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1.
The linear analysis of chemical shifts (LACS) has provided a robust method for identifying and correcting 13C chemical shift referencing problems in data from protein NMR spectroscopy. Unlike other approaches, LACS does not require
prior knowledge of the three-dimensional structure or inference of the secondary structure of the protein. It also does not
require extensive assignment of the NMR data. We report here a way of extending the LACS approach to 15N NMR data from proteins, so as to enable the detection and correction of inconsistencies in chemical shift referencing for
this nucleus. The approach is based on our finding that the secondary 15N chemical shift of the backbone nitrogen atom of residue i is strongly correlated with the secondary chemical shift difference (experimental minus random coil) between the alpha and
beta carbons of residue i − 1. Thus once alpha and beta 13C chemical shifts are available (their difference is referencing error-free), the 15N referencing can be validated, and an appropriate offset correction can be derived. This approach can be implemented prior
to a structure determination and can be used to analyze potential referencing problems in database data not associated with
three-dimensional structure. Application of the LACS algorithm to the current BMRB protein chemical shift database, revealed
that nearly 35% of the BMRB entries have δ
15N values mis-referenced by over 0.7 ppm and over 25% of them have δ
1HN values mis-referenced by over 0.12 ppm. One implication of the findings reported here is that a backbone 15N chemical shift provides a better indicator of the conformation of the preceding residue than of the residue itself.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
2.
Inconsistent 13C and 15N chemical shift referencing is a continuing problem associated with protein chemical shift assignments deposited in BioMagResBank (BMRB). Here we describe a simple and robust approach that can quantitatively determine the 13C and 15N referencing offsets solely from chemical shift assignment data and independently of 3D coordinate data. This novel structure-independent approach permitted the assessment and determination of 13C and 15N reference offsets for all protein entries deposited in the BMRB. Tests on 452 proteins with known 3D structures show that this structure-independent approach yields 13C and 15N referencing offsets that exhibit excellent agreement with those calculated on the basis of 3D structures. Furthermore, this protocol appears to improve the accuracy of chemical shift-derived secondary structural identification, and has been formally incorporated into a computer program called PSSI (http//www.pronmr.com).Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7441-3 相似文献
3.
ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface. 相似文献
4.
5.
Florian Celli Ambre Petitalot Camille Samson François-Xavier Theillet Sophie Zinn-Justin 《Biomolecular NMR assignments》2018,12(2):225-229
Lamins are the main components of the nucleoskeleton. They form a protein meshwork that underlies the inner nuclear membrane. Mutations in the LMNA gene coding for A-type lamins (lamins A and C) cause a large panel of human diseases, referred to as laminopathies. These diseases include muscular dystrophies, lipodystrophies and premature aging diseases. Lamin A exhibits a C-terminal region that is different from lamin C and is post-translationally modified. It is produced as prelamin A and it is then farnesylated, cleaved, carboxymethylated and cleaved again in order to become mature lamin A. In patients with the severe Hutchinson–Gilford progeria syndrome, a specific single point mutation in LMNA leads to an aberrant splicing of the LMNA gene preventing the post-translational processing of prelamin A. This leads to the accumulation of a permanently farnesylated lamin A mutant lacking 50 amino acids named progerin. We here report the NMR 1H, 15N, 13CO, 13Cα and 13Cβ chemical shift assignment of the C-terminal region that is specific to prelamin A, from amino acid 567 to amino acid 664. We also report the NMR 1H, 15N, 13CO, 13Cα and 13Cβ chemical shift assignment of the C-terminal region of the progerin variant, from amino acid 567 to amino acid 614. Analysis of these chemical shift data confirms that both prelamin A and progerin C-terminal domains are largely disordered and identifies a common partially populated α-helix from amino acid 576 to amino acid 585. This helix is well conserved from fishes to mammals. 相似文献
6.
Christian Richter Helena Kovacs Janina Buck Anna Wacker Boris Fürtig Wolfgang Bermel Harald Schwalbe 《Journal of biomolecular NMR》2010,47(4):259-269
We present here a set of 13C-direct detected NMR experiments to facilitate the resonance assignment of RNA oligonucleotides. Three experiments have been
developed: (1) the (H)CC-TOCSY-experiment utilizing a virtual decoupling scheme to assign the intraresidual ribose 13C-spins, (2) the (H)CPC-experiment that correlates each phosphorus with the C4′ nuclei of adjacent nucleotides via J(C,P)
couplings and (3) the (H)CPC-CCH-TOCSY-experiment that correlates the phosphorus nuclei with the respective C1′,H1′ ribose
signals. The experiments were applied to two RNA hairpin structures. The current set of 13C-direct detected experiments allows direct and unambiguous assignment of the majority of the hetero nuclei and the identification
of the individual ribose moieties following their sequential assignment. Thus, 13C-direct detected NMR methods constitute useful complements to the conventional 1H-detected approach for the resonance assignment of oligonucleotides that is often hindered by the limited chemical shift
dispersion. The developed methods can also be applied to large deuterated RNAs. 相似文献
7.
