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1.
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
2.
Osteopontin (OPN) is a 33.7 kDa intrinsically disordered protein and a member of the SIBLING family of proteins. OPN is bearing a signal peptide for secretion into the extracellular space, where it exerts its main physiological function, the control of calcium biomineralization. It is often involved in tumorigenic processes influencing proliferation, migration and survival, as well as the adhesive properties of cancer cells via CD44 and integrin signaling pathways. Here we report the nearly complete NMR chemical shift assignment of recombinant human osteopontin. 相似文献
3.
Nazimuddin Khan David Ban Pablo Trigo-Mourino Marta G. Carneiro Manfred Konrad Donghan Lee T. Michael Sabo 《Biomolecular NMR assignments》2018,12(1):11-14
Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain 1H, 13C, and 15N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution. 相似文献
4.
5.
Heavy water (H218O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been
reported for the 18O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% 18O for DNA extracted from soil incubated with the addition of H218O. Four experiments were conducted to test how the atom% 18O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1)
time, (2) nutrients, (3) soil moisture, and (4) atom% 18O of added H2O. In the time series experiment, the atom% 18O of DNA increased linearly (R
2 = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R
2 = 0.991, p = 0.006) between the log10 of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log10 of the atom% 18O of DNA. For the experiment where soil moisture was manipulated, the atom% 18O of DNA increased with higher soil moisture until soil moisture reached 30%, above which 18O enrichment of DNA declined as soils became more saturated. When the atom% 18O for H2O added was varied, there was a positive linear relationship between the atom% 18O of the added water and the atom% 18O of the DNA. Results indicate that quantification of 18O incorporated into DNA from H218O has potential to be used as a proxy for microbial growth in soil. 相似文献
6.
A comparison of three labeling strategies for studies involving side chain methyl groups in high molecular weight proteins,
using 13CH3,13CH2D, and 13CHD2 methyl isotopomers, is presented. For each labeling scheme, 1H–13C pulse sequences that give optimal resolution and sensitivity are identified. Three highly deuterated samples of a 723 residue
enzyme, malate synthase G, with 13CH3,13CH2D, and 13CHD2 labeling in Ile δ1 positions, are used to test the pulse sequences experimentally, and a rationalization of each sequence’s
performance based on a product operator formalism that focuses on individual transitions is presented. The HMQC pulse sequence
has previously been identified as a transverse relaxation optimized experiment for 13CH3-labeled methyl groups attached to macromolecules, and a zero-quantum correlation pulse scheme (13CH3 HZQC) has been developed to further improve resolution in the indirectly detected dimension. We present a modified version
of the 13CH3 HZQC sequence that provides improved sensitivity by using the steady-state magnetization of both 13C and 1H spins. The HSQC and HMQC spectra of 13CH2D-labeled methyl groups in malate synthase G are very poorly resolved, but we present a new pulse sequence, 13CH2D TROSY, that exploits cross-correlation effects to record 1H–13C correlation maps with dramatically reduced linewidths in both dimensions. Well-resolved spectra of 13CHD2-labeled methyl groups can be recorded with HSQC or HMQC; a new 13CHD2 HZQC sequence is described that provides improved resolution with no loss in sensitivity in the applications considered here.
When spectra recorded on samples prepared with the three isotopomers are compared, it is clear that the 13CH3 labeling strategy is the most beneficial from the perspective of sensitivity (gains ≥2.4 relative to either 13CH2D or 13CHD2 labeling), although excellent resolution can be obtained with any of the isotopomers using the pulse sequences presented
here. 相似文献
7.
Agnieszka?Sitarska Lukasz?Skora Julia?Klopp Susan?Roest César?Fernández Binesh?Shrestha Alvar?D.?Gossert
For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for 15N and 13C with yields comparable to expression in full media. For 2H,15N and 2H,13C,15N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins. 相似文献
8.
J. W. Mietelski M. A. Olech K. Sobiech-Matura B. J. Howard P. Gaca M. Zwolak S. Błażej E. Tomankiewicz 《Polar Biology》2008,31(9):1081-1089
There are few data reported on radionuclide contamination in Antarctica. The aim of this paper is to report 137Cs, 90Sr and 238,239+240Pu and 40K activity concentrations measured in biological samples collected from King George Island (Southern Shetlands, Antarctica),
mostly during 2001–2002. The samples included: bones, eggshells and feathers of penguin Pygoscelis papua, bones and feathers of petrel Daption capense, bones and fur of seal Mirounga leonina, algae Himantothallus grandifolius, Desmarestia anceps and Cystosphaera jacquinotii, fish Notothenia corriceps, sea invertebrates Amphipoda, shells of limpet Nacella concina, lichen Usnea aurantiaco-atra, vascular plants Deschampsia antarctica and Colobanthus quitensis, fungi Omphalina pyxidata, moss Sanionia uncinata and soil. The results show a large variation in some activity concentrations. Samples from the marine environment had lower
contamination levels than those from terrestrial ecosystems. The highest activity concentrations for all radionuclides were
found in lichen and, to a lesser extent, in mosses, probably because lichens take up atmospheric pollutants and retain them.
The only significant correlation (except for that expected between 238Pu and 239+240Pu) was noted for moss and lichen samples between plutonium and 90Sr. A tendency to a slow decrease with time seems to be occurring. Analyses of the activity ratios show varying fractionation
between various radionuclides in different organisms. Algae were relatively more highly contaminated with plutonium and radiostrontium,
and depleted with radiocesium. Feathers had the lowest plutonium concentrations. Radiostrontium and, to a lesser extent, Pu
accumulated in bones. The present low intensity of fallout in Antarctic has a lower 238Pu/239+240Pu activity ratio than that expected for global fallout. 相似文献
9.
