley}, title = {{Microsecond protein dynamics from combined Bloch-McConnell and Near-Rotary-Resonance R1p relaxation-dispersion MAS NMR}}, doi = {10.1002/cphc.201800935}, volume = {20}, year = {2019}, } @article{8409, abstract = {The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeneity, and dynamics, atomic-resolution studies are inherently complex. Solid-state NMR is an important technique to gain insight into its structure, dynamics and interactions. Here, we explore the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We demonstrate that highly resolved spectra can be obtained, and show strategies to obtain site-specific resonance assignments and distance information. We also explore the use of proton-proton correlation experiments, thus opening the way for NMR studies of intact cell walls without the need for isotope labeling.}, author = {Bougault, Catherine and Ayala, Isabel and Vollmer, Waldemar and Simorre, Jean-Pierre and Schanda, Paul}, issn = {1047-8477}, journal = {Journal of Structural Biology}, keywords = {Structural Biology}, number = {1}, pages = {66--72}, publisher = {Elsevier}, title = {{Studying intact bacterial peptidoglycan by proton-detected NMR spectroscopy at 100 kHz MAS frequency}}, doi = {10.1016/j.jsb.2018.07.009}, volume = {206}, year = {2019}, } @article{8407, author = {Schanda, Paul}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, keywords = {Nuclear and High Energy Physics, Biophysics, Biochemistry, Condensed Matter Physics}, pages = {180--186}, publisher = {Elsevier}, title = {{Relaxing with liquids and solids – A perspective on biomolecular dynamics}}, doi = {10.1016/j.jmr.2019.07.025}, volume = {306}, year = {2019}, } @article{8410, author = {Schanda, Paul and Chekmenev, Eduard Y.}, issn = {1439-4235}, journal = {ChemPhysChem}, number = {2}, pages = {177--177}, publisher = {Wiley}, title = {{NMR for Biological Systems}}, doi = {10.1002/cphc.201801100}, volume = {20}, year = {2019}, } @article{8408, abstract = {Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize dynamics of phenylalanine residues using 1H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artefact-free dynamics measurements, and allows probing motions effectively without molecular-weight limitations. The application to the TET2 enzyme assembly of ~0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (ps-ms). We quantitatively probe ring flip motions, and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first 13C R1ρ MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly 13C-labeled samples.}, author = {Gauto, Diego F. and Macek, Pavel and Barducci, Alessandro and Fraga, Hugo and Hessel, Audrey and Terauchi, Tsutomu and Gajan, David and Miyanoiri, Yohei and Boisbouvier, Jerome and Lichtenecker, Roman and Kainosho, Masatsune and Schanda, Paul}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, keywords = {Colloid and Surface Chemistry, Biochemistry, General Chemistry, Catalysis}, number = {28}, pages = {11183--11195}, publisher = {American Chemical Society}, title = {{Aromatic ring dynamics, thermal activation, and transient conformations of a 468 kDa enzyme by specific 1H–13C labeling and fast magic-angle spinning NMR}}, doi = {10.1021/jacs.9b04219}, volume = {141}, year = {2019}, } @article{8443, abstract = {Characterizing the structure of membrane proteins (MPs) generally requires extraction from their native environment, most commonly with detergents. Yet, the physicochemical properties of detergent micelles and lipid bilayers differ markedly and could alter the structural organization of MPs, albeit without general rules. Dodecylphosphocholine (DPC) is the most widely used detergent for MP structure determination by NMR, but the physiological relevance of several prominent structures has been questioned, though indirectly, by other biophysical techniques, e.g., functional/thermostability assay (TSA) and molecular dynamics (MD) simulations. Here, we resolve unambiguously this controversy by probing the functional relevance of three different mitochondrial carriers (MCs) in DPC at the atomic level, using an exhaustive set of solution-NMR experiments, complemented by functional/TSA and MD data. Our results provide atomic-level insight into the structure, substrate interaction and dynamics of the detergent–membrane protein complexes and demonstrates cogently that, while high-resolution NMR signals can be obtained for MCs in DPC, they systematically correspond to nonfunctional states.}, author = {Kurauskas, Vilius and Hessel, Audrey and Ma, Peixiang and Lunetti, Paola and Weinhäupl, Katharina and Imbert, Lionel and Brutscher, Bernhard and King, Martin S. and Sounier, Rémy and Dolce, Vincenza and Kunji, Edmund R. S. and Capobianco, Loredana and Chipot, Christophe and Dehez, François and Bersch, Beate and Schanda, Paul}, issn = {1948-7185}, journal = {The Journal of Physical Chemistry Letters}, keywords = {General Materials Science}, number = {5}, pages = {933--938}, publisher = {American Chemical Society}, title = {{How detergent impacts membrane proteins: Atomic-level views of mitochondrial carriers in dodecylphosphocholine}}, doi = {10.1021/acs.jpclett.8b00269}, volume = {9}, year = {2018}, } @article{8440, abstract = {Mycobacterium tuberculosis can remain dormant in the host, an ability that explains the failure of many current tuberculosis treatments. Recently, the natural products cyclomarin, ecumicin, and lassomycin have been shown to efficiently kill Mycobacterium tuberculosis persisters. Their target is the N-terminal domain of the hexameric AAA+ ATPase ClpC1, which recognizes, unfolds, and translocates protein substrates, such as proteins containing phosphorylated arginine residues, to the ClpP1P2 protease for degradation. Surprisingly, these antibiotics do not inhibit ClpC1 ATPase activity, and how they cause cell death is still unclear. Here, using NMR and small-angle X-ray scattering, we demonstrate that arginine-phosphate binding to the ClpC1 N-terminal domain induces millisecond dynamics. We show that these dynamics are caused by conformational changes and do not result from unfolding or oligomerization of this domain. Cyclomarin binding to this domain specifically blocked these N-terminal dynamics. On the basis of these results, we propose a mechanism of action involving cyclomarin-induced restriction of ClpC1 dynamics, which modulates the chaperone enzymatic activity leading eventually to cell death.}, author = {Weinhäupl, Katharina and Brennich, Martha and Kazmaier, Uli and Lelievre, Joel and Ballell, Lluis and Goldberg, Alfred and Schanda, Paul and Fraga, Hugo}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, keywords = {Cell Biology, Biochemistry, Molecular Biology}, number = {22}, pages = {8379--8393}, publisher = {American Society for Biochemistry & Molecular Biology}, title = {{The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis}}, doi = {10.1074/jbc.ra118.002251}, volume = {293}, year = {2018}, } @article{8442, abstract = {Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.}, author = {Chipot, Christophe and Dehez, François and Schnell, Jason R. and Zitzmann, Nicole and Pebay-Peyroula, Eva and Catoire, Laurent J. and Miroux, Bruno and Kunji, Edmund R. S. and Veglia, Gianluigi and Cross, Timothy A. and Schanda, Paul}, issn = {0009-2665}, journal = {Chemical Reviews}, keywords = {General Chemistry}, number = {7}, pages = {3559--3607}, publisher = {American Chemical Society}, title = {{Perturbations of native membrane protein structure in alkyl phosphocholine detergents: A critical assessment of NMR and biophysical studies}}, doi = {10.1021/acs.chemrev.7b00570}, volume = {118}, year = {2018}, } @article{8441, abstract = {Solid-state near-rotary-resonance measurements of the spin–lattice relaxation rate in the rotating frame (R1ρ) is a powerful NMR technique for studying molecular dynamics in the microsecond time scale. The small difference between the spin-lock (SL) and magic-angle-spinning (MAS) frequencies allows sampling very slow motions, at the same time it brings up some methodological challenges. In this work, several issues affecting correct measurements and analysis of 15N R1ρ data are considered in detail. Among them are signal amplitude as a function of the difference between SL and MAS frequencies, “dead time” in the initial part of the relaxation decay caused by transient spin-dynamic oscillations, measurements under HORROR condition and proper treatment of the multi-exponential relaxation decays. The multiple 15N R1ρ measurements at different SL fields and temperatures have been conducted in 1D mode (i.e. without site-specific resolution) for a set of four different microcrystalline protein samples (GB1, SH3, MPD-ubiquitin and cubic-PEG-ubiquitin) to study the overall protein rocking in a crystal. While the amplitude of this motion varies very significantly, its correlation time for all four sample is practically the same, 30–50 μs. The amplitude of the rocking motion correlates with the packing density of a protein crystal. It has been suggested that the rocking motion is not diffusive but likely a jump-like dynamic process.