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Journal of Biomolecular NMR
Journal Prestige (SJR): 1.371
Citation Impact (citeScore): 2
Number of Followers: 8  
 
  Hybrid Journal Hybrid journal (It can contain Open Access articles)
ISSN (Print) 1573-5001 - ISSN (Online) 0925-2738
Published by Springer-Verlag Homepage  [2348 journals]
  • Selective labeling and unlabeling strategies in protein solid-state NMR
           spectroscopy
    • Authors: Denis Lacabanne; Beat H. Meier; Anja Böckmann
      Pages: 141 - 150
      Abstract: 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. For amino acids selective labeling or unlabeling, specific amino acids are supplemented in the minimal medium. The aim is that they will be incorporated in the protein by the bacteria. However, E. coli amino-acid anabolism and catabolism tend to interconnect different pathways, remnant of a subway system. These connections lead to inter conversion between amino acids, called scrambling. A thorough understanding of the involved pathways is thus important to obtain the desired labeling schemes, as not all combinations of amino acids are adapted. We present here a detailed overview of amino-acid metabolism in this context. Each amino-acid pathway is described in order to define accessible combinations for 13C or 15N specific labeling or unlabeling. Using as example the ABC transporter BmrA, a membrane protein of 600 residues, we demonstrate how these strategies can be applied. Indeed, even though there is no size limit in solid-state NMR, large (membrane) proteins are still a challenge due to heavy signal overlap. To initiate resonance assignment in these large systems, we describe how selectively labeled samples can be obtained with the addition of labeled or unlabeled amino acids in the medium. The reduced spectral overlap enabled us to identify typical spectral fingerprints and to initiate sequential assignment using the more sensitive 2D DARR experiments with long mixing time showing inter-residue correlations. Graphical
      PubDate: 2018-07-01
      DOI: 10.1007/s10858-017-0156-z
      Issue No: Vol. 71, No. 3 (2018)
       
  • Isotope labeling for studying RNA by solid-state NMR spectroscopy
    • Authors: Alexander Marchanka; Christoph Kreutz; Teresa Carlomagno
      Pages: 151 - 164
      Abstract: 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. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.
      PubDate: 2018-07-01
      DOI: 10.1007/s10858-018-0180-7
      Issue No: Vol. 71, No. 3 (2018)
       
  • Deuteration and selective labeling of alanine methyl groups of β 2
           -adrenergic receptor expressed in a baculovirus-insect cell expression
           system
    • Authors: 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
      Pages: 185 - 192
      Abstract: 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-13C1H3-labeled alanine with > 45% efficiency in highly deuterated proteins expressed in BVES. Application of the method to the NMR observations of β2-adrenergic receptor in micelles and in nanodiscs revealed the ligand-induced conformational differences throughout the transmembrane region of the GPCR.
      PubDate: 2018-07-01
      DOI: 10.1007/s10858-018-0174-5
      Issue No: Vol. 71, No. 3 (2018)
       
  • Stable isotope labeling approaches for NMR characterization of
           glycoproteins using eukaryotic expression systems
    • Authors: Saeko Yanaka; Hirokazu Yagi; Rina Yogo; Maho Yagi-Utsumi; Koichi Kato
      Pages: 193 - 202
      Abstract: 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. Yeast genetic engineering has enabled the overexpression of homogeneous high-mannose-type oligosaccharides with 13C labeling for NMR characterization of their conformational dynamics. The utility of stable isotope-assisted NMR spectroscopy has also been demonstrated using the Fc fragment of immunoglobulin G (IgG) as a model glycoprotein, providing useful information regarding intramolecular carbohydrate–protein interactions. Transverse relaxation optimization of intact IgG with a molecular mass of 150 kDa has been achieved by tailored deuteration of selected amino acid residues using a mammalian expression system. This offers a useful probe for the characterization of molecular interaction networks in multimolecular crowded systems typified by serum. Perspectives regarding the development of techniques for tailoring glycoform designs and isotope labeling of recombinant glycoproteins are also discussed.
      PubDate: 2018-07-01
      DOI: 10.1007/s10858-018-0169-2
      Issue No: Vol. 71, No. 3 (2018)
       