10.
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
11.
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
12.
Bertini I Felli IC Kümmerle R Luchinat C Pierattelli R 《Journal of biomolecular NMR》2004,30(3):245-251
13C-13C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, 15N, 13C and 70% 2H labeled). 13C-13C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of 13C-13C NOESY spectroscopy. 相似文献
13.
Schwab S Ramos HJ Souza EM Pedrosa FO Yates MG Chubatsu LS Rigo LU 《Archives of microbiology》2007,187(5):379-386
14.
Naotaka Sekiyama Andras Boeszoermenyi Haribabu Arthanari Gerhard Wagner Mélissa Léger-Abraham 《Biomolecular NMR assignments》2017,11(2):187-191
The eukaryotic translational initiation factor 4G (eIF4G) interacts with the cap-binding protein eIF4E through a consensus binding motif, Y(X)4LΦ (where X is any amino acid and Φ is a hydrophobic residue). 4E binding proteins (4E-BPs), which also contain a Y(X)4LΦ motif, regulate the eIF4E/eIF4G interaction. The non- or minimally-phosphorylated form of 4E-BP1 binds eIF4E, preventing eIF4E from interacting with eIF4G, thus inhibiting translation initiation. 4EGI-1, a small molecule inhibitor of the eIF4E/eIF4G interaction that is under investigation as a novel anti-cancer drug, has a dual activity; it disrupts the eIF4E/eIF4G interaction and stabilizes the binding of 4E-BP1 to eIF4E. Here, we report the complete backbone NMR resonance assignment of an unliganded 4E-BP1 fragment (4E-BP144–87). We also report the near complete backbone assignment of the same fragment in complex to eIF4E/m7GTP (excluding the assignment of the last C-terminus residue, D87). The chemical shift data constitute a prerequisite to understanding the mechanism of action of translation initiation inhibitors, including 4EGI-1, that modulate the eIF4E/4E-BP1 interaction. 相似文献
15.
Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576. 相似文献
16.
Deepshikha Verma Alok Bhattacharya Kandala V. R. Chary 《Biomolecular NMR assignments》2016,10(1):67-70
We report almost complete sequence specific 1H, 13C and 15N NMR assignments of a 150-residue long calmodulin-like calcium-binding protein from Entamoeba histolytica (EhCaBP6), as a prelude to its structural and functional characterization. 相似文献
17.
The sequence-specific backbone assignment of hematopoietic protein tyrosine phosphatase (HePTP; PTPN7) in presence of vanadate has been determined, based on triple-resonance experiments using uniformly [13C,15N]-labeled protein. These assignments facilitate further studies of HePTP in the presence of inhibitors to target leukemia and provide further insights into the function of protein tyrosine phosphatases. 相似文献
18.
Carsten Krichel Oliver H. Weiergräber Marina Pavlidou Jeannine Mohrlüder Melanie Schwarten Dieter Willbold Philipp Neudecker 《Biomolecular NMR assignments》2016,10(1):41-43
Autophagy is a versatile catabolic pathway for lysosomal degradation of cytoplasmic material. While the phenomenological and molecular characteristics of autophagic non-selective (bulk) decomposition have been investigated for decades, the focus of interest is increasingly shifting towards the selective mechanisms of autophagy. Both, selective as well as bulk autophagy critically depend on ubiquitin-like modifiers belonging to the Atg8 (autophagy-related 8) protein family. During evolution, Atg8 has diversified into eight different human genes. While all human homologues participate in the formation of autophagosomal membrane compartments, microtubule-associated protein light chain 3C (LC3C) additionally plays a unique role in selective autophagic clearance of intracellular pathogens (xenophagy), which relies on specific protein–protein recognition events mediated by conserved motifs. The sequence-specific 1H, 15N, and 13C resonance assignments presented here form the stepping stone to investigate the high-resolution structure and dynamics of LC3C and to delineate LC3C’s complex network of molecular interactions with the autophagic machinery by NMR spectroscopy. 相似文献
19.
Juranić N Nemutlu E Zhang S Dzeja P Terzic A Macura S 《Journal of biomolecular NMR》2011,50(3):237-245
Intramolecular correlations among the 18O-labels of metabolic oligophosphates, mapped by J-decoupled 31P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the 18O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable
determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures
accurate determination of 18O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological
usefulness of the J-decoupled 31P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally 18O labeled in perfused mammalian hearts. 相似文献
20.
Fatty acid-binding proteins (FABPs) are a family of proteins that modulate the transfer of various fatty acids in the cytosol and constitute a significant portion in many energy-consuming cells. The ligand binding properties and specific functions of a particular type of FABP seem to be diverse and depend on the respective binding cavity as well as the cell type from which this protein is derived. Previously, a novel FABP (lcFABP; lc: Luciola cerata) was identified in the light organ of Taiwanese fireflies. The lcFABP was proved to possess fatty acids binding capabilities, especially for fatty acids of length C14–C18. However, the structural details are unknown, and the structure–function relationship has remained to be further investigated. In this study, we finished the 1H, 15N and 13C chemical shift assignments of 15N/13C-enriched lcFABP by solution NMR spectroscopy. In addition, the secondary structure distribution was revealed based on the backbone N, H, Cα, Hα, C and side chain Cβ assignments. These results can provide the basis for further structural exploration of lcFABP. 相似文献