Protocols for the Sequential Solid-State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET-s(1-227) (original) (raw)
Related papers
Structural determination of larger proteins using stable isotope labeling and NMR spectroscopy
1996
This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The Views and opinions of authors expressed herein United States Government or any agency th Los Alamos National Laboratory, an affirmative actioWequal opportunity employer, is operated by the University of Calilomia for the U.S. Department of Energy under contrad W-7405-ENG-36. By acceptance of this article, the publsher recognizes that the U.S. Government wains a nonexchrswe, royaRyfree license to publish or reproduce the published lorn of this contribution, or to allow others to do so, lor U.S. Government purposes. The Los A l m s National Laboratory requests that the publisher identify this artlcle as work performed nt of Energy. Form No. 836 R5 sr zea I woi
High-Resolution Solid-State NMR Structure of a 17.6 kDa Protein
Journal of The American Chemical Society, 2010
The use of pseudocontact shifts arising from paramagnetic metal ions in a microcrystalline protein sample is proposed as a strategy to obtain unambiguous signal assignments in solid-state NMR spectra enabling distance extraction for protein structure calculation. With this strategy, 777 unambiguous (281 sequential, 217 medium-range, and 279 long-range) distance restraints could be obtained from PDSD, DARR, CHHC, and the recently introduced PAR and PAIN-CP solid-state experiments for the cobalt(II)substituted catalytic domain of matrix metalloproteinase 12 (159 amino acids, 17.6 kDa). The obtained structure is a high resolution one, with backbone rmsd of 1.0 ( 0.2 Å, and is in good agreement with the X-ray structure (rmsd to X-ray 1.3 Å). The proposed strategy, which may be generalized for nonmetalloproteins with the use of paramagnetic tags, represents a significant step ahead in protein structure determination using solid-state NMR.
A selective experiment for the sequential protein backbone assignment from 3D heteronuclear spectra
2005
Two modifications of the triple-resonance CANCO sequence, designed for backbone assignment in proteins [Angew. Chem. Int. Ed. 43 (2004) 2257], are presented here. These two new sequences display the intra-residue Ca-CO correlation selectively, while in the original sequence both the inter-and the intra-residue correlations were present. In addition, one of the two variants benefits from an improved sensitivity. Both sequences are a useful complement to the CANCO sequence for facile sequence-specific protein assignment by protonless NMR.
Journal of Computational Chemistry, 2001
The sequence-specific assignment of resonances is still the most time-consuming procedure that is necessary as the first step in high-resolution NMR studies of proteins. In many cases a reliable three-dimensional (3D) structure of the protein is available, for example, from X-ray spectroscopy or homology modeling. Here we introduce the st2nmr program that uses the 3D structure and Nuclear Overhauser Effect spectroscopy (NOESY) peak list(s) to evaluate and optimize trial sequence-specific assignments of spin systems derived from correlation spectra to residues of the protein. A distance-dependent target function that scores trial assignments based on the presence of expected NOESY crosspeaks is optimized in a Monte Carlo fashion. The performance of the program st2nmr is tested on real NMR data of an α-helical (cytochrome c) and β-sheet (lipocalin) protein using homology models and/or X-ray structures; it succeeded in completely reproducing the correct sequence-specific assignments in most cases using 2D and/or 15 N/ 13 C Nuclear Overhauser Effect (NOE) data. Additionally to amino acid residues the program can also handle ligands that are bound to the protein, such as heme, and can be used as a complementary tool to fully automated assignment procedures.
Journal of the American Chemical Society, 1999
A novel isotopic labeling strategy is described for the structural analysis of proteins by NMR. Overexpression of a protein in a mammalian cell-line cultured in a medium containing amino acids labeled only in the backbone (N, C R , H R , C′) atoms leads to the formation of exclusively backbone-labeled protein. We demonstrate that the absence of the one bond scalar coupling between the 13 C R and 13 C atoms that is observed in uniformly 13 C enriched proteins offers a substantial sensitivity and resolution advantage in triple resonance NMR experiments that are commonly used to obtain backbone resonance assignments. This approach is illustrated in application to the subunit of human chorionic gonadotropin isotopically enriched with 13 C (97%), 15 N (97%), and 2 H (50%) exclusively in the backbone atoms of Phe, Val, and Leu residues.
Solid-state NMR triple-resonance backbone assignments in a protein
Journal of Biomolecular Nmr, 1999
Triple-resonance solid-state NMR spectroscopy is demonstrated to sequentially assign the 13C′ and 15N amide backbone resonances of adjacent residues in an oriented protein sample. The observed 13C′ chemical shift frequency provides an orientational constraint complementary to those measured from the 1H and 15N amide resonances in double-resonance experiments.
Journal of Biomolecular NMR, 2015
The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or un-curated (i.e. automatically generated) form. Ten structure calculation programs, using fully automated protocols only, generated a total of 164 three-dimensional structures (entries) for the ten targets, sometimes using both curated and un-curated lists to generate multiple entries for a single target. The accuracy of the entries could be established by comparing them to the corresponding manually solved structure of each target, which was not available at the time the data were provided. Across the entire data set, 71 % of all entries submitted achieved an accuracy relative to the reference NMR structure better than 1.5 Å. Methods based on NOESY peak lists achieved even better results with up to 100 % of the entries within the 1.5 Å threshold for some programs. However, some methods did not converge for some targets using un-curated NOESY peak lists. Over 90 % of the entries achieved an accuracy better than the more relaxed threshold of 2.5 Å that was used in the previous CASD-NMR-2010 round. Comparisons between entries generated with un-curated versus curated peaks show only marginal improvements for the latter in those cases where both calculations converged. Keywords Protein Á NMR Á Structure determination Á Automation Á Quality Á Validation Á Blind testing Á NOE Á Chemical shift Á CASD-NMR Á Accuracy Á Precision Antonio Rosato, Wim Vranken and Rasmus H. Fogh have been contributed equally to this work.