Journal Cover
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  [2351 journals]
  • Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein
           ligation
    • Authors: Kornelia M. Mikula; Luisa Krumwiede; Andreas Plückthun; Hideo Iwaï
      Pages: 225 - 235
      Abstract: 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.
      PubDate: 2018-08-01
      DOI: 10.1007/s10858-018-0175-4
      Issue No: Vol. 71, No. 4 (2018)
       
  • Cost-effective large-scale expression of proteins for NMR studies
    • Authors: Julia Klopp; Aurélie Winterhalter; Rémy Gébleux; Daniela Scherer-Becker; Christian Ostermeier; Alvar D. Gossert
      Pages: 247 - 262
      Abstract: 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 13C-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 2H, 13C and 15N 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. These protocols are based on high cell-density fermentation, but the key procedures are easily transferred to shake flask cultures.
      PubDate: 2018-08-01
      DOI: 10.1007/s10858-018-0179-0
      Issue No: Vol. 71, No. 4 (2018)
       
  • Genetically encoded amino acids with tert -butyl and trimethylsilyl groups
           for site-selective studies of proteins by NMR spectroscopy
    • Authors: Choy Theng Loh; Luke A. Adams; Bim Graham; Gottfried Otting
      Pages: 287 - 293
      Abstract: 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. The 1H-NMR resonance of the TMS group appeared near 0 ppm in a spectral region with few protein resonances, facilitating the observation of signal changes in response to ligand binding. In all proteins, the R 2 relaxation rate of the tert-butyl group of Tbf was only little greater than that of Tby (less than two-fold). Deuteration of the phenyl ring of Tbf made only a relatively small difference. The effective T 2 relaxation time of the TMS signal was longer than 140 ms even in the 54 kDa system.
      PubDate: 2018-08-01
      DOI: 10.1007/s10858-017-0157-y
      Issue No: Vol. 71, No. 4 (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)
       
  • Joint X-ray/NMR structure refinement of multidomain/multisubunit systems
    • Authors: Azzurra Carlon; Enrico Ravera; Giacomo Parigi; Garib N. Murshudov; Claudio Luchinat
      Abstract: Data integration in structural biology has become a paradigm for the characterization of biomolecular systems, and it is now accepted that combining different techniques can fill the gaps in each other’s blind spots. In this frame, one of the combinations, which we have implemented in REFMAC-NMR, is residual dipolar couplings from NMR together with experimental data from X-ray diffraction. The first are exquisitely sensitive to the local details but does not give any information about overall shape, whereas the latter encodes more the information about the overall shape but at the same time tends to miss the local details even at the highest resolutions. Once crystals are obtained, it is often rather easy to obtain a complete X-ray dataset, however it is time-consuming to obtain an exhaustive NMR dataset. Here, we discuss the effect of including a-priori knowledge on the properties of the system to reduce the number of experimental data needed to obtain a more complete picture. We thus introduce a set of new features of REFMAC-NMR that allow for improved handling of RDC data for multidomain proteins and multisubunit biomolecular complexes, and encompasses the use of pseudo-contact shifts as an additional source of NMR-based information. The new feature may either help in improving the refinement, or assist in spotting differences between the crystal and the solution data. We show three different examples where NMR and X-ray data can be reconciled to a unique structural model without invoking mobility.
      PubDate: 2018-10-11
      DOI: 10.1007/s10858-018-0212-3
       
  • 15 N transverse relaxation measurements for the characterization of
           µs–ms dynamics are deteriorated by the deuterium isotope effect on 15 N
           resulting from solvent exchange
    • Authors: Pratibha Kumari; Lukas Frey; Alexander Sobol; Nils-Alexander Lakomek; Roland Riek
      Abstract: 15N R2 relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide—water exchange can severely skew Hahn-echo based 15N R2 relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the 15N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent 15N R2 rate constants—which should not be mistaken with protein inherent chemical exchange contributions, Rex, to 15N R2. For measurements of 15N R2 rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D2O molar fraction in the sample buffer, as low as 1%, or the use of an external D2O reference along with a modified 15N R2 Hahn-echo based experiment. This combination allows for the measurement of Rex contributions to 15N R2 originating from conformational exchange in a time window from µs to ms.
      PubDate: 2018-10-10
      DOI: 10.1007/s10858-018-0211-4
       
  • A methyl 1 H double quantum CPMG experiment to study protein
           conformational exchange
    • Authors: Anusha B. Gopalan; Tairan Yuwen; Lewis E. Kay; Pramodh Vallurupalli
      Abstract: Protein conformational changes play crucial roles in enabling function. The Carr–Purcell–Meiboom–Gill (CPMG) experiment forms the basis for studying such dynamics when they involve the interconversion between highly populated and sparsely formed states, the latter having lifetimes ranging from ~ 0.5 to ~ 5 ms. Among the suite of experiments that have been developed are those that exploit methyl group probes by recording methyl 1H single quantum (Tugarinov and Kay in J Am Chem Soc 129:9514–9521, 2007) and triple quantum (Yuwen et al. in Angew Chem Int Ed Engl 55:11490–11494, 2016) relaxation dispersion profiles. Here we build upon these by developing a third experiment in which methyl 1H double quantum coherences evolve during a CPMG relaxation element. By fitting single, double, and triple quantum datasets, akin to recording the single quantum dataset at static magnetic fields of Bo, 2Bo and 3Bo, we show that accurate exchange values can be obtained even in cases where exchange rates exceed 10,000 s−1. The utility of the double quantum experiment is demonstrated with a pair of cavity mutants of T4 lysozyme (T4L) with ground and excited states interchanged and with exchange rates differing by fourfold (~ 900 s−1 and ~ 3600 s−1), as well as with a fast-folding domain where the unfolded state lifetime is ~ 80 µs.
      PubDate: 2018-10-01
      DOI: 10.1007/s10858-018-0208-z
       
