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Journal of Biomolecular NMR

Alexander A Shcherbakov, Mei Hong
The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are 13 C-15 N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~ 5 Å due to the low gyromagnetic ratios of 15 N and 13 C. Here we present a robust 2D 13 C-19 F REDOR experiment to measure multiple distances to ~ 10 Å...
May 21, 2018: Journal of Biomolecular NMR
Julia Klopp, Aurélie Winterhalter, Rémy Gébleux, Daniela Scherer-Becker, Christian Ostermeier, Alvar D Gossert
We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13 C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of 2 H, 13 C and 15 N using auto-induction or isopropyl-β-D-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression...
May 19, 2018: Journal of Biomolecular NMR
Piotr Klukowski, Michał Augoff, Maciej Zamorski, Adam Gonczarek, Michał J Walczak
Analysis of structure, function and interactions of proteins by NMR spectroscopy usually requires the assignment of resonances to the corresponding nuclei in protein. This task, although automated by methods such as FLYA or PINE, is still frequently performed manually. To facilitate the manual sequence-specific chemical shift assignment of complex proteins, we propose a method based on Dirichlet process mixture model (DPMM) that performs automated matching of groups of signals observed in NMR spectra to corresponding nuclei in protein sequence...
May 18, 2018: Journal of Biomolecular NMR
João M C Teixeira, Simon P Skinner, Miguel Arbesú, Alexander L Breeze, Miquel Pons
We present Farseer-NMR ( ), a software package to treat, evaluate and combine NMR spectroscopic data from sets of protein-derived peaklists covering a range of experimental conditions. The combined advances in NMR and molecular biology enable the study of complex biomolecular systems such as flexible proteins or large multibody complexes, which display a strong and functionally relevant response to their environmental conditions, e.g. the presence of ligands, site-directed mutations, post translational modifications, molecular crowders or the chemical composition of the solution...
May 11, 2018: Journal of Biomolecular NMR
Friedemann Flügge, Thomas Peters
Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions...
April 26, 2018: Journal of Biomolecular NMR
Bastian Franke, Christian Opitz, Shin Isogai, Anne Grahl, Leonildo Delgado, Alvar D Gossert, Stephan Grzesiek
Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host...
April 23, 2018: Journal of Biomolecular NMR
Honglue Shi, Mary C Clay, Atul Rangadurai, Bharathwaj Sathyamoorthy, David A Case, Hashim M Al-Hashimi
NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson-Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1 A) and guanine (m1 G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1 A-T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove...
April 19, 2018: Journal of Biomolecular NMR
Paul A O'Brien, Arthur G Palmer
A TROSY-based NMR experiment is described for simultaneous measurement of the 15 N longitudinal relaxation rate constant R1 and the {1 H}-15 N nuclear Overhauser enhancement. The experiment is based on the observation that the TROSY mixing pulse sequence element symmetrically exchanges 1 H and 15 N magnetizations. The accuracy of the proposed technique is validated by comparison to independent measurements of both relaxation parameters for the protein ubiquitin. The simultaneous experiment is approximately 20-33% shorter than conventional sequential measurements...
April 16, 2018: Journal of Biomolecular NMR
Alexander Marchanka, Christoph Kreutz, Teresa Carlomagno
Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion...
April 12, 2018: Journal of Biomolecular NMR
Łukasz Jaremko, Mariusz Jaremko, Andrzej Ejchart, Michał Nowakowski
Simple and convenient method of protein dynamics evaluation from the insufficient experimental15 N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse15 N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N-H vectors on two different time scales, S2 and Rex , can be elucidated...
March 28, 2018: Journal of Biomolecular NMR
Walter Becker, Luke A Adams, Bim Graham, Gabriel E Wagner, Klaus Zangger, Gottfried Otting, Christoph Nitsche
Protein-ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D1 H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B-NS3 protease and the human prolyl isomerase FK506 binding protein...
March 21, 2018: Journal of Biomolecular NMR
Anusha B Gopalan, Pramodh Vallurupalli
Carr-Purcell-Meiboom-Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the μs to millisecond (ms) timescale between a visible major state and 'invisible' minor states. The exchange rate(s) ([Formula: see text]), population(s) of the minor state(s) and the absolute value of the chemical shift difference [Formula: see text] (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of [Formula: see text] that is required to reconstruct the spectrum of the 'invisible' minor state(s) cannot be obtained from CPMG data alone...
March 21, 2018: Journal of Biomolecular NMR
Kornelia M Mikula, Luisa Krumwiede, Andreas Plückthun, Hideo Iwaï
Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling...
