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Journal of Biomolecular NMR

Natalia Brodaczewska, Zuzana Košťálová, Dušan Uhrín
Overlap of NMR signals is the major cause of difficulties associated with NMR structure elucidation of molecules contained in complex mixtures. A 2D homonuclear correlation spectroscopy in particular suffers from low dispersion of 1H chemical shifts; larger dispersion of 13C chemical shifts is often used to reduce this overlap, while still providing the proton-proton correlation information e.g. in the form of a 2D 1H, 13C HSQC-TOCSY experiment. For this methodology to work, 13C chemical shift must be resolved...
January 11, 2018: Journal of Biomolecular NMR
Alexandre A Arnold, Jean-Philippe Bourgouin, Bertrand Genard, Dror E Warschawski, Réjean Tremblay, Isabelle Marcotte
In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically 13C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents...
January 11, 2018: Journal of Biomolecular NMR
Naima G Sharaf, Andrew E Brereton, In-Ja L Byeon, P Andrew Karplus, Angela M Gronenborn
In the original publication of the article, the given name and family name of the author P. Andrew Karplus was published incorrectly. The name should read as "P. Andrew" - Given name and "Karplus" - Family name.
December 12, 2017: Journal of Biomolecular NMR
Koh Takeuchi, Haribabu Arthanari, Ichio Shimada, Gerhard Wagner
The authors regret a mistake appeared in the supplement of this paper.
December 11, 2017: Journal of Biomolecular NMR
Jia-Liang Chen, Yu Zhao, Yan-Jun Gong, Bin-Bin Pan, Xiao Wang, Xun-Cheng Su
Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond...
December 9, 2017: Journal of Biomolecular NMR
Marie Dujardin, François-Xavier Cantrelle, Guy Lippens, Xavier Hanoulle
The non structural protein 5A (NS5A) regulates the replication of the hepatitis C viral RNA through a direct molecular interaction of its domain 2 (NS5A-D2) with the RNA dependent RNA polymerase NS5B. Because of conflicting data in the literature, we study here this molecular interaction using fluorinated versions of the NS5A-D2 protein derived from the JFH1 Hepatitis C Virus strain. Two methods to prepare fluorine-labelled NS5A-D2 involving the biosynthetic incorporation of a 19F-tryptophan using 5-fluoroindole and the posttranslational introduction of fluorine by chemical conjugation of 2-iodo-N-(trifluoromethyl)acetamide with the NS5A-D2 cysteine side chains are presented...
December 7, 2017: Journal of Biomolecular NMR
Su-Jin Kang, Yasuto Todokoro, Suyeon Bak, Toshiharu Suzuki, Masasuke Yoshida, Toshimichi Fujiwara, Hideo Akutsu
FoF1-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H+ translocation in membranes. The Fo c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state 13C NMR (ssNMR) signals of the Fo c-subunit ring of thermophilic Bacillus PS3 (TFo c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly 13C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic...
December 2, 2017: Journal of Biomolecular NMR
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 1H-NMR signals of the tert-butyl and TMS groups were compared to the 1H-NMR signal of tert-butyltyrosine (Tby) in protein systems with molecular weights ranging from 8 to 54 kDa...
December 2, 2017: Journal of Biomolecular NMR
Denis Lacabanne, Beat H Meier, Anja Böckmann
Selective isotope labeling is central in NMR experiments and often allows to push the limits on the systems investigated. It has the advantage to supply additional resolution by diminishing the number of signals in the spectra. This is particularly interesting when dealing with the large protein systems which are currently becoming accessible to solid-state NMR studies. Isotope labeled proteins for NMR experiments are most often expressed in E. coli systems, where bacteria are grown in minimal media supplemented with 15NH4Cl and 13C-glucose as sole source of nitrogen and carbon...
December 2, 2017: Journal of Biomolecular NMR
Noor E Hafsa, Mark V Berjanskii, David Arndt, David S Wishart
Protein structure determination using nuclear magnetic resonance (NMR) spectroscopy can be both time-consuming and labor intensive. Here we demonstrate how chemical shift threading can permit rapid, robust, and accurate protein structure determination using only chemical shift data. Threading is a relatively old bioinformatics technique that uses a combination of sequence information and predicted (or experimentally acquired) low-resolution structural data to generate high-resolution 3D protein structures. The key motivations behind using NMR chemical shifts for protein threading lie in the fact that they are easy to measure, they are available prior to 3D structure determination, and they contain vital structural information...
December 1, 2017: Journal of Biomolecular NMR
Pushpa Mishra, C Ashley Barnes, Madeleine Strickland, Nico Tjandra
Protein structure determination using NMR is dependent on experimentally acquired distance restraints. Often, however, an insufficient number of these restraints are available for determining a protein's correct fold, much less its detailed three-dimensional structure. In consideration of this problem, we propose a simple means to acquire supplemental structural restraints from protein surface accessibilities using solvent saturation transfer to proteins (SSTP), based on the principles of paramagnetic chemical-exchange saturation transfer...
