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Journal of Biomolecular NMR

Masatsune Kainosho, Yohei Miyanoiri, Tsutomu Terauchi, Mitsuhiro Takeda
In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods...
June 22, 2018: Journal of Biomolecular NMR
Margaret Phillips, Janet To, Toshio Yamazaki, Toshio Nagashima, Jaume Torres, Konstantin Pervushin
Aquaporins are integral membrane proteins that facilitate water flow across biological membranes. Their involvement in multiple physiological functions and disease states has prompted intense research to discover water channel activity modulators. However, inhibitors found so far are weak and/or lack specificity. For organic compounds, which lack of high electron-dense atoms, the identification of binding sites is even more difficult. Nuclear magnetic resonance spectroscopy (NMR) requires large amounts of the protein, and expression and purification of mammalian aquaporins in large quantities is a difficult task...
June 18, 2018: Journal of Biomolecular NMR
Jerzy Sitkowski, Wojciech Bocian, Elżbieta Bednarek, Mateusz Urbańczyk, Wiktor Koźmiński, Piotr Borowicz, Grażyna Płucienniczak, Natalia Łukasiewicz, Iwona Sokołowska, Lech Kozerski
The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13 C,15 N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble...
June 12, 2018: Journal of Biomolecular NMR
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...
June 12, 2018: Journal of Biomolecular NMR
Benjamin Morash, Muzaddid Sarker, Jan K Rainey
Sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) is the most widely accepted internal standard for protein NMR studies in aqueous conditions. Since its introduction as a reference standard, however, concerns have been raised surrounding its propensity to interact with biological molecules through electrostatic and hydrophobic interactions. While DSS has been shown to interact with certain proteins, membrane protein studies by solution-state NMR require use of membrane mimetics such as detergent micelles and, to date, no study has explicitly examined the potential for interaction between membrane mimetics and DSS...
June 6, 2018: Journal of Biomolecular NMR
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...
June 5, 2018: Journal of Biomolecular NMR
Soumya Deep Chatterjee, Marcellus Ubbink, Hugo van Ingen
Understanding of the molecular mechanisms of protein function requires detailed insight into the conformational landscape accessible to the protein. Conformational changes can be crucial for biological processes, such as ligand binding, protein folding, and catalysis. NMR spectroscopy is exquisitely sensitive to such dynamic changes in protein conformations. In particular, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are a powerful tool to investigate protein dynamics on a millisecond time scale...
June 2, 2018: Journal of Biomolecular NMR
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...
June 2, 2018: Journal of Biomolecular NMR
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...
June 1, 2018: Journal of Biomolecular NMR
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...
May 28, 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
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
Alexey Krushelnitsky, Diego Gauto, Diana C Rodriguez Camargo, Paul Schanda, Kay Saalwächter
Solid-state near-rotary-resonance measurements of the spin-lattice relaxation rate in the rotating frame (R1ρ ) is a powerful NMR technique for studying molecular dynamics in the microsecond time scale. The small difference between the spin-lock (SL) and magic-angle-spinning (MAS) frequencies allows sampling very slow motions, at the same time it brings up some methodological challenges. In this work, several issues affecting correct measurements and analysis of 15 N R1ρ data are considered in detail. Among them are signal amplitude as a function of the difference between SL and MAS frequencies, "dead time" in the initial part of the relaxation decay caused by transient spin-dynamic oscillations, measurements under HORROR condition and proper treatment of the multi-exponential relaxation decays...
May 2018: Journal of Biomolecular NMR
Dan Nguyen, Junji Iwahara
NMR relaxation of arginine (Arg) 15 Nε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal 15 N-15 N scalar couplings on measurements of transverse relaxation rates (R 2 ) for Arg side-chain 15 Nε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal 15 N-15 N couplings ( 2 J NN ) of the 15 Nε nuclei and found that the magnitudes of the 2 J NN coupling constants were virtually uniform with an average of 1...
May 2018: Journal of Biomolecular NMR
Maureen Leninger, William M Marsiglia, Alexej Jerschow, Nathaniel J Traaseth
Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to 1 H, 15 N or 13 C...
May 2018: 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 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 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
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
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 2018: Journal of Biomolecular NMR
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