  • Conformationally locked lanthanide chelating tags for convenient
           pseudocontact shift protein nuclear magnetic resonance spectroscopy
    • Abstract: Abstract 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. The Dy-, Tm-, Tb- and Lu-complexes of DOTA-M8-(4R4S)-SSPy were conjugated to ubiquitin S57C and selectively 15N leucine labeled human carbonic anhydrase II S50C, resulting in only one set of signals. Furthermore, we investigated the conformation of the thulium- and dysprosium-complexes in vacuo and with implicit water solvent using density functional theory calculations. The calculated energy differences between the two different conformations (7.0–50.5 kJ/mol) and experimental evidence from the corresponding ytterbium- and yttrium-complexes clearly suggest a SAP [Λ(δδδδ)] geometry for the complexes presented in this study. The lanthanide chelating tag studied in this work offer insights into the solution structure of proteins by inducing strong pseudocontact shifts, show different tensor properties compared to its predecessor, enables a convenient assignment procedure, is accessed by a more economic synthesis than its predecessor and constitutes a highly promising starting point for further developments of lanthanide chelating tags.
      PubDate: 2018-08-16
       
  • Automatic 13 C chemical shift reference correction for unassigned protein
           NMR spectra
    • Authors: Xi Chen; Andrey Smelter; Hunter N. B. Moseley
      Abstract: Poor chemical shift referencing, especially for 13C 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 13C chemical shift referencing errors before the protein resonance assignment step of analysis and without three-dimensional structure. By combining the BaMORC methodology with a new intra-peaklist grouping algorithm, we created a combined method called Unassigned BaMORC that utilizes only unassigned experimental peak lists and the amino acid sequence. Unassigned BaMORC kept all experimental three-dimensional HN(CO)CACB-type peak lists tested within ± 0.4 ppm of the correct 13C reference value. On a much larger unassigned chemical shift test set, the base method kept 13C chemical shift referencing errors to within ± 0.45 ppm at a 90% confidence interval. With chemical shift assignments, Assigned BaMORC can detect and correct 13C chemical shift referencing errors to within ± 0.22 at a 90% confidence interval. Therefore, Unassigned BaMORC can correct 13C chemical shift referencing errors when it will have the most impact, right before protein resonance assignment and other downstream analyses are started. After assignment, chemical shift reference correction can be further refined with Assigned BaMORC. These new methods will allow non-NMR experts to detect and correct 13C referencing error at critical early data analysis steps, lowering the bar of NMR expertise required for effective protein NMR analysis.
      PubDate: 2018-08-10
      DOI: 10.1007/s10858-018-0202-5
       
  • Improving yields of deuterated, methyl labeled protein by growing in H 2 O
    • Authors: Evan S. O’Brien; Danny W. Lin; Brian Fuglestad; Matthew A. Stetz; Travis Gosse; Cecilia Tommos; A. Joshua Wand
      Abstract: Solution NMR continues to make strides in addressing protein systems of significant size and complexity. A fundamental requirement to fully exploit the 15N–1H TROSY and 13C–1H3 methyl TROSY effects is highly deuterated protein. Unfortunately, traditional overexpression in Escherichia coli (E. coli) during growth on media prepared in D2O 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. Expression of protein during growth in H2O, with the addition of 2H-labeled amino acids derived from algal extract, can potentially avoid these issues. We demonstrate a novel fermentation methodology for high-density bacterial growth in H2O M9 medium that allows for appropriate isotopic labeling and deuteration. Yields are significantly higher than those achieved in D2O M9 for a variety of protein targets while still achieving 75–80% deuteration. Because the procedure does not require bulk D2O or deuterated glucose, the cost per liter of growth medium is significantly decreased; taking into account improvements in yield, these savings can be quite dramatic. Triple-labeled protein is also efficiently produced including specific 13CH3 labeling of isoleucine, leucine, and valine using the traditional ILV precursors in combination with an ILV-depleted mix of 2H/15N amino acids. These results are demonstrated for the membrane protein sensory rhodopsin II and the soluble proteins human aldoketoreductase AKR1c3, human ubiquitin, and bacterial flavodoxin. Limitations of the approach in the context of very large molecular weight proteins are illustrated using the bacterial Lac repressor transcription factor.
      PubDate: 2018-08-02
      DOI: 10.1007/s10858-018-0200-7
       
  • Structural investigation of glycan recognition by the ERAD quality control
           lectin Yos9
    • Authors: Andreas Kniss; Sina Kazemi; Frank Löhr; Maren Berger; Vladimir V. Rogov; Peter Güntert; Thomas Sommer; Ernst Jarosch; Volker Dötsch
      Abstract: 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. We show that binding is achieved by loops between β-strands performing an inward movement and that this movement also affects the entire β-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.
      PubDate: 2018-07-31
      DOI: 10.1007/s10858-018-0201-6
       