  • Conformational exchange of aromatic side chains by 1 H CPMG relaxation
           dispersion
    • Authors: Heiner N. Raum; Matthias Dreydoppel; Ulrich Weininger
      Abstract: 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 13C CPMG relaxation dispersion experiments. Here we investigate the possibility of aromatic 1H CPMG relaxation dispersion experiments as a complementary method. Artifact-free dispersions are possible on uniformly 1H and 13C labeled samples for histidine δ2 and ε1, as well as for tryptophan δ1. The method has been validated by measuring fast folding–unfolding kinetics of the small protein CspB under native conditions. The determined rate constants and populations agree well with previous results from 13C CPMG relaxation dispersion experiments. The CPMG-derived chemical shift differences between the folded and unfolded states are in good agreement with those obtained directly from the spectra. In contrast, the 1H relaxation dispersion profiles in phenylalanine, tyrosine and the six-ring moiety of tryptophan, display anomalous behavior caused by 3J 1H–1H couplings and, if present, strong 13C–13C couplings. Therefore they require site-selective 1H/2H and, in case of strong couplings, 13C/12C labeling. In summary, aromatic 1H CPMG relaxation dispersion experiments work on certain positions (His δ2, His ε1 and Trp δ1) in uniformly labeled samples, while other positions require site-selective isotope labeling. Graphical abstract
      PubDate: 2018-09-18
      DOI: 10.1007/s10858-018-0210-5
       
  • Direct amide 15 N to 13 C transfers for solid-state assignment experiments
           in deuterated proteins
    • Authors: Alons Lends; Francesco Ravotti; Giorgia Zandomeneghi; Anja Böckmann; Matthias Ernst; Beat H. Meier
      Abstract: 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. The proposed pulse sequence uses only 1H–15N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than 1H–13C CP transfers, and employs a dipolar version of the INEPT experiment for N–C transfer. By avoiding HN–C (HN stands for amide protons) and C–C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired 13C spins.
      PubDate: 2018-09-11
      DOI: 10.1007/s10858-018-0207-0
       
  • 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
    • Authors: Michal Rivlin; Gil Navon
      Abstract: 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 1H and 13C 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 6.14 and a temperature of 4 °C, were in the range of 360–670 s−1.
      PubDate: 2018-09-10
      DOI: 10.1007/s10858-018-0209-y
       
  • Rapid automated determination of chemical shift anisotropy values in the
           carbonyl and carboxyl groups of fd-y21m bacteriophage using solid state
           NMR
    • Authors: Tom Aharoni; Amir Goldbourt
      Abstract: 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 13C CSA with the corresponding isotropic 13C′–13C cross-peaks in order to probe backbone and sidechain dynamics in an intact fd-y21m filamentous phage viral capsid. The assignment of the isotropic cross-peaks and the analysis were conducted automatically using a new software named ‘Raven’. The software can be utilized to auto-assign any 2D 13C–13C or 15N–13C spectrum given a previously-determined assignment table and generates simultaneously all intensity curves acquired in the third dimension. Here, all CSA spectra were automatically generated, and subsequently matched against a simulated set of CSA curves to yield their values. For the multi-copy, 50-residue-long protein capsid of fd-y21m, the backbone of the helical region is rigid, with reduced CSA values of ~ 12.5 kHz (~ 83 ppm). The N-terminus shows motionally-averaged CSA lineshapes and the carboxylic sidechain groups of four residues indicate large amplitude motions for D4, D5, D12 and E20. The current results further strengthen our previous studies of 15N CSA values and are in agreement with qualitative analysis of 13C–13C dipolar build-up curves, which were automatically obtained using our software. Our automated analysis technique is general and can be applied to study protein structure and dynamics, with data resulting from experiments that probe different variables such as relaxation rates and scaled anisotropic interactions. Graphical abstract
      PubDate: 2018-08-23
      DOI: 10.1007/s10858-018-0206-1
       
  • Correlated motions of C′–N and C α –C β pairs in protonated and
           per-deuterated GB3
    • Authors: Liliya Vugmeyster; Aaron Griffin; Dmitry Ostrovsky; Shibani Bhattacharya; Parker J. Nichols; C. James McKnight; Beat Vögeli
      Abstract: We investigated correlated µs-ms time scale motions of neighboring 13C′–15N and 13Cα–13Cβ 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. The time scale of motions falls into the intermediate to fast regime (with respect to the chemical shift time scale, 100–400 s−1 range) for the 13C′–15N pairs and into the slow to intermediate regime for the 13Cα–13Cβ pairs (about 150 s−1). Comparison of the results obtained for protonated and deuterated GB3 suggests that deuteration has a tendency to reduce these slow scale correlated motions, especially for the 13Cα–13Cβ pairs.
      PubDate: 2018-08-18
      DOI: 10.1007/s10858-018-0205-2
       
  • On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR
           studies
    • Authors: Lindsay Clark; Igor Dikiy; Daniel M. Rosenbaum; Kevin H. Gardner
      Abstract: 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). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.
      PubDate: 2018-08-18
      DOI: 10.1007/s10858-018-0204-3
       
  • Conformationally locked lanthanide chelating tags for convenient
           pseudocontact shift protein nuclear magnetic resonance spectroscopy
    • Authors: Daniel Joss; Roché M. Walliser; Kaspar Zimmermann; Daniel Häussinger
      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
      DOI: 10.1007/s10858-018-0203-4
       
  • 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
       
  • 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
       
  • 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
       
 
 
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