}, author = {Krushelnitsky, Alexey and Gauto, Diego and Rodriguez Camargo, Diana C. and Schanda, Paul and Saalwächter, Kay}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, number = {1}, pages = {53--67}, publisher = {Springer Nature}, title = {{Microsecond motions probed by near-rotary-resonance R1ρ 15N MAS NMR experiments: The model case of protein overall-rocking in crystals}}, doi = {10.1007/s10858-018-0191-4}, volume = {71}, year = {2018}, } @article{8439, abstract = {Lipopolysaccharides (LPS) are complex glycolipids forming the outside layer of Gram-negative bacteria. Their hydrophobic and heterogeneous nature greatly hampers their structural study in an environment similar to the bacterial surface. We have studied LPS purified from E. coli and pathogenic P. aeruginosa with long O-antigen polysaccharides assembled in solution as vesicles or elongated micelles. Solid-state NMR with magic-angle spinning permitted the identification of NMR signals arising from regions with different flexibilities in the LPS, from the lipid components to the O-antigen polysaccharides. Atomic scale data on the LPS enabled the study of the interaction of gentamicin antibiotic bound to P. aeruginosa LPS, for which we could confirm that a specific oligosaccharide is involved in the antibiotic binding. The possibility to study LPS alone and bound to a ligand when it is assembled in membrane-like structures opens great prospects for the investigation of proteins and antibiotics that specifically target such an important molecule at the surface of Gram-negative bacteria.}, author = {Laguri, Cedric and Silipo, Alba and Martorana, Alessandra M. and Schanda, Paul and Marchetti, Roberta and Polissi, Alessandra and Molinaro, Antonio and Simorre, Jean-Pierre}, issn = {1554-8929}, journal = {ACS Chemical Biology}, keywords = {Molecular Medicine, Biochemistry, General Medicine}, number = {8}, pages = {2106--2113}, publisher = {American Chemical Society}, title = {{Solid state NMR studies of intact lipopolysaccharide endotoxin}}, doi = {10.1021/acschembio.8b00271}, volume = {13}, year = {2018}, } @article{8437, abstract = {Chaperonins are ubiquitous protein assemblies present in bacteria, eukaryota, and archaea, facilitating the folding of proteins, preventing protein aggregation, and thus participating in maintaining protein homeostasis in the cell. During their functional cycle, they bind unfolded client proteins inside their double ring structure and promote protein folding by closing the ring chamber in an adenosine 5′-triphosphate (ATP)–dependent manner. Although the static structures of fully open and closed forms of chaperonins were solved by x-ray crystallography or electron microscopy, elucidating the mechanisms of such ATP-driven molecular events requires studying the proteins at the structural level under working conditions. We introduce an approach that combines site-specific nuclear magnetic resonance observation of very large proteins, enabled by advanced isotope labeling methods, with an in situ ATP regeneration system. Using this method, we provide functional insight into the 1-MDa large hsp60 chaperonin while processing client proteins and reveal how nucleotide binding, hydrolysis, and release control switching between closed and open states. While the open conformation stabilizes the unfolded state of client proteins, the internalization of the client protein inside the chaperonin cavity speeds up its functional cycle. This approach opens new perspectives to study structures and mechanisms of various ATP-driven biological machineries in the heat of action.}, author = {Mas, Guillaume and Guan, Jia-Ying and Crublet, Elodie and Debled, Elisa Colas and Moriscot, Christine and Gans, Pierre and Schoehn, Guy and Macek, Pavel and Schanda, Paul and Boisbouvier, Jerome}, issn = {2375-2548}, journal = {Science Advances}, number = {9}, publisher = {American Association for the Advancement of Science}, title = {{Structural investigation of a chaperonin in action reveals how nucleotide binding regulates the functional cycle}}, doi = {10.1126/sciadv.aau4196}, volume = {4}, year = {2018}, } @article{8436, abstract = {The exchange of metabolites between the mitochondrial matrix and the cytosol depends on β-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and β-barrel protein biogenesis. Our work reveals how a single mitochondrial “transfer-chaperone” system is able to guide α-helical and β-barrel membrane proteins in a “nascent chain-like” conformation through a ribosome-free compartment.}, author = {Weinhäupl, Katharina and Lindau, Caroline and Hessel, Audrey and Wang, Yong and Schütze, Conny and Jores, Tobias and Melchionda, Laura and Schönfisch, Birgit and Kalbacher, Hubert and Bersch, Beate and Rapaport, Doron and Brennich, Martha and Lindorff-Larsen, Kresten and Wiedemann, Nils and Schanda, Paul}, issn = {0092-8674}, journal = {Cell}, keywords = {General Biochemistry, Genetics and Molecular Biology}, number = {5}, pages = {1365--1379.e25}, publisher = {Elsevier}, title = {{Structural basis of membrane protein chaperoning through the mitochondrial intermembrane space}}, doi = {10.1016/j.cell.2018.10.039}, volume = {175}, year = {2018}, } @article{8438, author = {Kurauskas, Vilius and Hessel, Audrey and Dehez, François and Chipot, Christophe and Bersch, Beate and Schanda, Paul}, issn = {1545-9993}, journal = {Nature Structural & Molecular Biology}, keywords = {Molecular Biology, Structural Biology}, number = {9}, pages = {745--747}, publisher = {Springer Nature}, title = {{Dynamics and interactions of AAC3 in DPC are not functionally relevant}}, doi = {10.1038/s41594-018-0127-4}, volume = {25}, year = {2018}, } @article{8446, abstract = {Solid‐state NMR spectroscopy can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid‐state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here the use of two sets of experiments is shown to expand the tool kit of 1H‐detected assignment approaches, which correlate a given amide pair either to the two adjacent CO–CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross‐polarization for heteronuclear transfers. The utility of these experiments is exemplified with application to assemblies of deuterated, fully amide‐protonated proteins from approximately 20 to 60 kDa monomer, at magic‐angle spinning (MAS) frequencies from approximately 40 to 55 kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. The resonance assignment of a domain within the 50.4 kDa bacteriophage T5 tube protein pb6 is reported, and this is compared to NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly.}, author = {Fraga, Hugo and Arnaud, Charles‐Adrien and Gauto, Diego F. and Audin, Maxime and Kurauskas, Vilius and Macek, Pavel and Krichel, Carsten and Guan, Jia‐Ying and Boisbouvier, Jerome and Sprangers, Remco and Breyton, Cécile and Schanda, Paul}, issn = {1439-4235}, journal = {ChemPhysChem}, keywords = {Physical and Theoretical Chemistry, Atomic and Molecular Physics, and Optics}, number = {19}, pages = {2697--2703}, publisher = {Wiley}, title = {{Solid‐state NMR H–N–(C)–H and H–N–C–C 3D/4D correlation experiments for resonance assignment of large proteins}}, doi = {10.1002/cphc.201700572}, volume = {18}, year = {2017}, } @article{8445, abstract = {Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, near-rotary-resonance relaxation dispersion (NERRD) experiments probe angular backbone motion, while Bloch–McConnell relaxation dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with an ~3–5° amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.}, author = {Kurauskas, Vilius and Izmailov, Sergei A. and Rogacheva, Olga N. and Hessel, Audrey and Ayala, Isabel and Woodhouse, Joyce and Shilova, Anastasya and Xue, Yi and Yuwen, Tairan and Coquelle, Nicolas and Colletier, Jacques-Philippe and Skrynnikov, Nikolai R. and Schanda, Paul}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Slow conformational exchange and overall rocking motion in ubiquitin protein crystals}}, doi = {10.1038/s41467-017-00165-8}, volume = {8}, year = {2017}, } @article{8444, abstract = {Biophysical investigation of membrane proteins generally requires their extraction from native sources using detergents, a step that can lead, possibly irreversibly, to protein denaturation. The propensity of dodecylphosphocholine (DPC), a detergent widely utilized in NMR studies of membrane proteins, to distort their structure has been the subject of much controversy. It has been recently proposed that the binding specificity of the yeast mitochondrial ADP/ATP carrier (yAAC3) toward cardiolipins is preserved in DPC, thereby suggesting that DPC is a suitable environment in which to study membrane proteins. In this communication, we used all-atom molecular dynamics simulations to investigate the specific binding of cardiolipins to yAAC3. Our data demonstrate that the interaction interface observed in a native-like environment differs markedly from that inferred from an NMR investigation in DPC, implying that in this detergent, the protein structure is distorted. We further investigated yAAC3 solubilized in DPC and in the milder dodecylmaltoside with thermal-shift assays. The loss of thermal transition observed in DPC confirms that the protein is no longer properly folded in this environment.}, author = {Dehez, François and Schanda, Paul and King, Martin S. and Kunji, Edmund R.S. and Chipot, Christophe}, issn = {0006-3495}, journal = {Biophysical Journal}, keywords = {Biophysics}, number = {11}, pages = {2311--2315}, publisher = {Elsevier}, title = {{Mitochondrial ADP/ATP carrier in dodecylphosphocholine binds cardiolipins with non-native affinity}}, doi = {10.1016/j.bpj.2017.09.019}, volume = {113}, year = {2017}, } @article{8449, abstract = {Ensuring the correct folding of RNA molecules in the cell is of major importance for a large variety of biological functions. Therefore, chaperone proteins that assist RNA in adopting their functionally active states are abundant in all living organisms. An important feature of RNA chaperone proteins is that they do not require an external energy source to perform their activity, and that they interact transiently and non-specifically with their RNA targets. So far, little is known about the mechanistic details of the RNA chaperone activity of these proteins. Prominent examples of RNA chaperones are bacterial cold shock proteins (Csp) that have been reported to bind single-stranded RNA and DNA. Here, we have used advanced NMR spectroscopy techniques to investigate at atomic resolution the RNA-melting activity of CspA, the major cold shock protein of Escherichia coli, upon binding to different RNA hairpins. Real-time NMR provides detailed information on the folding kinetics and folding pathways. Finally, comparison of wild-type CspA with single-point mutants and small peptides yields insights into the complementary roles of aromatic and positively charged amino-acid side chains for the RNA chaperone activity of the protein.