November 30, 2017: Journal of Biomolecular NMR
Jithender G Reddy, Supriya Pratihar, David Ban, Sebastian Frischkorn, Stefan Becker, Christian Griesinger, Donghan Lee
Molecular dynamics play a significant role in how molecules perform their function. A critical method that provides information on dynamics, at the atomic level, is NMR-based relaxation dispersion (RD) experiments. RD experiments have been utilized for understanding multiple biological processes occurring at micro-to-millisecond time, such as enzyme catalysis, molecular recognition, ligand binding and protein folding. Here, we applied the recently developed high-power RD concept to the Carr-Purcell-Meiboom-Gill sequence (extreme CPMG; E-CPMG) for the simultaneous detection of fast and slow dynamics...
November 29, 2017: Journal of Biomolecular NMR
Mathilde Lescanne, Simon P Skinner, Anneloes Blok, Monika Timmer, Linda Cerofolini, Marco Fragai, Claudio Luchinat, Marcellus Ubbink
A new version of the program PARAssign has been evaluated for assignment of NMR resonances of the 76 methyl groups in leucines, isoleucines and valines in a 25 kDa protein, using only the structure of the protein and pseudocontact shifts (PCS) generated with a lanthanoid tag at up to three attachment sites. The number of reliable assignments depends strongly on two factors. The principle axes of the magnetic susceptibility tensors of the paramagnetic centers should not be parallel so as to avoid correlated PCS...
November 27, 2017: Journal of Biomolecular NMR
Azamat R Galiakhmetov, Elizaveta A Kovrigina, Chuanwu Xia, Jung-Ja P Kim, Evgenii L Kovrigin
NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70 kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240 kDa). To overcome paramagnetic relaxation in the reduced state of POR as well as the signal broadening due to its high molecular weight, we utilized the methyl-TROSY approach...
November 22, 2017: Journal of Biomolecular NMR
Youlin Xia, Paolo Rossi, Manu V Subrahmanian, Chengdong Huang, Tamjeed Saleh, Cristina Olivieri, Charalampos G Kalodimos, Gianluigi Veglia
In multidimensional solution NMR experiments, π pulses are used extensively for inversion and refocusing operations on (1)H, (13)C and (15)N nuclei. Pulse miscalibration, off-resonance effects, and J-coupling evolution during π pulse execution result in severe signal losses that are exacerbated at high magnetic fields. Here, we report the implementation of a triply-compensated π pulse (G5) optimized for both inversion and refocusing in widely used 2- and 3-dimensional experiments. By replacing most of the hard π pulses, adiabatic or composite pulses on the (1)H, (13)C and (15)N channels with G5 pulses, we obtained signal enhancements ranging from 80 to 240%...
November 21, 2017: Journal of Biomolecular NMR
Pablo Trigo-Mouriño, Christian Griesinger, Donghan Lee
Understanding the dissociation of molecules is the basis to modulate interactions of biomedical interest. Optimizing drugs for dissociation rates is found to be important for their efficacy, selectivity, and safety. Here, we show an application of the high-power relaxation dispersion (RD) method to the determination of the dissociation rates of weak binding ligands from receptors. The experiment probes proton RD on the ligand and, therefore, avoids the need for any isotopic labeling. The large ligand excess eases the detection significantly...
November 16, 2017: Journal of Biomolecular NMR
Sabrina Berkamp, Sang Ho Park, Anna A De Angelis, Francesca M Marassi, Stanley J Opella
The structure of monomeric human chemokine IL-8 (residues 1-66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1-72), with the main differences being the location of the N-loop (residues 10-22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67-72). NMR was used to analyze the interactions of monomeric IL-8 (1-66) with ND-CXCR1 (residues 1-38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1-72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs...
November 15, 2017: Journal of Biomolecular NMR
Rob Kaptein, Rolf Boelens, Claudio Luchinat
No abstract text is available yet for this article.
November 13, 2017: Journal of Biomolecular NMR
Harold W Mackenzie, D Flemming Hansen
Arginine side-chains are often key for enzyme catalysis, protein-ligand and protein-protein interactions. The importance of arginine stems from the ability of the terminal guanidinium group to form many key interactions, such as hydrogen bonds and salt bridges, as well as its perpetual positive charge. We present here an arginine (13)C(ζ)-detected NMR experiment in which a double-quantum coherence involving the two (15)N(η) nuclei is evolved during the indirect chemical shift evolution period. As the precession frequency of the double-quantum coherence is insensitive to exchange of the two (15)N(η); this new approach is shown to eliminate the previously deleterious line broadenings of (15)N(η) resonances caused by the partially restricted rotation about the C(ζ)-N(ε) bond...
November 10, 2017: Journal of Biomolecular NMR
Thomas Wiegand, Wei-Chih Liao, Ta Chung Ong, Alexander Däpp, Riccardo Cadalbert, Christophe Copéret, Anja Böckmann, Beat H Meier
DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein-DNA and protein-ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein-DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy...
November 8, 2017: Journal of Biomolecular NMR
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