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Journal of Biomolecular NMR

Stephan B Azatian, Navneet Kaur, Michael P Latham
We describe a general and simple modification to the standard M9 minimal medium recipe that leads to an approximate twofold increase in the yield of heterologously expressed proteins in Escherichia coli BL21(DE3) bacteria. We monitored the growth of bacteria transformed with plasmids for three different test proteins in five minimal media with different concentrations of buffering salts and/or initial media pH. After purification of the over-expressed proteins, we found a clear correlation between the protein yield and change in media pH over time, where the minimal media that were the most buffered and therefore most resistant to change in pH produced the most protein...
January 7, 2019: Journal of Biomolecular NMR
Daniel Lane, Thomas E Skinner, Naum I Gershenzon, Wolfgang Bermel, Ronald Soong, Rudraksha Dutta Majumdar, Yalda Liaghati Mobarhan, Sebastian Schmidt, Hermann Heumann, Martine Monette, Myrna J Simpson, André J Simpson
In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D 1 H-13 C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo...
January 2, 2019: Journal of Biomolecular NMR
Eldon L Ulrich, Kumaran Baskaran, Hesam Dashti, Yannis E Ioannidis, Miron Livny, Pedro R Romero, Dimitri Maziuk, Jonathan R Wedell, Hongyang Yao, Hamid R Eghbalnia, Jeffrey C Hoch, John L Markley
The growth of the biological nuclear magnetic resonance (NMR) field and the development of new experimental technology have mandated the revision and enlargement of the NMR-STAR ontology used to represent experiments, spectral and derived data, and supporting metadata. We present here a brief description of the NMR-STAR ontology and software tools for manipulating NMR-STAR data files, editing the files, extracting selected data, and creating data visualizations. Detailed information on these is accessible from the links provided...
December 22, 2018: Journal of Biomolecular NMR
Thomas Wiegand, Andreas Hunkeler, Alexander Däpp, Joeri Verasdonck, Riccardo Cadalbert, Luc Bousset, Ronald Melki, Anja Böckmann, Beat H Meier
Magic-angle spinning (MAS) is mandatory in solid-state NMR experiments to achieve resolved spectra. In rare cases, instabilities in the rotation or damage of either the rotor or the rotor cap can lead to a so called "rotor crash" involving a disintegration of the sample container and possibly the release of an aerosol or of dust. We present a modified design of a 3.2 mm probe with a confining chamber which in case of a rotor crash prevents the release of aerosols and possibly hazardous materials...
December 10, 2018: Journal of Biomolecular NMR
Carolina Lixa, Amanda Mujo, Mariana T Q de Magalhães, Fabio C L Almeida, Luis Mauricio T R Lima, Anderson S Pinheiro
Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1...
December 8, 2018: Journal of Biomolecular NMR
Luke W Arbogast, Frank Delaglio, Joel R Tolman, John P Marino
While the use of 1 H-13 C methyl correlated NMR spectroscopy at natural isotopic abundance has been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies, spectral interference from aliphatic excipients remains a significant obstacle to its widespread application. These signals can cause large baseline artifacts, obscure protein resonances, and cause dynamic range suppression of weak peaks in non-uniform sampling applications, thus hampering both traditional peak-based spectral analyses as well as emerging chemometric methods of analysis...
November 27, 2018: Journal of Biomolecular NMR
Nina Eleni Christou, Bernhard Brutscher
Aromatic amino-acid side chains are essential components for the structure and function of proteins. We present herein a set of NMR experiments for time-efficient resonance assignment of histidine and tyrosine side chains in uniformly 13 C/15 N-labeled proteins. The use of band-selective 13 C pulses allows to deal with linear chains of coupled spins, thus avoiding signal loss that occurs in branched spin systems during coherence transfer. Furthermore, our pulse schemes make use of longitudinal 1 H relaxation enhancement, Ernst-angle excitation, and simultaneous detection of 1 H and 13 C steady-state polarization to achieve significant signal enhancements...
November 21, 2018: Journal of Biomolecular NMR
Alexander Klein, Suresh K Vasa, Rasmus Linser
Given that solid-state NMR is being used for protein samples of increasing molecular weight and complexity, higher-dimensionality methods are likely to be more and more indispensable for unambiguous chemical shift assignments in the near future. In addition, solid-state NMR spectral properties are increasingly comparable with solution NMR, allowing adaptation of more sophisticated solution NMR strategies for the solid state in addition to the conventional methodology. Assessing first principles, here we demonstrate the application of automated projection spectroscopy for a micro-crystalline protein in the solid state...
November 14, 2018: Journal of Biomolecular NMR
Belén Chaves-Arquero, David Pantoja-Uceda, Alicia Roque, Inmaculada Ponte, Pedro Suau, M Angeles Jiménez
The C-terminal domain of histone H1.0 (C-H1.0) is involved in DNA binding and is a main determinant of the chromatin condensing properties of histone H1.0. Phosphorylation at the (S/T)-P-X-(K/R) motifs affects DNA binding and is crucial for regulation of C-H1.0 function. Since C-H1.0 is an intrinsically disordered domain, solution NMR is an excellent approach to characterize the effect of phosphorylation on the structural and dynamic properties of C-H1.0. However, its very repetitive, low-amino acid-diverse and Pro-rich sequence, together with the low signal dispersion observed at the 1 H-15 N HSQC spectra of both non- and tri-phosphorylated C-H1...
November 9, 2018: Journal of Biomolecular NMR
Azzurra Carlon, Enrico Ravera, Giacomo Parigi, Garib N Murshudov, Claudio Luchinat
Data integration in structural biology has become a paradigm for the characterization of biomolecular systems, and it is now accepted that combining different techniques can fill the gaps in each other's blind spots. In this frame, one of the combinations, which we have implemented in REFMAC-NMR, is residual dipolar couplings from NMR together with experimental data from X-ray diffraction. The first are exquisitely sensitive to the local details but does not give any information about overall shape, whereas the latter encodes more the information about the overall shape but at the same time tends to miss the local details even at the highest resolutions...