Edith Buchinger Siv Å. Wiik Anna Kusnierczyk Renana Rabe Per. A. Aas Bodil Kavli Geir Slupphaug Finn L. Aachmann 《Biomolecular NMR assignments》2018,12(1):15-22
Human uracil N-glycosylase isoform 2—UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1–92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93–313, 25.1 kDa). Here, we report the backbone 1H, 13C, and 15N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein. 相似文献
8.
Natural Abundance Fourier Transform of <Superscript>13</Superscript>C Nuclear Magnetic Resonance Spectra of Lysozyme 总被引:1,自引:0,他引:1
NUCLEAR magnetic resonance (NMR) studies of biopolymers have so far been limited to proton resonances. On the other hand, 13C NMR analysis is potentially a more powerful technique because of the larger range of chemical shifts (300 p.p.m. compared with 10 p.p.m. for XH NMR) and because the chemical shift of 13C NMR is more sensitive to the nature of the chemical bonds. The chief limitations of 13C NMR are low natural abundance (1·1%), low relative sensitivity (1·59% of proton) and long relaxation times (?1 min). These can be largely overcome, however, by the combined techniques of nuclear Overhauser enhancement and fourier transform spectroscopy. Thus, Gibbons et al.1 obtained a simple and elegant spectral analysis of ‘Gramicidin S-A’, a decapeptide. We report here the first natural abundance 13C NMR spectrum of an enzyme. Lysozyme was chosen because its primary structure is known2,3 and because its 1H NMR spectrum has already been described4. 相似文献
9.
This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion 13C–13C correlation experiments with mixing times of 20 and 500 ms and band selective 13C–15N correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long -helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the 13C–13C correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins. 相似文献
10.
Thioredoxins (Trx) are ubiquitous proteins that regulate several biochemical processes inside the cell. Trx is an important player, displaying oxidoreductase activity and helping to keep and regulate the oxidative state of the cellular environment. Trx also participates in the regulation of many cellular functions, such as DNA synthesis, protection against oxidative stress, cell cycle and signal transduction. The oxidized Trx is the target for another set of proteins, such as thioredoxin reductase (TrR), which used the reductive potential of NADPH. The oxidized state of Trx also plays important role in regulation of redox state in the cells. In this regard, the oxidized form of Trx is a putative conformer that contributes to the cellular redox environment. Here we report the chemical shift assignments (1H, 13C and 15N) in solution at 15 °C. We also showed the secondary structure analysis of the oxidized form of yeast thioredoxin (yTrx1) as basis for future NMR studies of protein–target interactions and dynamics. The assignment was done at low concentration (200 µM) because it is important to keep intact the water cavity. 相似文献
11.
Sosuke Yoshinaga Norihito Ishida Tatsuichiro Tsuji Akihiro Sonoda Kaori Yunoki Mitsuhiro Takeda Etsuko Toda Yuya Terashima Kouji Matsushima Hiroaki Terasawa 《Biomolecular NMR assignments》2018,12(2):259-262
FROUNT is a cytoplasmic protein that interacts with the membrane-proximal C-terminal regions (Pro-Cs) of the CCR2 and CCR5 chemokine receptors. The interactions between FROUNT and the chemokine receptors play an important role in the migration of inflammatory immune cells. Therefore, FROUNT is a potential drug target for inflammatory diseases. However, the structural basis of the interactions between FROUNT and the chemokine receptors remains to be elucidated. We previously identified the C-terminal region (residues 532–656) of FROUNT as the structural domain responsible for the Pro-C binding, referred to as the chemokine receptor-binding domain (CRBD), and then constructed its mutant, bearing L538E/P612S mutations, with improved NMR spectral quality, referred to as CRBD_LEPS. We now report the main-chain and side-chain 1H, 13C, and 15N resonance assignments of CRBD_LEPS. The NMR signals of CRBD_LEPS were well dispersed and their intensities were uniform on the 1H–15N HSQC spectrum, and thus almost all of the main-chain and side-chain resonances were assigned. This assignment information provides the foundation for NMR studies of the three-dimensional structure of CRBD_LEPS in solution and its interactions with chemokine receptors. 相似文献
12.
Maxim Mayzel Alexandra Ahlner Patrik Lundström Vladislav Y. Orekhov 《Journal of biomolecular NMR》2017,67(1):1-13
The 31P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the 31P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH2p and GTPγS was measured in the absence and presence of Mg2+-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B 1 was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg2+·GMP and Mg2+·GppNHp the second order pressure coefficients are positive. To describe the data of Mg2+·GppCH2p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg2+ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure 31P NMR data on free Mg2+-GDP and Mg2+-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms. 相似文献
13.