}, author = {Rennella, Enrico and Sára, Tomáš and Juen, Michael and Wunderlich, Christoph and Imbert, Lionel and Solyom, Zsofia and Favier, Adrien and Ayala, Isabel and Weinhäupl, Katharina and Schanda, Paul and Konrat, Robert and Kreutz, Christoph and Brutscher, Bernhard}, issn = {0305-1048}, journal = {Nucleic Acids Research}, number = {7}, pages = {4255--4268}, publisher = {Oxford University Press}, title = {{RNA binding and chaperone activity of the E.coli cold-shock protein CspA}}, doi = {10.1093/nar/gkx044}, volume = {45}, year = {2017}, } @article{8447, abstract = {Solid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45–60 kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a β-turn region in the wild-type protein. We use 15N R1ρ relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-1H R1ρ relaxation to obtain insight into dynamics. We show that while quantitative interpretation of 1H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.}, author = {Gauto, Diego F. and Hessel, Audrey and Rovó, Petra and Kurauskas, Vilius and Linser, Rasmus and Schanda, Paul}, issn = {0926-2040}, journal = {Solid State Nuclear Magnetic Resonance}, keywords = {Nuclear and High Energy Physics, Instrumentation, General Chemistry, Radiation}, number = {10}, pages = {86--95}, publisher = {Elsevier}, title = {{Protein conformational dynamics studied by 15N and 1HR1ρ relaxation dispersion: Application to wild-type and G53A ubiquitin crystals}}, doi = {10.1016/j.ssnmr.2017.04.002}, volume = {87}, year = {2017}, } @article{8448, abstract = {We present an improved fast mixing device based on the rapid mixing of two solutions inside the NMR probe, as originally proposed by Hore and coworkers (J. Am. Chem. Soc. 125 (2003) 12484–12492). Such a device is important for off-equilibrium studies of molecular kinetics by multidimensional real-time NMR spectrsocopy. The novelty of this device is that it allows removing the injector from the NMR detection volume after mixing, and thus provides good magnetic field homogeneity independently of the initial sample volume placed in the NMR probe. The apparatus is simple to build, inexpensive, and can be used without any hardware modification on any type of liquid-state NMR spectrometer. We demonstrate the performance of our fast mixing device in terms of improved magnetic field homogeneity, and show an application to the study of protein folding and the structural characterization of transiently populated folding intermediates.}, author = {Franco, Rémi and Favier, Adrien and Schanda, Paul and Brutscher, Bernhard}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, keywords = {Nuclear and High Energy Physics, Biophysics, Biochemistry, Condensed Matter Physics}, number = {8}, pages = {125--129}, publisher = {Elsevier}, title = {{Optimized fast mixing device for real-time NMR applications}}, doi = {10.1016/j.jmr.2017.05.016}, volume = {281}, year = {2017}, } @article{8451, abstract = {The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane‐mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co‐polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high‐resolution solid‐state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.}, author = {Bersch, Beate and Dörr, Jonas M. and Hessel, Audrey and Killian, J. Antoinette and Schanda, Paul}, issn = {1433-7851}, journal = {Angewandte Chemie International Edition}, number = {9}, pages = {2508--2512}, publisher = {Wiley}, title = {{Proton-detected solid-state NMR spectroscopy of a Zinc diffusion facilitator protein in native nanodiscs}}, doi = {10.1002/anie.201610441}, volume = {56}, year = {2017}, } @inbook{8450, abstract = {Methyl groups are very useful probes of structure, dynamics, and interactions in protein NMR spectroscopy. In particular, methyl-directed experiments provide high sensitivity even in very large proteins, such as membrane proteins in a membrane-mimicking environment. In this chapter, we discuss the approach for labeling methyl groups in E. coli-based protein expression, as exemplified with the mitochondrial carrier GGC.}, author = {Kurauskas, Vilius and Schanda, Paul and Sounier, Remy}, booktitle = {Membrane protein structure and function characterization}, isbn = {9781493971497}, issn = {1064-3745}, pages = {109--123}, publisher = {Springer Nature}, title = {{Methyl-specific isotope labeling strategies for NMR studies of membrane proteins}}, doi = {10.1007/978-1-4939-7151-0_6}, volume = {1635}, year = {2017}, } @article{8455, abstract = {Solid-state NMR spectroscopy allows the characterization of the structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths increase only marginally. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus.}, author = {Kurauskas, Vilius and Crublet, Elodie and Macek, Pavel and Kerfah, Rime and Gauto, Diego F. and Boisbouvier, Jérôme and Schanda, Paul}, issn = {1359-7345}, journal = {Chemical Communications}, keywords = {Materials Chemistry, Electronic, Optical and Magnetic Materials, General Chemistry, Surfaces, Coatings and Films, Metals and Alloys, Ceramics and Composites, Catalysis}, number = {61}, pages = {9558--9561}, publisher = {Royal Society of Chemistry}, title = {{Sensitive proton-detected solid-state NMR spectroscopy of large proteins with selective CH3labelling: Application to the 50S ribosome subunit}}, doi = {10.1039/c6cc04484k}, volume = {52}, year = {2016}, } @article{8453, abstract = {Transverse relaxation rate measurements in magic-angle spinning solid-state nuclear magnetic resonance provide information about molecular motions occurring on nanosecond-to-millisecond (ns–ms) time scales. The measurement of heteronuclear (13C, 15N) relaxation rate constants in the presence of a spin-lock radiofrequency field (R1ρ relaxation) provides access to such motions, and an increasing number of studies involving R1ρ relaxation in proteins have been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely, (1) the role of CSA/dipolar cross-correlated relaxation (CCR) and (2) the impact of fast proton spin flips (i.e., proton spin diffusion and relaxation). We show that CSA/D CCR in R1ρ experiments is measurable and that the CCR rate constant depends on ns–ms motions; it can thus provide insight into dynamics. We find that proton spin diffusion attenuates this CCR due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R1ρ rate constants has practical advantages over the use of CCR rates, and this article reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation.}, author = {Kurauskas, Vilius and Weber, Emmanuelle and Hessel, Audrey and Ayala, Isabel and Marion, Dominique and Schanda, Paul}, issn = {1520-6106}, journal = {The Journal of Physical Chemistry B}, keywords = {Physical and Theoretical Chemistry, Materials Chemistry, Surfaces, Coatings and Films}, number = {34}, pages = {8905--8913}, publisher = {American Chemical Society}, title = {{Cross-correlated relaxation of dipolar coupling and chemical-shift anisotropy in magic-angle spinning R1ρ NMR measurements: Application to protein backbone dynamics measurements}}, doi = {10.1021/acs.jpcb.6b06129}, volume = {120}, year = {2016}, } @article{8452, abstract = {During spore formation in Bacillus subtilis a transenvelope complex is assembled across the double membrane that separates the mother cell and forespore. This complex (called the “A–Q complex”) is required to maintain forespore development and is composed of proteins with remote homology to components of type II, III, and IV secretion systems found in Gram-negative bacteria. Here, we show that one of these proteins, SpoIIIAG, which has remote homology to ring-forming proteins found in type III secretion systems, assembles into an oligomeric ring in the periplasmic-like space between the two membranes. Three-dimensional reconstruction of images generated by cryo-electron microscopy indicates that the SpoIIIAG ring has a cup-and-saucer architecture with a 6-nm central pore. Structural modeling of SpoIIIAG generated a 24-member ring with dimensions similar to those of the EM-derived saucer. Point mutations in the predicted oligomeric interface disrupted ring formation in vitro and impaired forespore gene expression and efficient spore formation in vivo. Taken together, our data provide strong support for the model in which the A–Q transenvelope complex contains a conduit that connects the mother cell and forespore. We propose that a set of stacked rings spans the intermembrane space, as has been found for type III secretion systems.