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1.
1H, 13C, and 15N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain from the domain of unknown function 5 (DUF5) effector (MLDVvDUF5) of the MARTX toxin from Vibrio vulnificus in its solution state. We have assigned 97 % of all backbone and side-chain carbon atoms, including 96 % of all backbone residues. Secondary chemical shift analysis using TALOS+ demonstrates four helices that align with those predicted by structure homology modeling using the MLDs of Pasteurella multocida toxin (PMT) and the clostridial TcdB and TcsL toxins as templates. Future studies will be towards solving the structure and determining the dynamics in the solution state.  相似文献   

2.
Phosphoenolpyruvate binding to the C-terminal domain (EIC) of enzyme I of the bacterial phosphotransferase system (PTS) initiates a phosphorylation cascade that results in sugar translocation across the cell membrane and controls a large number of essential pathways in bacterial metabolism. EIC undergoes an expanded to compact conformational equilibrium that is regulated by ligand binding and determines the phosphorylation state of the overall PTS. Here, we report the backbone 1H, 15N and 13C chemical shift assignments of the 70 kDa EIC dimer from the thermophilic bacterium Thermoanaerobacter tengcongensis. Assignments were obtained at 70 °C by heteronuclear multidimensional NMR spectroscopy. In total, 90% of all backbone resonances were assigned, with 264 out of a possible 299 residues assigned in the 1H–15N TROSY spectrum. The secondary structure predicted from the assigned backbone resonance using the program TALOS+ is in good agreement with the X-ray crystal structure of T. tengcongensis EIC. The reported assignments will allow detailed structural and thermodynamic investigations on the coupling between ligand binding and conformational dynamics in EIC.  相似文献   

3.
The staphylococcal enterotoxin H (SEH; 217 aa, 25 kD) belongs to a family of superantigens that cause a massive immune response upon simultaneous binding to the T cell receptor (TCR) and the major histocompatibility complex class II. The SEH-TCR interaction is weak and amenable to studies using NMR methodology. Essentially, 2 mg of U{2H, 13C,15N}-labeled SEH was used for the complete sequential backbone assignment of SEH at 900 MHz. The protein secondary structure inferred from the chemical shift index (Cα and Cβ) is in very good agreement with the secondary structure elements of the X-ray structure.  相似文献   

4.
Sequence specific resonance assignment of proteins forms the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone (1H, 15N, 13Cα and 13C′) resonances of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality experiment—(4,3)D-hnCOCANH and exploits the linear combinations of backbone (13Cα and 13C′) chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text). The resulted increased dispersion of peaks—which is different in sum (CA + CO) and difference (CA ? CO) frequency regions—greatly facilitates the analysis of the spectrum by resolving the problems (associated with routine assignment strategies) arising because of degenerate amide 15N and backbone 13C chemical shifts. Further, the spectrum provides direct distinction between intra- and inter-residue correlations because of their opposite peak signs. The other beneficial feature of the spectrum is that it provides: (a) multiple unidirectional sequential (ii + 1) 15N and 13C correlations and (b) facile identification of certain specific triplet sequences which serve as check points for mapping the stretches of sequentially connected HSQC cross peaks on to the primary sequence for assigning the resonances sequence specifically. On top of all this, the F 2F 3 planes of the spectrum corresponding to sum (CA + CO) and difference (CA ? CO) chemical shifts enable rapid and unambiguous identification of sequential HSQC peaks through matching their coordinates in these two planes (see the text). Overall, the experiment presented here will serve as an important backbone assignment tool for variety of structural and functional proteomics and drug discovery research programs by NMR involving well behaved small folded proteins (MW < 15 kDa) or a range of intrinsically disordered proteins.   相似文献   

5.
Androcam is a calmodulin-like protein that acts as a testis-specific light chain to myosin VI during spermatogenesis in D. melanogaster. Modest, localized chemical shift changes that accompany Ca2+ binding to the androcam N-terminal lobe indicate that unlike calmodulin, androcam does not undergo a dramatic conformational change upon binding calcium. Here we report the 1H, 15N and 13C resonances of androcam in the high calcium (10 mM) state and show the extent of chemical shift changes for backbone resonances relative to the low calcium state.  相似文献   