We describe an efficient NMR triple resonance approach for fast assignment of backbone amide resonance peaks in the 15N-HSQC spectrum. The exceptionally high resolutions achieved in the 3D HncocaNH and hNcocaNH experiments together with non-uniform
sampling facilitate error-free sequential connection of backbone amides. Data required for the complete backbone amide assignment
of the 56-residue protein GB1 domain were obtained in 14 h. Data analysis was vastly streamlined using a ‘backbone NH walk’
method to determine sequential connectivities without the need for 13C chemical shifts comparison. Amino acid residues in the sequentially connected NH chains are classified into two groups by
a simple variation of the NMR pulse sequence, and the resulting ‘ZeBra’ stripe patterns are useful for mapping these chains
to the protein sequence. In addition to resolving ambiguous assignments derived from conventional backbone experiments, this
approach can be employed to rapidly assign small proteins or flexible regions in larger proteins, and to transfer assignments
to mutant proteins or proteins in different ligand-binding states. 相似文献
14.
David P. Bowles Alexandar L. Hansen Calvin A. Rhoads Sidharth Mohan Chunhua Yuan Thomas J. Magliery 《Biomolecular NMR assignments》2018,12(2):291-295
Er-23 is a small, 51 amino acid, disulfide-rich pheromone protein used for cell signaling by Euplotes raikovi. Ten of the 51 amino acids are cysteine, allowing up to five disulfide bonds. Previous NMR work with Er-23 utilized homologously expressed protein, prohibiting isotopic labeling, and consequently the chemical shift assignments were incomplete. We have expressed uniformly 15N and 13C-labeled Er-23 in an E. coli expression system. Here we report the full backbone and side chain resonance assignments for recombinant Er-23. 相似文献
15.
RefDB is a secondary database of reference-corrected protein chemical shifts derived from the BioMagResBank (BMRB). The database was assembled by using a recently developed program (SHIFTX) to predict protein 1H, 13C and 15N chemical shifts from X-ray or NMR coordinate data of previously assigned proteins. The predicted shifts were then compared with the corresponding observed shifts and a variety of statistical evaluations performed. In this way, potential mis-assignments, typographical errors and chemical referencing errors could be identified and, in many cases, corrected. This approach allows for an unbiased, instrument-independent solution to the problem of retrospectively re-referencing published protein chemical shifts. Results from this study indicate that nearly 25% of BMRB entries with 13C protein assignments and 27% of BMRB entries with 15N protein assignments required significant chemical shift reference readjustments. Additionally, nearly 40% of protein entries deposited in the BioMagResBank appear to have at least one assignment error. From this study it evident that protein NMR spectroscopists are increasingly adhering to recommended IUPAC 13C and 15N chemical shift referencing conventions, however, approximately 20% of newly deposited protein entries in the BMRB are still being incorrectly referenced. This is cause for some concern. However, the utilization of RefDB and its companion programs may help mitigate this ongoing problem. RefDB is updated weekly and the database, along with its associated software, is freely available at http://redpoll.pharmacy.ualberta.ca and the BMRB website. 相似文献
16.
Alons Lends Francesco Ravotti Giorgia Zandomeneghi Anja Böckmann Matthias Ernst Beat H. Meier 《Journal of biomolecular NMR》2018,72(1-2):69-78
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. 相似文献
17.
Paolo Rossi Youlin Xia Nandish Khanra Gianluigi Veglia Charalampos G. Kalodimos 《Journal of biomolecular NMR》2016,66(4):259-271
The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and 15N, methyl labeled samples in H2O. The experiments benefit from a combination of selective T 1 relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear 15N,13C-edited, or with diagonal-free 13C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination. 相似文献
18.
Significant resolution improvement in 13C,13C-TOCSY spectra of uniformly deuterated and 13C, 15N-labeled protein and 13C,15N-labeled RNA samples is achieved by introduction of multiple-band-selective 13C-homodecoupling applied simultaneously with 1H- or 2H- and 15N-decoupling at all stages of multidimensional experiments including signal acquisition period. The application of single, double or triple band-selective 13C-decoupling in 2D-[13C,13C]-TOCSY experiments during acquisition strongly simplifies the homonuclear splitting pattern. The technical aspects of complex multiple-band homonuclear decoupling and hardware requirements are discussed. The use of this technique (i) facilitates the resonance assignment process as it reduces signal overlap in homonuclear 13C-spectra and (ii) possibly improves the signal-to-noise ratio through multiplet collapse. It can be applied in any 13C-detected experiment. 相似文献
19.
Aromatic amino-acid side chains are essential components for the structure and function of proteins. We present herein a set of NMR experiments for time-efficient resonance assignment of histidine and tyrosine side chains in uniformly 13C/15N-labeled proteins. The use of band-selective 13C pulses allows to deal with linear chains of coupled spins, thus avoiding signal loss that occurs in branched spin systems during coherence transfer. Furthermore, our pulse schemes make use of longitudinal 1H relaxation enhancement, Ernst-angle excitation, and simultaneous detection of 1H and 13C steady-state polarization to achieve significant signal enhancements. 相似文献
20.
Daniel Hatlem Julie E. Heggelund Daniel Burschowsky Ute Krengel Per E. Kristiansen 《Biomolecular NMR assignments》2017,11(1):99-104
The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB5 toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM1-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the 1H, 13C, 15N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on 13C′, 13Cα, 13Cβ, 1HN and 15N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM1-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens. 相似文献