}, author = {Rodrigues, Christopher D. A. and Henry, Xavier and Neumann, Emmanuelle and Kurauskas, Vilius and Bellard, Laure and Fichou, Yann and Schanda, Paul and Schoehn, Guy and Rudner, David Z. and Morlot, Cecile}, issn = {0027-8424}, journal = {Proceedings of the National Academy of Sciences}, number = {41}, pages = {11585--11590}, publisher = {National Academy of Sciences}, title = {{A ring-shaped conduit connects the mother cell and forespore during sporulation in Bacillus subtilis}}, doi = {10.1073/pnas.1609604113}, volume = {113}, year = {2016}, } @article{8454, abstract = {Magic-angle spinning solid-state NMR spectroscopy is an important technique to study molecular structure, dynamics and interactions, and is rapidly gaining importance in biomolecular sciences. Here we provide an overview of experimental approaches to study molecular dynamics by MAS solid-state NMR, with an emphasis on the underlying theoretical concepts and differences of MAS solid-state NMR compared to solution-state NMR. The theoretical foundations of nuclear spin relaxation are revisited, focusing on the particularities of spin relaxation in solid samples under magic-angle spinning. We discuss the range of validity of Redfield theory, as well as the inherent multi-exponential behavior of relaxation in solids. Experimental challenges for measuring relaxation parameters in MAS solid-state NMR and a few recently proposed relaxation approaches are discussed, which provide information about time scales and amplitudes of motions ranging from picoseconds to milliseconds. We also discuss the theoretical basis and experimental measurements of anisotropic interactions (chemical-shift anisotropies, dipolar and quadrupolar couplings), which give direct information about the amplitude of motions. The potential of combining relaxation data with such measurements of dynamically-averaged anisotropic interactions is discussed. Although the focus of this review is on the theoretical foundations of dynamics studies rather than their application, we close by discussing a small number of recent dynamics studies, where the dynamic properties of proteins in crystals are compared to those in solution.}, author = {Schanda, Paul and Ernst, Matthias}, issn = {0079-6565}, journal = {Progress in Nuclear Magnetic Resonance Spectroscopy}, number = {8}, pages = {1--46}, publisher = {Elsevier}, title = {{Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules}}, doi = {10.1016/j.pnmrs.2016.02.001}, volume = {96}, year = {2016}, } @article{8456, abstract = {The large majority of three-dimensional structures of biological macromolecules have been determined by X-ray diffraction of crystalline samples. High-resolution structure determination crucially depends on the homogeneity of the protein crystal. Overall ‘rocking’ motion of molecules in the crystal is expected to influence diffraction quality, and such motion may therefore affect the process of solving crystal structures. Yet, so far overall molecular motion has not directly been observed in protein crystals, and the timescale of such dynamics remains unclear. Here we use solid-state NMR, X-ray diffraction methods and μs-long molecular dynamics simulations to directly characterize the rigid-body motion of a protein in different crystal forms. For ubiquitin crystals investigated in this study we determine the range of possible correlation times of rocking motion, 0.1–100 μs. The amplitude of rocking varies from one crystal form to another and is correlated with the resolution obtainable in X-ray diffraction experiments.}, author = {Ma, Peixiang and Xue, Yi and Coquelle, Nicolas and Haller, Jens D. and Yuwen, Tairan and Ayala, Isabel and Mikhailovskii, Oleg and Willbold, Dieter and Colletier, Jacques-Philippe and Skrynnikov, Nikolai R. and Schanda, Paul}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Biochemistry, Genetics and Molecular Biology, General Physics and Astronomy, General Chemistry}, publisher = {Springer Nature}, title = {{Observing the overall rocking motion of a protein in a crystal}}, doi = {10.1038/ncomms9361}, volume = {6}, year = {2015}, } @article{8457, abstract = {We review recent advances in methodologies to study microseconds‐to‐milliseconds exchange processes in biological molecules using magic‐angle spinning solid‐state nuclear magnetic resonance (MAS ssNMR) spectroscopy. The particularities of MAS ssNMR, as compared to solution‐state NMR, are elucidated using numerical simulations and experimental data. These simulations reveal the potential of MAS NMR to provide detailed insight into short‐lived conformations of biological molecules. Recent studies of conformational exchange dynamics in microcrystalline ubiquitin are discussed.}, author = {Ma, Peixiang and Schanda, Paul}, isbn = {9780470034590}, journal = {eMagRes}, number = {3}, pages = {699--708}, publisher = {Wiley}, title = {{Conformational exchange processes in biological systems: Detection by solid-state NMR}}, doi = {10.1002/9780470034590.emrstm1418}, volume = {4}, year = {2015}, } @article{8459, abstract = {Nuclear magnetic resonance (NMR) is a powerful tool for observing the motion of biomolecules at the atomic level. One technique, the analysis of relaxation dispersion phenomenon, is highly suited for studying the kinetics and thermodynamics of biological processes. Built on top of the relax computational environment for NMR dynamics is a new dispersion analysis designed to be comprehensive, accurate and easy-to-use. The software supports more models, both numeric and analytic, than current solutions. An automated protocol, available for scripting and driving the graphical user interface (GUI), is designed to simplify the analysis of dispersion data for NMR spectroscopists. Decreases in optimization time are granted by parallelization for running on computer clusters and by skipping an initial grid search by using parameters from one solution as the starting point for another —using analytic model results for the numeric models, taking advantage of model nesting, and using averaged non-clustered results for the clustered analysis.}, author = {Morin, Sébastien and Linnet, Troels E and Lescanne, Mathilde and Schanda, Paul and Thompson, Gary S and Tollinger, Martin and Teilum, Kaare and Gagné, Stéphane and Marion, Dominique and Griesinger, Christian and Blackledge, Martin and d’Auvergne, Edward J}, issn = {1367-4803}, journal = {Bioinformatics}, keywords = {Statistics and Probability, Computational Theory and Mathematics, Biochemistry, Molecular Biology, Computational Mathematics, Computer Science Applications}, number = {15}, pages = {2219--2220}, publisher = {Oxford University Press}, title = {{Relax: The analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data}}, doi = {10.1093/bioinformatics/btu166}, volume = {30}, year = {2014}, } @article{8458, abstract = {The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance, to date no structure of a protein in complex with an intact bacterial peptidoglycan has been resolved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spectroscopy to derive for the first time an atomic model of an l,d-transpeptidase from Bacillus subtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains—the catalytic domain as well as the proposed peptidoglycan recognition domain—are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the implication of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein–peptidoglycan complex paves the way for the design of new antibiotic drugs targeting l,d-transpeptidases. The strategy developed here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.}, author = {Schanda, Paul and Triboulet, Sébastien and Laguri, Cédric and Bougault, Catherine M. and Ayala, Isabel and Callon, Morgane and Arthur, Michel and Simorre, Jean-Pierre}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, number = {51}, pages = {17852--17860}, publisher = {American Chemical Society}, title = {{Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan}}, doi = {10.1021/ja5105987}, volume = {136}, year = {2014}, } @article{8460, abstract = {The function of proteins depends on their ability to sample a variety of states differing in structure and free energy. Deciphering how the various thermally accessible conformations are connected, and understanding their structures and relative energies is crucial in rationalizing protein function. Many biomolecular reactions take place within microseconds to milliseconds, and this timescale is therefore of central functional importance. Here we show that R1ρ relaxation dispersion experiments in magic‐angle‐spinning solid‐state NMR spectroscopy make it possible to investigate the thermodynamics and kinetics of such exchange process, and gain insight into structural features of short‐lived states.}, author = {Ma, Peixiang and Haller, Jens D. and Zajakala, Jérémy and Macek, Pavel and Sivertsen, Astrid C. and Willbold, Dieter and Boisbouvier, Jérôme and Schanda, Paul}, issn = {1433-7851}, journal = {Angewandte Chemie International Edition}, number = {17}, pages = {4312--4317}, publisher = {Wiley}, title = {{Probing transient conformational states of proteins by solid-state R1ρ relaxation-dispersion NMR spectroscopy}}, doi = {10.1002/anie.201311275}, volume = {53}, year = {2014}, } @article{8461, abstract = {Solid-state NMR provides insight into protein motion over time scales ranging from picoseconds to seconds. While in solution state the methodology to measure protein dynamics is well established, there is currently no such consensus protocol for measuring dynamics in solids. In this article, we perform a detailed investigation of measurement protocols for fast motions, i.e. motions ranging from picoseconds to a few microseconds, which is the range covered by dipolar coupling and relaxation experiments. We perform a detailed theoretical investigation how dipolar couplings and relaxation data can provide information about amplitudes and time scales of local motion. We show that the measurement of dipolar couplings is crucial for obtaining accurate motional parameters, while systematic errors are found when only relaxation data are used. Based on this realization, we investigate how the REDOR experiment can provide such data in a very accurate manner. We identify that with accurate rf calibration, and explicit consideration of rf field inhomogeneities, one can obtain highly accurate absolute order parameters. We then perform joint model-free analyses of 6 relaxation data sets and dipolar couplings, based on previously existing, as well as new data sets on microcrystalline ubiquitin. We show that nanosecond motion can be detected primarily in loop regions, and compare solid-state data to solution-state relaxation and RDC analyses. The protocols investigated here will serve as a useful basis towards the establishment of a routine protocol for the characterization of ps–μs motions in proteins by solid-state NMR.