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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
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 8, 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
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 3, 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 in13 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...
February 28, 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
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...
February 5, 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 (15N or 13C). 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...
February 2, 2018: Journal of Biomolecular NMR
Tairan Yuwen, Lewis E Kay
Chemical exchange saturation transfer (CEST) experiments are becoming increasingly popular for investigating biomolecular exchange dynamics with rates on the order of approximately 50-500 s-1 and a rich toolkit of different methods has emerged over the past few years. Typically, experiments are based on the evolution of longitudinal magnetization, or in some cases two-spin order, during a fixed CEST relaxation delay, with the same class of magnetization prepared at the start and selected at end of the CEST period...
January 19, 2018: Journal of Biomolecular NMR
Sebanti Gupta, Robert Tycko
Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716-730, 2010; J Mol Biol 426:1109-1127, 2014; J Biol Chem 291:13098-13112, 2016; J Am Chem Soc 138:8538-8546, 2016; J Am Chem Soc 138:12029-12032, 2016; J Am Chem Soc 134:6455-6466, 2012; J Am Chem Soc 132:1976-1987, 2010; J Am Chem Soc 135:17793-17803, 2013; Proc Natl Acad Sci USA 112:14617-14622, 2015; J Am Chem Soc 138:14066-14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements...
January 18, 2018: 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
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...
February 2018: 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 a19 F-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...
January 2018: Journal of Biomolecular NMR
Su-Jin Kang, Yasuto Todokoro, Suyeon Bak, Toshiharu Suzuki, Masasuke Yoshida, Toshimichi Fujiwara, Hideo Akutsu
Fo F1 -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-state13 C 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 uniformly13 C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic...
January 2018: 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...
January 2018: 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...
January 2018: 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...
January 2018: 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...
January 2018: 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. The1 H-NMR signals of the tert-butyl and TMS groups were compared to the1 H-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
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