  • Advanced isotopic labeling for the NMR investigation of challenging
           proteins and nucleic acids
    • Authors: Jerome Boisbouvier; Lewis E. Kay
      PubDate: 2018-07-24
      DOI: 10.1007/s10858-018-0199-9
       
  • Perspective: next generation isotope-aided methods for protein NMR
           spectroscopy
    • Authors: Masatsune Kainosho; Yohei Miyanoiri; Tsutomu Terauchi; Mitsuhiro Takeda
      Abstract: 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. The idea of “TROSY by isotope labeling” has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52–57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12C-atoms give profoundly narrow 1H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the 1H-signals are acquired via the spin-coupled 13C- and/or 15N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional 1H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.
      PubDate: 2018-06-22
      DOI: 10.1007/s10858-018-0198-x
       
  • Binding of a small molecule water channel inhibitor to aquaporin Z
           examined by solid-state MAS NMR
    • Authors: Margaret Phillips; Janet To; Toshio Yamazaki; Toshio Nagashima; Jaume Torres; Konstantin Pervushin
      Abstract: 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. However, since aquaporin Z (AqpZ) can be purified and expressed in good quantities and has a high similarity to human AQP1 (~ 40% identity), it can be used as a model for studying the structure and function of human aquaporins. In the present study, we have used solid-state MAS NMR to investigate the binding of a lead compound [1-(4-methylphenyl)1H-pyrrole-2,5-dione] to AqpZ, through mapping of chemical shift perturbations in the presence of the compound.
      PubDate: 2018-06-18
      DOI: 10.1007/s10858-018-0195-0
       
  • Segmental isotope labelling and solid-state NMR of a 12 × 59 kDa
           motor protein: identification of structural variability
    • Authors: Thomas Wiegand; Riccardo Cadalbert; Christine von Schroetter; Frédéric H.-T. Allain; Beat H. Meier
      Abstract: 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. We uniformly 13C/15N labeled the N-terminal domain (147 residues) of the protein, while the C-terminal domain (311 residues) remained in natural abundance. The reduced signal overlap in solid-state NMR spectra allowed to identify structural “hotspots” for which the structure of the N-terminal domain in the context of the oligomeric full-length protein differs from the one in the isolated form. They are located near the linker between the two domains, in an α-helical hairpin.
      PubDate: 2018-06-12
      DOI: 10.1007/s10858-018-0196-z
       
  • Insight into human insulin aggregation revisited using NMR derived
           translational diffusion parameters
    • Authors: Jerzy Sitkowski; Wojciech Bocian; Elżbieta Bednarek; Mateusz Urbańczyk; Wiktor Koźmiński; Piotr Borowicz; Grażyna Płucienniczak; Natalia Łukasiewicz; Iwona Sokołowska; Lech Kozerski
      Abstract: The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N 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. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration. Graphical
      PubDate: 2018-06-12
      DOI: 10.1007/s10858-018-0197-y
       
  • Concentration-dependent changes to diffusion and chemical shift of
           internal standard molecules in aqueous and micellar solutions
    • Abstract: 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. Consistent with its amphipathic character, we show DSS to self-associate at elevated concentrations using pulsed field gradient-based diffusion NMR measurements. More critically, DSS diffusion is significantly attenuated in the presence of either like-charged sodium dodecyl sulfate or zwitterionic dodecylphosphocholine micelles, the two most commonly used detergent-based membrane mimetic systems used in solution-state NMR. Binding to oppositely charged dodecyltrimethylammonium bromide micelles is also highly favourable. DSS-micelle interactions are accompanied by a systematic, concentration- and binding propensity-dependent change in the chemical shift of the DSS reference signal by up to 60 ppb. The alternative reference compound 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA) exhibits highly similar behaviour, with reversal of the relative magnitude of chemical shift perturbation and proportion bound in comparison to DSS. Both DSS and DSA, thus, interact with micelles, and self-assemble at high concentration. Chemical shift perturbation of and modulation of micellar properties by these molecules has clear implications for their use as reference standards.
      PubDate: 2018-06-06
      DOI: 10.1007/s10858-018-0194-1
       