}, author = {Haller, Jens D. and Schanda, Paul}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, keywords = {Spectroscopy, Biochemistry}, number = {3}, pages = {263--280}, publisher = {Springer Nature}, title = {{Amplitudes and time scales of picosecond-to-microsecond motion in proteins studied by solid-state NMR: a critical evaluation of experimental approaches and application to crystalline ubiquitin}}, doi = {10.1007/s10858-013-9787-x}, volume = {57}, year = {2013}, } @article{8462, abstract = {The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.}, author = {Rennella, E. and Cutuil, T. and Schanda, Paul and Ayala, I. and Gabel, F. and Forge, V. and Corazza, A. and Esposito, G. and Brutscher, B.}, issn = {0022-2836}, journal = {Journal of Molecular Biology}, keywords = {Molecular Biology}, number = {15}, pages = {2722--2736}, publisher = {Elsevier}, title = {{Oligomeric states along the folding pathways of β2-microglobulin: Kinetics, thermodynamics, and structure}}, doi = {10.1016/j.jmb.2013.04.028}, volume = {425}, year = {2013}, } @article{8463, abstract = {The 1H dipolar network, which is the major obstacle for applying proton detection in the solid-state, can be reduced by deuteration, employing the RAP (Reduced Adjoining Protonation) labeling scheme, which yields random protonation at non-exchangeable sites. We present here a systematic study on the optimal degree of random sidechain protonation in RAP samples as a function of the MAS (magic angle spinning) frequency. In particular, we compare 1H sensitivity and linewidth of a microcrystalline protein, the SH3 domain of chicken α-spectrin, for samples, prepared with 5–25 % H2O in the E. coli growth medium, in the MAS frequency range of 20–60 kHz. At an external field of 19.96 T (850 MHz), we find that using a proton concentration between 15 and 25 % in the M9 medium yields the best compromise in terms of sensitivity and resolution, with an achievable average 1H linewidth on the order of 40–50 Hz. Comparing sensitivities at a MAS frequency of 60 versus 20 kHz, a gain in sensitivity by a factor of 4–4.5 is observed in INEPT-based 1H detected 1D 1H,13C correlation experiments. In total, we find that spectra recorded with a 1.3 mm rotor at 60 kHz have almost the same sensitivity as spectra recorded with a fully packed 3.2 mm rotor at 20 kHz, even though ~20× less material is employed. The improved sensitivity is attributed to 1H line narrowing due to fast MAS and to the increased efficiency of the 1.3 mm coil.}, author = {Asami, Sam and Szekely, Kathrin and Schanda, Paul and Meier, Beat H. and Reif, Bernd}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, number = {2}, pages = {155--168}, publisher = {Springer Nature}, title = {{Optimal degree of protonation for 1H detection of aliphatic sites in randomly deuterated proteins as a function of the MAS frequency}}, doi = {10.1007/s10858-012-9659-9}, volume = {54}, year = {2012}, } @article{8465, abstract = {We demonstrate that conformational exchange processes in proteins on microsecond-to-millisecond time scales can be detected and quantified by solid-state NMR spectroscopy. We show two independent approaches that measure the effect of conformational exchange on transverse relaxation parameters, namely Carr–Purcell–Meiboom–Gill relaxation-dispersion experiments and measurement of differential multiple-quantum coherence decay. Long coherence lifetimes, as required for these experiments, are achieved by the use of highly deuterated samples and fast magic-angle spinning. The usefulness of the approaches is demonstrated by application to microcrystalline ubiquitin. We detect a conformational exchange process in a region of the protein for which dynamics have also been observed in solution. Interestingly, quantitative analysis of the data reveals that the exchange process is more than 1 order of magnitude slower than in solution, and this points to the impact of the crystalline environment on free energy barriers.}, author = {Tollinger, Martin and Sivertsen, Astrid C. and Meier, Beat H. and Ernst, Matthias and Schanda, Paul}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, number = {36}, pages = {14800--14807}, publisher = {American Chemical Society}, title = {{Site-resolved measurement of microsecond-to-millisecond conformational-exchange processes in proteins by solid-state NMR spectroscopy}}, doi = {10.1021/ja303591y}, volume = {134}, year = {2012}, } @article{8466, abstract = {Recent advances in NMR spectroscopy and the availability of high magnetic field strengths now offer the possibility to record real-time 3D NMR spectra of short-lived protein states, e.g., states that become transiently populated during protein folding. Here we present a strategy for obtaining sequential NMR assignments as well as atom-resolved information on structural and dynamic features within a folding intermediate of the amyloidogenic protein β2-microglobulin that has a half-lifetime of only 20 min.}, author = {Rennella, Enrico and Cutuil, Thomas and Schanda, Paul and Ayala, Isabel and Forge, Vincent and Brutscher, Bernhard}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, number = {19}, pages = {8066--8069}, publisher = {American Chemical Society}, title = {{Real-time NMR characterization of structure and dynamics in a transiently populated protein folding intermediate}}, doi = {10.1021/ja302598j}, volume = {134}, year = {2012}, } @article{8467, abstract = {Partial deuteration is a powerful tool to increase coherence life times and spectral resolution in proton solid-state NMR. The J coupling to deuterium needs, however, to be decoupled to maintain the good resolution in the (usually indirect) 13C dimension(s). We present a simple and reversible way to expand a commercial 1.3 mm HCN MAS probe with a 2H channel with sufficient field strength for J-decoupling of deuterium, namely 2–3 kHz. The coil is placed at the outside of the stator and requires no significant modifications to the probe. The performance and the realizable gains in sensitivity and resolution are demonstrated using perdeuterated ubiquitin, with selectively CHD2-labeled methyl groups.}, author = {Huber, Matthias and With, Oliver and Schanda, Paul and Verel, René and Ernst, Matthias and Meier, Beat H.}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, pages = {76--80}, publisher = {Elsevier}, title = {{A supplementary coil for 2H decoupling with commercial HCN MAS probes}}, doi = {10.1016/j.jmr.2011.10.010}, volume = {214}, year = {2012}, } @article{8469, abstract = {The accurate experimental determination of dipolar-coupling constants for one-bond heteronuclear dipolar couplings in solids is a key for the quantification of the amplitudes of motional processes. Averaging of the dipolar coupling reports on motions on time scales up to the inverse of the coupling constant, in our case tens of microseconds. Combining dipolar-coupling derived order parameters that characterize the amplitudes of the motion with relaxation data leads to a more precise characterization of the dynamical parameters and helps to disentangle the amplitudes and the time scales of the motional processes, which impact relaxation rates in a highly correlated way. Here. we describe and characterize an improved experimental protocol – based on REDOR – to measure these couplings in perdeuterated proteins with a reduced sensitivity to experimental missettings. Because such effects are presently the dominant source of systematic errors in experimental dipolar-coupling measurements, these compensated experiments should help to significantly improve the precision of such data. A detailed comparison with other commonly used pulse sequences (T-MREV, phase-inverted CP,R18 5/2, and R18 7/1) is provided.}, author = {Schanda, Paul and Meier, Beat H. and Ernst, Matthias}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, keywords = {Nuclear and High Energy Physics, Biophysics, Biochemistry, Condensed Matter Physics}, number = {2}, pages = {246--259}, publisher = {Elsevier}, title = {{Accurate measurement of one-bond H–X heteronuclear dipolar couplings in MAS solid-state NMR}}, doi = {10.1016/j.jmr.2011.03.015}, volume = {210}, year = {2011}, } @article{8470, abstract = {Adding a new dimension: 4D or 3D proton‐detected spectra of perdeuterated protein samples with 1H labelled amides and methyl groups permit collecting unambiguous distance restraints with high sensitivity and determining protein structure by solid‐state NMR (see picture).}, author = {Huber, Matthias and Hiller, Sebastian and Schanda, Paul and Ernst, Matthias and Böckmann, Anja and Verel, René and Meier, Beat H.}, issn = {1439-4235}, journal = {ChemPhysChem}, keywords = {Physical and Theoretical Chemistry, Atomic and Molecular Physics, and Optics}, number = {5}, pages = {915--918}, publisher = {Wiley}, title = {{A proton-detected 4D solid-state NMR experiment for protein structure determination}}, doi = {10.1002/cphc.201100062}, volume = {12}, year = {2011}, } @article{8464, abstract = {Nonsymmetric motion: Solid‐state NMR measurements of dipolar coupling tensors provide insight into protein dynamics. The hitherto ignored asymmetry of the dipolar coupling tensor contains valuable information about motional asymmetry, which was used in the first direct site‐resolved measurement of such tensors. Important motions such as rotamer jumps can now be directly detected in the solid state.