October 11, 2018: Journal of Biomolecular NMR
Pratibha Kumari, Lukas Frey, Alexander Sobol, Nils-Alexander Lakomek, Roland Riek
15 N R2 relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide-water exchange can severely skew Hahn-echo based 15 N R2 relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the 15 N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data...
October 10, 2018: Journal of Biomolecular NMR
Anusha B Gopalan, Tairan Yuwen, Lewis E Kay, Pramodh Vallurupalli
Protein conformational changes play crucial roles in enabling function. The Carr-Purcell-Meiboom-Gill (CPMG) experiment forms the basis for studying such dynamics when they involve the interconversion between highly populated and sparsely formed states, the latter having lifetimes ranging from ~ 0.5 to ~ 5 ms. Among the suite of experiments that have been developed are those that exploit methyl group probes by recording methyl 1 H single quantum (Tugarinov and Kay in J Am Chem Soc 129:9514-9521, 2007) and triple quantum (Yuwen et al...
October 2018: Journal of Biomolecular NMR
Heiner N Raum, Matthias Dreydoppel, Ulrich Weininger
Aromatic side chains are attractive probes of protein dynamics on the millisecond time scale, because they are often key residues in enzyme active sites and protein binding sites. Further they allow to study specific processes, like histidine tautomerization and ring flips. Till now such processes have been studied by aromatic 13 C CPMG relaxation dispersion experiments. Here we investigate the possibility of aromatic 1 H CPMG relaxation dispersion experiments as a complementary method. Artifact-free dispersions are possible on uniformly 1 H and 13 C labeled samples for histidine δ2 and ε1, as well as for tryptophan δ1...
October 2018: Journal of Biomolecular NMR
Michal Rivlin, Gil Navon
3-O-Methyl-D-glucose (3OMG) was recently suggested as an agent to image tumors using chemical exchange saturation transfer (CEST) MRI. To characterize the properties of 3OMG in solution, the anomeric equilibrium and the mutarotation rates of 3OMG were studied by 1 H and 13 C NMR. This information is essential in designing the in vivo CEST experiments. At room temperature, the ratio of α and β 3OMG anomers at equilibrium was 1:1.4, and the time to reach 95% equilibrium was 6 h. The chemical exchange rates between the hydroxyl protons of 3OMG and water, measured by CEST and spin lock at pH 6...
October 2018: Journal of Biomolecular NMR
Tom Aharoni, Amir Goldbourt
Determination of chemical shift anisotropy (CSA) in immobilized proteins and protein assemblies is one of several tools to determine protein dynamics on the timescales of microseconds and faster. The large CSA values of C=O groups in the rigid limit makes them in particular attractive for measurements of large amplitude motions, or their absence. In this study, we implement a 3D R-symmetry-based sequence that recouples the second spatial component of the 13 C CSA with the corresponding isotropic 13 C'-13 C cross-peaks in order to probe backbone and sidechain dynamics in an intact fd-y21m filamentous phage viral capsid...
October 2018: Journal of Biomolecular NMR
Liliya Vugmeyster, Aaron Griffin, Dmitry Ostrovsky, Shibani Bhattacharya, Parker J Nichols, C James McKnight, Beat Vögeli
We investigated correlated µs-ms time scale motions of neighboring 13 C'-15 N and 13 Cα -13 Cβ nuclei in both protonated and perdeuterated samples of GB3. The techniques employed, NMR relaxation due to cross-correlated chemical shift modulations, specifically target concerted changes in the isotropic chemical shifts of the two nuclei associated with spatial fluctuations. Field-dependence of the relaxation rates permits identification of the parameters defining the chemical exchange rate constant under the assumption of a two-site exchange...
October 2018: Journal of Biomolecular NMR
Daniel Joss, Roché M Walliser, Kaspar Zimmermann, Daniel Häussinger
Pseudocontact shifts (PCS) generated by lanthanide chelating tags yield valuable restraints for investigating protein structures, dynamics and interactions in solution. In this work, dysprosium-, thulium- and terbium-complexes of eight-fold methylated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tags [DOTA-M8-(4R4S)-SSPy] are presented that induce large pseudocontact shifts up to 5.5 ppm and adopt exclusively the square antiprismatic conformation. This is in contrast to our earlier findings on complexes of the stereoisomeric DOTA-M8-(8S)-SSPy, where significant amounts of the twisted square antiprismatic conformer for the Dy tag were observed...
October 2018: Journal of Biomolecular NMR
Xi Chen, Andrey Smelter, Hunter N B Moseley
Poor chemical shift referencing, especially for 13 C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR, including protein structure determination and analysis of protein dynamics. To solve this problem, we constructed a Bayesian probabilistic framework that circumvents the limitations of previous reference correction methods that required protein resonance assignment and/or three-dimensional protein structure. Our algorithm named Bayesian Model Optimized Reference Correction (BaMORC) can detect and correct 13 C chemical shift referencing errors before the protein resonance assignment step of analysis and without three-dimensional structure...
October 2018: Journal of Biomolecular NMR
Andreas Kniss, Sina Kazemi, Frank Löhr, Maren Berger, Vladimir V Rogov, Peter Güntert, Thomas Sommer, Ernst Jarosch, Volker Dötsch
Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose...
October 2018: Journal of Biomolecular NMR
Alons Lends, Francesco Ravotti, Giorgia Zandomeneghi, Anja Böckmann, Matthias Ernst, Beat H Meier
The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons...
September 11, 2018: Journal of Biomolecular NMR
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