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Journal of Biomolecular NMR

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https://www.readbyqxmd.com/read/30229369/conformational-exchange-of-aromatic-side-chains-by-1-h-cpmg-relaxation-dispersion
#1
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...
September 18, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30206780/direct-amide-15-n-to-13-c-transfers-for-solid-state-assignment-experiments-in-deuterated-proteins
#2
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
https://www.readbyqxmd.com/read/30203383/3-o-methyl-d-glucose-mutarotation-and-proton-exchange-rates-assessed-by-13-c-1-h-nmr-and-by-chemical-exchange-saturation-transfer-and-spin-lock-measurements
#3
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...
September 10, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30141148/rapid-automated-determination-of-chemical-shift-anisotropy-values-in-the-carbonyl-and-carboxyl-groups-of-fd-y21m-bacteriophage-using-solid-state-nmr
#4
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...
August 23, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30121872/correlated-motions-of-c-n-and-c-%C3%AE-c-%C3%AE-pairs-in-protonated-and-per-deuterated-gb3
#5
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...
August 18, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30117038/conformationally-locked-lanthanide-chelating-tags-for-convenient-pseudocontact-shift-protein-nuclear-magnetic-resonance-spectroscopy
#6
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...
August 16, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30097912/automatic-13-c-chemical-shift-reference-correction-for-unassigned-protein-nmr-spectra
#7
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...
August 10, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30066206/structural-investigation-of-glycan-recognition-by-the-erad-quality-control-lectin-yos9
#8
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...
July 31, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30121871/on-the-use-of-pichia-pastoris-for-isotopic-labeling-of-human-gpcrs-for-nmr-studies
#9
Lindsay Clark, Igor Dikiy, Daniel M Rosenbaum, Kevin H Gardner
NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design)...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30073492/improving-yields-of-deuterated-methyl-labeled-protein-by-growing-in-h-2-o
#10
Evan S O'Brien, Danny W Lin, Brian Fuglestad, Matthew A Stetz, Travis Gosse, Cecilia Tommos, A Joshua Wand
Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the 15 N-1 H TROSY and 13 C-1 H3 methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in D2 O leads to many difficulties and limitations, such as cell toxicity, decreased yield, and the need to unfold or destabilize proteins for back exchange of amide protons. These issues are exacerbated for non-ideal systems such as membrane proteins...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29948439/segmental-isotope-labelling-and-solid-state-nmr-of-a-12-%C3%A3-59-kda-motor-protein-identification-of-structural-variability
#11
Thomas Wiegand, Riccardo Cadalbert, Christine von Schroetter, Frédéric H-T Allain, Beat H Meier
Segmental isotope labelling enables the NMR study of an individual domain within a multidomain protein, but still in the context of the entire full-length protein. Compared to the fully labelled protein, spectral overlap can be greatly reduced. We here describe segmental labelling of the (double-) hexameric DnaB helicase from Helicobacter pylori using a ligation approach. Solid-state spectra demonstrate that the ligated protein has the same structure and structural order as the directly expressed full-length protein...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29869771/methyl-selective-isotope-labeling-using-%C3%AE-ketoisovalerate-for-the-yeast-pichia-pastoris-recombinant-protein-expression-system
#12
Rika Suzuki, Masayoshi Sakakura, Masaki Mori, Moe Fujii, Satoko Akashi, Hideo Takahashi
Methyl-detected NMR spectroscopy is a useful tool for investigating the structures and interactions of large macromolecules such as membrane proteins. The procedures for preparation of methyl-specific isotopically-labeled proteins were established for the Escherichia coli (E. coli) expression system, but typically it is not feasible to express eukaryotic proteins using E. coli. The Pichia pastoris (P. pastoris) expression system is the most common yeast expression system, and is known to be superior to the E...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29860649/methyl-group-reorientation-under-ligand-binding-probed-by-pseudocontact-shifts
#13
Mathilde Lescanne, Puneet Ahuja, Anneloes Blok, Monika Timmer, Tomas Akerud, Marcellus Ubbink
Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29779067/cost-effective-large-scale-expression-of-proteins-for-nmr-studies
#14
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...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29536230/segmental-isotopic-labeling-by-asparaginyl-endopeptidase-mediated-protein-ligation
#15
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...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29197976/genetically-encoded-amino-acids-with-tert-butyl-and-trimethylsilyl-groups-for-site-selective-studies-of-proteins-by-nmr-spectroscopy
#16
Choy Theng Loh, Luke A Adams, Bim Graham, Gottfried Otting
The amino acids 4-(tert-butyl)phenylalanine (Tbf) and 4-(trimethylsilyl)phenylalanine (TMSf), as well as a partially deuterated version of Tbf (dTbf), were chemically synthesized and site-specifically incorporated into different proteins, using an amber stop codon, suppressor tRNA and the broadband aminoacyl-tRNA synthetase originally evolved for the incorporation of p-cyano-phenylalanine. The 1 H-NMR signals of the tert-butyl and TMS groups were compared to the 1 H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54 kDa...
August 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/30043256/advanced-isotopic-labeling-for-the-nmr-investigation-of-challenging-proteins-and-nucleic-acids
#17
EDITORIAL
Jerome Boisbouvier, Lewis E Kay
No abstract text is available yet for this article.
July 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29858959/nmr-probing-of-invisible-excited-states-using-selectively-labeled-rnas
#18
Regan M LeBlanc, Andrew P Longhini, Vitali Tugarinov, T Kwaku Dayie
Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are invaluable for probing sparsely and transiently populated biomolecular states that cannot be directly detected by traditional NMR experiments and that are invisible by other biophysical approaches. A notable gap for RNA is the absence of CPMG experiments for measurement of methine base 1 H and methylene C5' chemical shifts of ribose moieties in the excited state, partly because of complications from homonuclear 13 C-13 C scalar couplings...
July 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29808436/late-metabolic-precursors-for-selective-aromatic-residue-labeling
#19
Julia Schörghuber, Leonhard Geist, Gerald Platzer, Michael Feichtinger, Marilena Bisaccia, Lukas Scheibelberger, Frederik Weber, Robert Konrat, Roman J Lichtenecker
In recent years, we developed a toolbox of heavy isotope containing compounds, which serve as metabolic amino acid precursors in the E. coli-based overexpression of aromatic residue labeled proteins. Our labeling techniques show excellent results both in terms of selectivity and isotope incorporation levels. They are additionally distinguished by low sample production costs and meet the economic demands to further implement protein NMR spectroscopy as a routinely used method in drug development processes. Different isotopologues allow for the assembly of optimized protein samples, which fulfill the requirements of various NMR experiments to elucidate protein structures, analyze conformational dynamics, or probe interaction surfaces...
July 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29687312/production-of-isotope-labeled-proteins-in-insect-cells-for-nmr
#20
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...
July 2018: Journal of Biomolecular NMR
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