Journal of Biomolecular NMR

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Amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies
Journal of Biomolecular NMR - Tập 26 - Trang 367-372 - 2003
André Strauss, Francis Bitsch, Brian Cutting, Gabriele Fendrich, Patrick Graff, Janis Liebetanz, Mauro Zurini, Wolfgang Jahnke
Culture conditions for successful amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with 15N-phenylalanine, 15N-glycine, 15N-tyrosine or 15N-valine. For the essential amino acids phenylalanine, tyrosine and valine high 15N-label incorporation rates of ≥90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.
Determination of methyl order parameters using solid state NMR under off magic angle spinning
Journal of Biomolecular NMR - Tập 73 - Trang 471-475 - 2019
Kai Xue, Salvatore Mamone, Benita Koch, Riddhiman Sarkar, Bernd Reif
Quantification of dipolar couplings in biological solids is important for the understanding of dynamic processes. Under Magic Angle Spinning (MAS), order parameters are normally obtained by recoupling of anisotropic interactions involving the application of radio frequency pulses. We have recently shown that amide backbone order parameters can be estimated accurately in a spin-echo experiment in case the rotor spinning angle is slightly mis-calibrated. In this work, we apply this method to determine methyl order parameters in a deuterated sample of the SH3 domain of chicken α-spectrin in which the methyl containing side chains valine and leucine are selectively protonated.
Letter to the Editor:1H,13C and15N resonance assignments for the N-cadherin prodomain
Journal of Biomolecular NMR - Tập 28 Số 1 - Trang 87-88 - 2004
Alexander W. Koch, Amjad Farooq, Lei Zeng, David Colman, Ming Zhou
Heteronuclear relayed E.COSY applied to the determination of accurate 3J(HN,C′) and 3J(Hβ,C′) coupling constants in Desulfovibrio vulgaris flavodoxin
Journal of Biomolecular NMR - - 1996
Jürgen M. Schmidt, Frank Löhr, Heinz Rüterjans
A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings 3J(HN,C′) and 3J(Hβ,C′) in uniformly 13C-enriched proteins. The protocols for measuring accurate coupling constants are based on 1H,13C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95–105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of ϕ and X 1 dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant 13C, 15N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 3J(HN,C′) and 100 3J(Hβ,C′) coupling constants are obtained from a single spectrum.
Myristoylation as a general method for immobilization and alignment of soluble proteins for solid-state NMR structural studies
Journal of Biomolecular NMR - Tập 25 - Trang 55-61 - 2003
M.F. Mesleh, K.G. Valentine, S.J. Opella, J.M. Louis, A.M. Gronenborn
N-terminal myristoylation of the immunoglobulin-binding domain of protein G (GB1) from group G Streptococcus provides the means to bind the protein to aligned phospholipid bilayers for solid-state NMR structural studies. The myristoylated protein is immobilized by its interactions with bilayers, and the sample alignment enables orientationally dependent 15N chemical shifts and 1H-15N-dipolar couplings to be measured. Spectra calculated for the average solution NMR structure of the protein at various orientations with respect to the magnetic field direction were compared to the experimental spectrum. The best fit identified the orientation of the myristoylated protein on the lipid bilayers, and demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms, and that the structure is not grossly distorted by its interaction with the phosholipid bilayer surface or by its location in the restricted aqueous space between bilayer leaflets. The protein is oriented such that its charged sides face the phosphatidylcholine headgroups of the lipids with the single amphiphilic helix running parallel to the bilayer surface.
A general Bayesian method for an automated signal class recognition in 2D NMR spectra combined with a multivariate discriminant analysis
Journal of Biomolecular NMR - - 1995
C. Antz, Klaus−Peter Neidig, Hans Robert Kalbitzer
1H, 13C and 15N NMR backbone assignments of 25.5 kDa metallo-β-lactamase from shape Bacteroides fragilis
Journal of Biomolecular NMR - Tập 12 - Trang 201-202 - 1998
Sergio D.B. Scrofani, Peter E. Wright, H. Jane Dyson
[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1
Journal of Biomolecular NMR - Tập 3 - Trang 325-334 - 1993
Stefan Highsmith, Mark Kubinec, Devendra K. Jaiswal, Hiromi Morimoto, Philip G. Williams, David E. Wemmer
The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes. The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0°C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-3H]ADP.Vi shifts both peaks slightly more upfield without chaning their widths or relative areas.
Combination of 15N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE
Journal of Biomolecular NMR - Tập 55 - Trang 391-399 - 2013
James R. Banigan, Anindita Gayen, Nathaniel J. Traaseth
Magic-angle-spinning (MAS) solid-state NMR spectroscopy has emerged as a viable method to characterize membrane protein structure and dynamics. Nevertheless, the spectral resolution for uniformly labeled samples is often compromised by redundancy of the primary sequence and the presence of helical secondary structure that results in substantial resonance overlap. The ability to simplify the spectrum in order to obtain unambiguous site-specific assignments is a major bottleneck for structure determination. To address this problem, we used a combination of 15N reverse labeling, afterglow spectroscopic techniques, and frequency-selective dephasing experiments that dramatically improved the ability to resolve peaks in crowded spectra. This was demonstrated using the polytopic membrane protein EmrE, an efflux pump involved in multidrug resistance. Residues preceding the 15N reverse labeled amino acid were imaged using a 3D NCOCX afterglow experiment and those following were recorded using a frequency-selective dephasing experiment. Our approach reduced the spectral congestion and provided a sensitive way to obtain chemical shift assignments for a membrane protein where no high-resolution structure is available. This MAS methodology is widely applicable to the study of other polytopic membrane proteins in functional lipid bilayer environments.
Erratum to: Correlation of chemical shifts predicted by molecular dynamics simulations for partially disordered proteins
Journal of Biomolecular NMR - Tập 62 - Trang 119-119 - 2015
Jerome M. Karp, Ertan Eryilmaz, David Cowburn
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