  • Methyl-selective isotope labeling using α-ketoisovalerate for the yeast
           Pichia pastoris recombinant protein expression system
    • Authors: Rika Suzuki; Masayoshi Sakakura; Masaki Mori; Moe Fujii; Satoko Akashi; Hideo Takahashi
      Abstract: 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. coli system for the expression of mammalian proteins, including secretory and membrane proteins. However, this system has not yet been optimized for methyl-specific isotope labeling, especially for Val/Leu-methyl specific isotope incorporation. To overcome this difficulty, we explored various culture conditions for the yeast cells to efficiently uptake Val/Leu precursors. Among the searched conditions, we found that the cultivation pH has a critical effect on Val/Leu precursor uptake. At an acidic cultivation pH, the uptake of the Val/Leu precursor was increased, and methyl groups of Val and Leu in the synthesized recombinant protein yielded intense 1H–13C correlation signals. Based on these results, we present optimized protocols for the Val/Leu-methyl-selective 13C incorporation by the P. pastoris expression system.
      PubDate: 2018-06-05
      DOI: 10.1007/s10858-018-0192-3
       
  • Methyl group reorientation under ligand binding probed by pseudocontact
           shifts
    • Authors: Mathilde Lescanne; Puneet Ahuja; Anneloes Blok; Monika Timmer; Tomas Akerud; Marcellus Ubbink
      Abstract: 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. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1–3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.
      PubDate: 2018-06-02
      DOI: 10.1007/s10858-018-0190-5
       
  • Removal of slow-pulsing artifacts in in-phase 15 N relaxation dispersion
           experiments using broadband 1 H decoupling
    • Authors: Soumya Deep Chatterjee; Marcellus Ubbink; Hugo van Ingen
      Abstract: 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. CPMG experiments that probe the chemical shift modulation of 15N in-phase magnetization are particularly attractive, due to their high sensitivity. These experiments require high power 1H decoupling during the CPMG period to keep the 15N magnetization in-phase. Recently, an improved version of the in-phase 15N-CPMG experiment was introduced, offering greater ease of use by employing a single 1H decoupling power for all CPMG pulsing rates. In these experiments however, incomplete decoupling of off-resonance amide 1H spins introduces an artefactual dispersion of relaxation rates, the so-called slow-pulsing artifact. Here, we analyze the slow-pulsing artifact in detail and demonstrate that it can be suppressed through the use of composite pulse decoupling (CPD). We report the performances of various CPD schemes and show that CPD decoupling based on the 90 x –240 y –90 x element results in high-quality dispersion curves free of artifacts, even for amides with high 1H offset.
      PubDate: 2018-06-02
      DOI: 10.1007/s10858-018-0193-2
       
  • NMR probing of invisible excited states using selectively labeled RNAs
    • Authors: Regan M. LeBlanc; Andrew P. Longhini; Vitali Tugarinov; T. Kwaku Dayie
      Abstract: 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 1H and methylene C5′ chemical shifts of ribose moieties in the excited state, partly because of complications from homonuclear 13C–13C scalar couplings. Here we present site-specific 13C labeling that makes possible the design of pulse sequences for recording accurate 1H–13C MQ and SQ CPMG experiments for ribose methine H1′–C1′ and H2′–C2′, base and ribose 1H CPMG, as well as a new 1H–13C TROSY-detected methylene (CH2) C5′ CPMG relaxation pulse schemes. We demonstrate the utility of these experiments for two RNAs, the A-Site RNA known to undergo exchange and the IRE RNA suspected of undergoing exchange on microseconds to millisecond time-scale. We anticipate the new labeling approaches will facilitate obtaining structures of invisible states and provide insights into the relevance of such states for RNA-drug interactions.
      PubDate: 2018-06-01
      DOI: 10.1007/s10858-018-0184-3
       
  • Late metabolic precursors for selective aromatic residue labeling
    • Authors: Julia Schörghuber; Leonhard Geist; Gerald Platzer; Michael Feichtinger; Marilena Bisaccia; Lukas Scheibelberger; Frederik Weber; Robert Konrat; Roman J. Lichtenecker
      Abstract: 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. In the present article, we want to summarize the precursors we developed so far and give examples of their special value in the probing of protein–ligand interaction.
      PubDate: 2018-05-28
      DOI: 10.1007/s10858-018-0188-z
       
  • Production of isotope-labeled proteins in insect cells for NMR
    • Authors: Bastian Franke; Christian Opitz; Shin Isogai; Anne Grahl; Leonildo Delgado; Alvar D. Gossert; Stephan Grzesiek
      Abstract: 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.
      PubDate: 2018-04-23
      DOI: 10.1007/s10858-018-0172-7
       
 
 
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