}, author = {Schanda, Paul and Huber, Matthias and Boisbouvier, Jérôme and Meier, Beat H. and Ernst, Matthias}, issn = {1433-7851}, journal = {Angewandte Chemie International Edition}, number = {46}, pages = {11005--11009}, publisher = {Wiley}, title = {{Solid-state NMR measurements of asymmetric dipolar couplings provide insight into protein side-chain motion}}, doi = {10.1002/anie.201103944}, volume = {50}, year = {2011}, } @article{8468, author = {Lalli, Daniela and Schanda, Paul and Chowdhury, Anup and Retel, Joren and Hiller, Matthias and Higman, Victoria A. and Handel, Lieselotte and Agarwal, Vipin and Reif, Bernd and van Rossum, Barth and Akbey, Ümit and Oschkinat, Hartmut}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, number = {4}, pages = {477--485}, publisher = {Springer Nature}, title = {{Three-dimensional deuterium-carbon correlation experiments for high-resolution solid-state MAS NMR spectroscopy of large proteins}}, doi = {10.1007/s10858-011-9578-1}, volume = {51}, year = {2011}, } @article{8471, abstract = {Despite the importance of protein fibrils in the context of conformational diseases, information on their structure is still sparse. Hydrogen/deuterium exchange measurements of backbone amide protons allow the identification hydrogen-bonding patterns and reveal pertinent information on the amyloid β-sheet architecture. However, they provide only little information on the identity of residues exposed to solvent or buried inside the fibril core. NMR spectroscopy is a potent method for identifying solvent-accessible residues in proteins via observation of polarization transfer between chemically exchanging side-chain protons and water protons. We show here that the combined use of highly deuterated samples and fast magic-angle spinning greatly attenuates unwanted spin diffusion and allows identification of polarization exchange with the solvent in a site-specific manner. We apply this measurement protocol to HET-s(218–289) prion fibrils under different conditions (including physiological pH, where protofibrils assemble together into thicker fibrils) and demonstrate that each protofibril of HET-s(218–289), is surrounded by water, thus excluding the existence of extended dry interfibril contacts. We also show that exchangeable side-chain protons inside the hydrophobic core of HET-s(218–289) do not exchange over time intervals of weeks to months. The experiments proposed in this study can provide insight into the detailed structural features of amyloid fibrils in general.}, author = {Van Melckebeke, Hélène and Schanda, Paul and Gath, Julia and Wasmer, Christian and Verel, René and Lange, Adam and Meier, Beat H. and Böckmann, Anja}, issn = {0022-2836}, journal = {Journal of Molecular Biology}, number = {3}, pages = {765--772}, publisher = {Elsevier}, title = {{Probing water accessibility in HET-s(218–289) amyloid fibrils by solid-state NMR}}, doi = {10.1016/j.jmb.2010.11.004}, volume = {405}, year = {2011}, } @article{8473, abstract = {β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.}, author = {Corazza, Alessandra and Rennella, Enrico and Schanda, Paul and Mimmi, Maria Chiara and Cutuil, Thomas and Raimondi, Sara and Giorgetti, Sofia and Fogolari, Federico and Viglino, Paolo and Frydman, Lucio and Gal, Maayan and Bellotti, Vittorio and Brutscher, Bernhard and Esposito, Gennaro}, issn = {0021-9258}, journal = {Journal of Biological Chemistry}, keywords = {Cell Biology, Biochemistry, Molecular Biology}, number = {8}, pages = {5827--5835}, publisher = {American Society for Biochemistry & Molecular Biology}, title = {{Native-unlike long-lived intermediates along the folding pathway of the amyloidogenic protein β2-Microglobulin revealed by real-time two-dimensional NMR}}, doi = {10.1074/jbc.m109.061168}, volume = {285}, year = {2010}, } @article{8472, abstract = {Characterization of protein dynamics by solid-state NMR spectroscopy requires robust and accurate measurement protocols, which are not yet fully developed. In this study, we investigate the backbone dynamics of microcrystalline ubiquitin using different approaches. A rotational-echo double-resonance type (REDOR-type) methodology allows one to accurately measure 1H−15N order parameters in highly deuterated samples. We show that the systematic errors in the REDOR experiment are as low as 1% or even less, giving access to accurate data for the amplitudes of backbone mobility. Combining such dipolar-coupling-derived order parameters with autocorrelated and cross-correlated 15N relaxation rates, we are able to quantitate amplitudes and correlation times of backbone dynamics on picosecond and nanosecond time scales in a residue-resolved manner. While the mobility on picosecond time scales appears to have rather uniform amplitude throughout the protein, we unambiguously identify and quantitate nanosecond mobility with order parameters S2 as low as 0.8 in some regions of the protein, where nanosecond dynamics has also been revealed in solution state. The methodology used here, a combination of accurate dipolar-coupling measurements and different relaxation parameters, yields details about dynamics on different time scales and can be applied to solid protein samples such as amyloid fibrils or membrane proteins.}, author = {Schanda, Paul and Meier, Beat H. and Ernst, Matthias}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, number = {45}, pages = {15957--15967}, publisher = {American Chemical Society}, title = {{Quantitative analysis of protein backbone dynamics in microcrystalline ubiquitin by solid-state NMR spectroscopy}}, doi = {10.1021/ja100726a}, volume = {132}, year = {2010}, } @article{8479, abstract = {Multidimensional NMR spectroscopy is a well-established technique for the characterization of structure and fast-time-scale dynamics of highly populated ground states of biological macromolecules. The investigation of short-lived excited states that are important for molecular folding, misfolding and function, however, remains a challenge for modern biomolecular NMR techniques. Off-equilibrium real-time kinetic NMR methods allow direct observation of conformational or chemical changes by following peak positions and intensities in a series of spectra recorded during a kinetic event. Because standard multidimensional NMR methods required to yield sufficient atom-resolution are intrinsically time-consuming, many interesting phenomena are excluded from real-time NMR analysis. Recently, spatially encoded ultrafast 2D NMR techniques have been proposed that allow one to acquire a 2D NMR experiment within a single transient. In addition, when combined with the SOFAST technique, such ultrafast experiments can be repeated at high rates. One of the problems detected for such ultrafast protein NMR experiments is related to the heteronuclear decoupling during detection with interferences between the pulses and the oscillatory magnetic field gradients arising in this scheme. Here we present a method for improved ultrafast data acquisition yielding higher signal to noise and sharper lines in single-scan 2D NMR spectra. In combination with a fast-mixing device, the recording of 1H–15N correlation spectra with repetition rates of up to a few Hertz becomes feasible, enabling real-time studies of protein kinetics occurring on time scales down to a few seconds.}, author = {Gal, Maayan and Kern, Thomas and Schanda, Paul and Frydman, Lucio and Brutscher, Bernhard}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, keywords = {Spectroscopy, Biochemistry}, pages = {1--10}, publisher = {Springer Nature}, title = {{An improved ultrafast 2D NMR experiment: Towards atom-resolved real-time studies of protein kinetics at multi-Hz rates}}, doi = {10.1007/s10858-008-9284-9}, volume = {43}, year = {2009}, } @article{8478, abstract = {Allosteric regulation is an effective mechanism of control in biological processes. In allosteric proteins a signal originating at one site in the molecule is communicated through the protein structure to trigger a specific response at a remote site. Using NMR relaxation dispersion techniques we directly observe the dynamic process through which the KIX domain of CREB binding protein communicates allosteric information between binding sites. KIX mediates cooperativity between pairs of transcription factors through binding to two distinct interaction surfaces in an allosteric manner. We show that binding the activation domain of the mixed lineage leukemia (MLL) transcription factor to KIX induces a redistribution of the relative populations of KIX conformations toward a high-energy state in which the allosterically activated second binding site is already preformed, consistent with the Monod−Wyman−Changeux (WMC) model of allostery. The structural rearrangement process that links the two conformers and by which allosteric information is communicated occurs with a time constant of 3 ms at 27 °C. Our dynamic NMR data reveal that an evolutionarily conserved network of hydrophobic amino acids constitutes the pathway through which information is transmitted.}, author = {Brüschweiler, Sven and Schanda, Paul and Kloiber, Karin and Brutscher, Bernhard and Kontaxis, Georg and Konrat, Robert and Tollinger, Martin}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, number = {8}, pages = {3063--3068}, publisher = {American Chemical Society}, title = {{Direct observation of the dynamic process underlying allosteric signal transmission}}, doi = {10.1021/ja809947w}, volume = {131}, year = {2009}, } @article{8476, abstract = {Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein−nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA−ligand titration experiments, measurement of residual dipolar 15N−1H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*GA RNA kissing complex and a 26 kDa tRNA.