March 13, 2018: Journal of Biomolecular NMR
Yutaka Kofuku, Tomoki Yokomizo, Shunsuke Imai, Yutaro Shiraishi, Mei Natsume, Hiroaki Itoh, Masayuki Inoue, Kunio Nakata, Shunsuke Igarashi, Hideyuki Yamaguchi, Toshimi Mizukoshi, Ei-Ichiro Suzuki, Takumi Ueda, Ichio Shimada
G protein-coupled receptors (GPCRs) exist in equilibrium between multiple conformations, and their populations and exchange rates determine their functions. However, analyses of the conformational dynamics of GPCRs in lipid bilayers are still challenging, because methods for observations of NMR signals of large proteins expressed in a baculovirus-insect cell expression system (BVES) are limited. Here, we report a method to incorporate methyl-13 C1 H3 -labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES...
March 8, 2018: Journal of Biomolecular NMR
Saeko Yanaka, Hirokazu Yagi, Rina Yogo, Maho Yagi-Utsumi, Koichi Kato
Glycoproteins are characterized by the heterogeneous and dynamic nature of their glycan moieties, which hamper crystallographic analysis. NMR spectroscopy provides potential advantages in dealing with such complicated systems, given that the target molecules can be isotopically labeled. Methods of metabolic isotope labeling in recombinant glycoproteins have been developed recently using a variety of eukaryotic production vehicles, including mammalian, yeast, insect, and plant cells, each of which has a distinct N-glycan diversification pathway...
February 28, 2018: Journal of Biomolecular NMR
Albert A Smith, Emilie Testori, Riccardo Cadalbert, Beat H Meier, Matthias Ernst
In our recent publication (Smith et al., J Biomol NMR 65:171-191, 2016) on the dynamics of HET-s(218-289), we reported on page 176, that calculation of solid-state NMR R1ρ rate constants using analytical equations based on Redfield theory (Kurbanov et al., J Chem Phys 135:184104:184101-184109, 2011) failed when the correlation time of motion becomes too long.
March 2018: Journal of Biomolecular NMR
James Tolchard, Manoj Kumar Pandey, Mélanie Berbon, Abdelmajid Noubhani, Sven J Saupe, Yusuke Nishiyama, Birgit Habenstein, Antoine Loquet
We present a new solid-state NMR proton-detected three-dimensional experiment dedicated to the observation of protein proton side chain resonances in nano-liter volumes. The experiment takes advantage of very fast magic angle spinning and double quantum 13C-13C transfer to establish efficient (H)CCH correlations detected on side chain protons. Our approach is demonstrated on the HET-s prion domain in its functional amyloid fibrillar form, fully protonated, with a sample amount of less than 500 µg using a MAS frequency of 70 kHz...
March 2018: Journal of Biomolecular NMR
Maria Grazia Murrali, Marco Schiavina, Valerio Sainati, Wolfgang Bermel, Roberta Pierattelli, Isabella C Felli
The increasingly recognized biological relevance of intrinsically disordered proteins requires a continuous expansion of the tools for their characterization via NMR spectroscopy, the only technique so far able to provide atomic-resolution information on these highly mobile macromolecules. Here we present the implementation of projection spectroscopy in 13 C-direct detected NMR experiments to achieve the sequence specific assignment of IDPs. The approach was used to obtain the complete backbone assignment at high temperature of α-synuclein, a paradigmatic intrinsically disordered protein...
March 2018: Journal of Biomolecular NMR
Jakob Toudahl Nielsen, Frans A A Mulder
Chemical shifts contain important site-specific information on the structure and dynamics of proteins. Deviations from statistical average values, known as random coil chemical shifts (RCCSs), are extensively used to infer these relationships. Unfortunately, the use of imprecise reference RCCSs leads to biased inference and obstructs the detection of subtle structural features. Here we present a new method, POTENCI, for the prediction of RCCSs that outperforms the currently most authoritative methods. POTENCI is parametrized using a large curated database of chemical shifts for protein segments with validated disorder; It takes pH and temperature explicitly into account, and includes sequence-dependent nearest and next-nearest neighbor corrections as well as second-order corrections...
March 2018: Journal of Biomolecular NMR
Cristina Olivieri, Manu Veliparambil Subrahmanian, Youlin Xia, Jonggul Kim, Fernando Porcelli, Gianluigi Veglia
Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein-protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (15 N or 13 C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein-protein complexes, making this approach time consuming and expensive...
March 2018: Journal of Biomolecular NMR
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