}, author = {Farjon, Jonathan and Boisbouvier, Jérôme and Schanda, Paul and Pardi, Arthur and Simorre, Jean-Pierre and Brutscher, Bernhard}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, number = {24}, pages = {8571--8577}, publisher = {American Chemical Society}, title = {{Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution}}, doi = {10.1021/ja901633y}, volume = {131}, year = {2009}, } @article{8477, abstract = {An optimized NMR experiment that combines the advantages of methyl-TROSY and SOFAST-HMQC has been developed. It allows the recording of high quality methyl 1H−13C correlation spectra of protein assemblies of several hundreds of kDa in a few seconds. The SOFAST-methyl-TROSY-based experiment offers completely new opportunities for the study of structural and dynamic changes occurring in molecular nanomachines while they perform their biological function in vitro.}, author = {Amero, Carlos and Schanda, Paul and Durá, M. Asunción and Ayala, Isabel and Marion, Dominique and Franzetti, Bruno and Brutscher, Bernhard and Boisbouvier, Jérôme}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, number = {10}, pages = {3448--3449}, publisher = {American Chemical Society}, title = {{Fast two-dimensional NMR spectroscopy of high molecular weight protein assemblies}}, doi = {10.1021/ja809880p}, volume = {131}, year = {2009}, } @article{8474, abstract = {Hydrogen bonds are ubiquitous interactions in proteins, and are important for their folding and functionality. Scalar coupling constants across hydrogen bonds in the protein backbone, some as small as 0.5 Hz, can be directly measured in the solid state by NMR spectroscopy (see figure). The nuclei on both sides of the hydrogen bond can be identified and the size of the coupling constant can be measured accurately.}, author = {Schanda, Paul and Huber, Matthias and Verel, René and Ernst, Matthias and Meier, Beat H.}, issn = {1433-7851}, journal = {Angewandte Chemie International Edition}, keywords = {General Chemistry, Catalysis}, number = {49}, pages = {9322--9325}, publisher = {Wiley}, title = {{Direct detection of 3hJN' hydrogen-bond scalar couplings in proteins by solid-state NMR spectroscopy}}, doi = {10.1002/anie.200904411}, volume = {48}, year = {2009}, } @article{8475, author = {Schanda, Paul}, issn = {0079-6565}, journal = {Progress in Nuclear Magnetic Resonance Spectroscopy}, number = {3}, pages = {238--265}, publisher = {Elsevier}, title = {{Fast-pulsing longitudinal relaxation optimized techniques: Enriching the toolbox of fast biomolecular NMR spectroscopy}}, doi = {10.1016/j.pnmrs.2009.05.002}, volume = {55}, year = {2009}, } @article{8481, abstract = {The copK gene is localized on the pMOL30 plasmid of Cupriavidus metallidurans CH34 within the complex cop cluster of genes, for which 21 genes have been identified. The expression of the corresponding periplasmic CopK protein is strongly upregulated in the presence of copper, leading to a high periplasmic accumulation. The structure and metal-binding properties of CopK were investigated by NMR and mass spectrometry. The protein is dimeric in the apo state with a dissociation constant in the range of 10- 5 M estimated from analytical ultracentrifugation. Mass spectrometry revealed that CopK has two high-affinity Cu(I)-binding sites per monomer with different Cu(I) affinities. Binding of Cu(II) was observed but appeared to be non-specific. The solution structure of apo-CopK revealed an all-β fold formed of two β-sheets in perpendicular orientation with an unstructured C-terminal tail. The dimer interface is formed by the surface of the C-terminal β-sheet. Binding of the first Cu(I)-ion induces a major structural modification involving dissociation of the dimeric apo-protein. Backbone chemical shifts determined for the 1Cu(I)-bound form confirm the conservation of the N-terminal β-sheet, while the last strand of the C-terminal sheet appears in slow conformational exchange. We hypothesize that the partial disruption of the C-terminal β-sheet is related to dimer dissociation. NH-exchange data acquired on the apo-protein are consistent with a lower thermodynamic stability of the C-terminal sheet. CopK contains seven methionine residues, five of which appear highly conserved. Chemical shift data suggest implication of two or three methionines (Met54, Met38, Met28) in the first Cu(I) site. Addition of a second Cu(I) ion further increases protein plasticity. Comparison of the structural and metal-binding properties of CopK with other periplasmic copper-binding proteins reveals two conserved features within these functionally related proteins: the all-β fold and the methionine-rich Cu(I)-binding site.}, author = {Bersch, Beate and Favier, Adrien and Schanda, Paul and van Aelst, Sébastien and Vallaeys, Tatiana and Covès, Jacques and Mergeay, Max and Wattiez, Ruddy}, issn = {0022-2836}, journal = {Journal of Molecular Biology}, keywords = {Molecular Biology}, number = {2}, pages = {386--403}, publisher = {Elsevier}, title = {{Molecular structure and metal-binding properties of the periplasmic CopK protein expressed in Cupriavidus metallidurans CH34 during copper challenge}}, doi = {10.1016/j.jmb.2008.05.017}, volume = {380}, year = {2008}, } @article{8480, abstract = {The KIX domain of the transcription co-activator CBP is a three-helix bundle protein that folds via rapid accumulation of an intermediate state, followed by a slower folding phase. Recent NMR relaxation dispersion studies revealed the presence of a low-populated (excited) state of KIX that exists in equilibrium with the natively folded form under non-denaturing conditions, and likely represents the equilibrium analog of the folding intermediate. Here, we combine amide hydrogen/deuterium exchange measurements using rapid NMR data acquisition techniques with backbone 15N and 13C relaxation dispersion experiments to further investigate the equilibrium folding of the KIX domain. Residual structure within the folding intermediate is detected by both methods, and their combination enables reliable quantification of the amount of persistent residual structure. Three well-defined folding subunits are found, which display variable stability and correspond closely to the individual helices in the native state. While two of the three helices (α2 and α3) are partially formed in the folding intermediate (to ∼ 50% and ∼ 80%, respectively, at 20 °C), the third helix is disordered. The observed helical content within the excited state exceeds the helical propensities predicted for the corresponding peptide regions, suggesting that the two helices are weakly mutually stabilized, while methyl 13C relaxation dispersion data indicate that a defined packing arrangement is unlikely. Temperature-dependent experiments reveal that the largest enthalpy and entropy changes along the folding reaction occur during the final transition from the intermediate to the native state. Our experimental data are consistent with a folding mechanism where helices α2 and α3 form rapidly, although to different extents, while helix α1 consolidates only as folding proceeds to complete the native state-structure.}, author = {Schanda, Paul and Brutscher, Bernhard and Konrat, Robert and Tollinger, Martin}, issn = {0022-2836}, journal = {Journal of Molecular Biology}, keywords = {Molecular Biology}, number = {4}, pages = {726--741}, publisher = {Elsevier}, title = {{Folding of the KIX domain: Characterization of the equilibrium analog of a folding intermediate using 15N/13C relaxation dispersion and fast 1H/2H amide exchange NMR spectroscopy}}, doi = {10.1016/j.jmb.2008.05.040}, volume = {380}, year = {2008}, } @article{8482, abstract = {The SOFAST-HMQC experiment [P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015] allows recording two-dimensional correlation spectra of macromolecules such as proteins in only a few seconds acquisition time. To achieve the highest possible sensitivity, SOFAST-HMQC experiments are preferably performed on high-field NMR spectrometers equipped with cryogenically cooled probes. The duty cycle of over 80% in fast-pulsing SOFAST-HMQC experiments, however, may cause problems when using a cryogenic probe. Here we introduce SE-IPAP-SOFAST-HMQC, a new pulse sequence that provides comparable sensitivity to standard SOFAST-HMQC, while avoiding heteronuclear decoupling during 1H detection, and thus significantly reducing the radiofrequency load of the probe during the experiment. The experiment is also attractive for fast and sensitive measurement of heteronuclear one-bond spin coupling constants.}, author = {Kern, Thomas and Schanda, Paul and Brutscher, Bernhard}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, keywords = {Nuclear and High Energy Physics, Biophysics, Biochemistry, Condensed Matter Physics}, number = {2}, pages = {333--338}, publisher = {Elsevier}, title = {{Sensitivity-enhanced IPAP-SOFAST-HMQC for fast-pulsing 2D NMR with reduced radiofrequency load}}, doi = {10.1016/j.jmr.2007.11.015}, volume = {190}, year = {2008}, } @article{8483, abstract = {Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of α-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of α-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.}, author = {Schanda, Paul and Forge, V. and Brutscher, B.}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, keywords = {Multidisciplinary}, number = {27}, pages = {11257--11262}, publisher = {National Academy of Sciences}, title = {{Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy}}, doi = {10.1073/pnas.0702069104}, volume = {104}, year = {2007}, } @article{8487, abstract = {Following unidirectional biophysical events such as the folding of proteins or the equilibration of binding interactions, requires experimental methods that yield information at both atomic-level resolution and at high repetition rates. Toward this end a number of different approaches enabling the rapid acquisition of 2D NMR spectra have been recently introduced, including spatially encoded “ultrafast” 2D NMR spectroscopy and SOFAST HMQC NMR. Whereas the former accelerates acquisitions by reducing the number of scans that are necessary for completing arbitrary 2D NMR experiments, the latter operates by reducing the delay between consecutive scans while preserving sensitivity. Given the complementarities between these two approaches it seems natural to combine them into a single tool, enabling the acquisition of full 2D protein NMR spectra at high repetition rates. We demonstrate here this capability with the introduction of “ultraSOFAST” HMQC NMR, a spatially encoded and relaxation-optimized approach that can provide 2D protein correlation spectra at ∼1 s repetition rates for samples in the ∼2 mM concentration range. The principles, relative advantages, and current limitations of this new approach are discussed, and its application is exemplified with a study of the fast hydrogen−deuterium exchange characterizing amide sites in Ubiquitin.}, author = {Gal, Maayan and Schanda, Paul and Brutscher, Bernhard and Frydman, Lucio}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, keywords = {Colloid and Surface Chemistry, Biochemistry, General Chemistry, Catalysis}, number = {5}, pages = {1372--1377}, publisher = {American Chemical Society}, title = {{UltraSOFAST HMQC NMR and the repetitive acquisition of 2D protein spectra at Hz rates}}, doi = {10.1021/ja066915g}, volume = {129}, year = {2007}, } @article{8485, abstract = {High signal to noise is a necessity for the quantification of NMR spectral parameters to be translated into accurate and precise restraints on protein structure and dynamics. An important source of long-range structural information is obtained from 1H–1H residual dipolar couplings (RDCs) measured for weakly aligned molecules. For sensitivity reasons, such measurements are generally performed on highly deuterated protein samples. Here we show that high sensitivity is also obtained for protonated protein samples if the pulse schemes are optimized in terms of longitudinal relaxation efficiency and J-mismatch compensated coherence transfer. The new sensitivity-optimized quantitative J-correlation experiment yields important signal gains reaching factors of 1.5 to 8 for individual correlation peaks when compared to previously proposed pulse schemes.}, author = {Schanda, Paul and Lescop, Ewen and Falge, Mirjam and Sounier, Rémy and Boisbouvier, Jérôme and Brutscher, Bernhard}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, keywords = {Spectroscopy, Biochemistry}, pages = {47--55}, publisher = {Springer Nature}, title = {{Sensitivity-optimized experiment for the measurement of residual dipolar couplings between amide protons}}, doi = {10.1007/s10858-006-9138-2}, volume = {38}, year = {2007}, } @article{8486, abstract = {A technique is described that allows reducing acquisition times of multidimensional NMR experiments by extensive spectral folding. The method is simple and has many interesting applications for NMR studies of molecular structure, dynamics, and kinetics.}, author = {Lescop, Ewen and Schanda, Paul and Rasia, Rodolfo and Brutscher, Bernhard}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, keywords = {Colloid and Surface Chemistry, Biochemistry, General Chemistry, Catalysis}, number = {10}, pages = {2756--2757}, publisher = {American Chemical Society}, title = {{Automated spectral compression for fast multidimensional NMR and increased time resolution in real-time NMR spectroscopy}}, doi = {10.1021/ja068949u}, volume = {129}, year = {2007}, } @article{8484, abstract = {A series of sequential, intra-residue, and bi-directional BEST H–N–CA, H–N–CO, and H–N–CB pulse sequences is presented that extends the BEST concept introduced recently for fast multidimensional protein NMR [Schanda et al., J. Am. Chem. Soc. 128 (2006) 9042] to the complete set of experiments required for sequential resonance assignment. We demonstrate for the protein ubiquitin that 3D BEST H–N–C correlation spectra can be recorded on a 600 MHz NMR spectrometer equipped with a cryogenic probe in only a few minutes of acquisition time with sufficient sensitivity to detect all expected cross peaks.}, author = {Lescop, Ewen and Schanda, Paul and Brutscher, Bernhard}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, number = {1}, pages = {163--169}, publisher = {Elsevier}, title = {{A set of BEST triple-resonance experiments for time-optimized protein resonance assignment}}, doi = {10.1016/j.jmr.2007.04.002}, volume = {187}, year = {2007}, } @article{8489, abstract = {Structure elucidation of proteins by either NMR or X‐ray crystallography often requires the screening of a large number of samples for promising protein constructs and optimum solution conditions. For large‐scale screening of protein samples in solution, robust methods are needed that allow a rapid assessment of the folding of a polypeptide under diverse sample conditions. Here we present HET‐SOFAST NMR, a highly sensitive new method for semi‐quantitative characterization of the structural compactness and heterogeneity of polypeptide chains in solution. On the basis of one‐dimensional 1H HET‐SOFAST NMR data, obtained on well‐folded, molten globular, partially‐ and completely unfolded proteins, we define empirical thresholds that can be used as quantitative benchmarks for protein compactness. For 15N‐enriched protein samples, two‐dimensional 1H‐15N HET‐SOFAST correlation spectra provide site‐specific information about the structural heterogeneity along the polypeptide chain.}, author = {Schanda, Paul and Forge, Vincent and Brutscher, Bernhard}, issn = {0749-1581}, journal = {Magnetic Resonance in Chemistry}, number = {S1}, pages = {S177--S184}, publisher = {Wiley}, title = {{HET-SOFAST NMR for fast detection of structural compactness and heterogeneity along polypeptide chains}}, doi = {10.1002/mrc.1825}, volume = {44}, year = {2006}, } @article{8488, abstract = {We demonstrate for different protein samples that three-dimensional HNCO and HNCA correlation spectra may be recorded in a few minutes acquisition time using the band-selective excitation short-transient sequences presented here. This opens new perspectives for the NMR structural investigation of unstable protein samples and real-time site-resolved studies of protein kinetics.}, author = {Schanda, Paul and Van Melckebeke, Hélène and Brutscher, Bernhard}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, keywords = {Colloid and Surface Chemistry, Biochemistry, General Chemistry, Catalysis}, number = {28}, pages = {9042--9043}, publisher = {American Chemical Society}, title = {{Speeding up three-dimensional protein NMR experiments to a few minutes}}, doi = {10.1021/ja062025p}, volume = {128}, year = {2006}, } @article{8490, abstract = {We demonstrate the feasibility of recording 1H–15N correlation spectra of proteins in only one second of acquisition time. The experiment combines recently proposed SOFAST-HMQC with Hadamard-type 15N frequency encoding. This allows site-resolved real-time NMR studies of kinetic processes in proteins with an increased time resolution. The sensitivity of the experiment is sufficient to be applicable to a wide range of molecular systems available at millimolar concentration on a high magnetic field spectrometer.}, author = {Schanda, Paul and Brutscher, Bernhard}, issn = {1090-7807}, journal = {Journal of Magnetic Resonance}, keywords = {Nuclear and High Energy Physics, Biophysics, Biochemistry, Condensed Matter Physics}, number = {2}, pages = {334--339}, publisher = {Elsevier}, title = {{Hadamard frequency-encoded SOFAST-HMQC for ultrafast two-dimensional protein NMR}}, doi = {10.1016/j.jmr.2005.10.007}, volume = {178}, year = {2006}, } @article{8491, abstract = {Fast multidimensional NMR with a time resolution of a few seconds provides a new tool for high throughput screening and site-resolved real-time studies of kinetic molecular processes by NMR. Recently we have demonstrated the feasibility to record protein 1H–15N correlation spectra in a few seconds of acquisition time using a new SOFAST-HMQC experiment (Schanda and Brutscher (2005) J. Am. Chem. Soc. 127, 8014). Here, we investigate in detail the performance of SOFAST-HMQC to record 1H–15N and 1H−13C correlation spectra of proteins of different size and at different magnetic field strengths. Compared to standard 1H–15N correlation experiments SOFAST-HMQC provides a significant gain in sensitivity, especially for fast repetition rates. Guidelines are provided on how to set up SOFAST-HMQC experiments for a given protein sample. In addition, an alternative pulse scheme, IPAP-SOFAST-HMQC is presented that allows application on NMR spectrometers equipped with cryogenic probes, and fast measurement of one-bond 1H–13C and 1H–15N scalar and residual dipolar coupling constants.}, author = {Schanda, Paul and Kupče, Ēriks and Brutscher, Bernhard}, issn = {0925-2738}, journal = {Journal of Biomolecular NMR}, keywords = {Spectroscopy, Biochemistry}, number = {4}, pages = {199--211}, publisher = {Springer Nature}, title = {{SOFAST-HMQC experiments for recording two-dimensional deteronuclear correlation spectra of proteins within a few seconds}}, doi = {10.1007/s10858-005-4425-x}, volume = {33}, year = {2005}, } @article{8492, abstract = {We demonstrate for different protein samples that 2D 1H−15N correlation NMR spectra can be recorded in a few seconds of acquisition time using a new band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence experiment. This has enabled us to measure fast hydrogen−deuterium exchange rate constants along the backbone of a small globular protein fragment by real-time 2D NMR.}, author = {Schanda, Paul and Brutscher, Bernhard}, issn = {0002-7863}, journal = {Journal of the American Chemical Society}, keywords = {Colloid and Surface Chemistry, Biochemistry, General Chemistry, Catalysis}, number = {22}, pages = {8014--8015}, publisher = {American Chemical Society}, title = {{Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds}}, doi = {10.1021/ja051306e}, volume = {127}, year = {2005}, }