6.
A simple and fast nuclear magnetic resonance method for docking proteins using pseudo-contact shift (PCS) and 1HN/15N chemical shift perturbation is presented. PCS is induced by a paramagnetic lanthanide ion that is attached to a target protein using a lanthanide binding peptide tag anchored at two points. PCS provides long-range (~40 Å) distance and angular restraints between the lanthanide ion and the observed nuclei, while the 1HN/15N chemical shift perturbation data provide loose contact-surface information. The usefulness of this method was demonstrated through the structure determination of the p62 PB1-PB1 complex, which forms a front-to-back 20 kDa homo-oligomer. As p62 PB1 does not intrinsically bind metal ions, the lanthanide binding peptide tag was attached to one subunit of the dimer at two anchoring points. Each monomer was treated as a rigid body and was docked based on the backbone PCS and backbone chemical shift perturbation data. Unlike NOE-based structural determination, this method only requires resonance assignments of the backbone 1HN/15N signals and the PCS data obtained from several sets of two-dimensional 15N-heteronuclear single quantum coherence spectra, thus facilitating rapid structure determination of the protein–protein complex.  相似文献   

7.
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.
A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, ≥90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed (?, ψ) torsion angles of ca 12º. TALOS-N also reports sidechain χ1 rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.  相似文献   

9.
The iron-regulated FrpD protein is a unique lipoprotein embedded into the outer membrane of the Gram-negative bacterium Neisseria meningitidis. The biological function of FrpD remains unknown but might consist in anchoring to the bacterial cell surface the Type I-secreted FrpC protein, which belongs to a Repeat in ToXins (RTX) protein family and binds FrpD with very high affinity (K d  = 0.2 nM). Here, we report the backbone 1H, 13C, and 15N chemical shift assignments for the FrpD43–271 protein that allow us to characterize the intimate interaction between FrpD and the N-terminal domain of FrpC.  相似文献   

10.
Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue—HN, N, CA and CO chemical shifts—from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i ? 1) amide 1H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135–147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.  相似文献   

11.
NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ HN, Hα or 13C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with 1HN(i ? 1), 13C′(i ? 1) and 15N(i), and 1HN(i + 1), 13C′(i) and 15N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone.  相似文献   

12.
Heavy chain antibodies differ in structure to conventional antibodies lacking both the light chain and the first heavy chain constant domain (CH1). Characteristics of the antigen-binding variable heavy domain of the heavy chain antibody (VHH) including the smaller size, high solubility and stability make them an attractive alternative to more traditional antibody fragments for detailed NMR-based structural analysis. Here we report essentially complete backbone and side chain 15N, 13C and 1H assignments for a free VHH. Analysis of the backbone chemical shift data obtained indicates that the VHH is comprised predominantly of β-sheets corresponding to nearly 60 % of the protein backbone.  相似文献   

13.
An ascomycetous yeast strain isolated from activated sludge could decolorize Reactive Black 5 azo dye at 200 mg l?1 up to 90 % within 12–18 h under agitated condition. Yeast decolorization ability was investigated at different RB5 concentrations and, at higher dye concentration, 500 mg l?1, the decolorization was found to be 98 % after 36 h incubation time. Extensive decolorization (95–99 %) was obtained in presence of five other azo dyes, Reactive Orange 16, Reactive Red 198, Direct Blue 71, Direct Yellow 12, and Direct Black 22, by isolated yeast. HPLC analysis, UV–vis spectra and colorless biomass obtained after complete decolorization showed that the decolorization occured through a biodegradation mechanism. Decolorization was occurred during the exponential growth phase which is associated to primary metabolism. Laccase production by the yeast cells was not detected. The isolated yeast was characterized according to phenotypical and molecular procedures and was closely related (99 % identity) to Issatchenkia orientalis.  相似文献   

14.
Membrane localization domain (MLD) was first proposed for a 4‐helix‐bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1‐specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats‐in‐toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1‐C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4‐helix‐bundle structure in solution.  相似文献   

15.
The taxonomic position of a Gram-positive, endospore-forming bacterium isolated from soil sample collected from an industrial site was analyzed by a polyphasic approach. The strain designated as IITR-54T matched most of the phenotypic and chemical characteristics of the genus Bacillus and represents a novel species. It was found to biodegrade 4-chlorobiphenyl through dechlorination and was isolated through enrichment procedure from an aged polychlorinated biphenyl-contaminated soil. Both resting cell assay and growth under aerobic liquid conditions using 4-chlorobiphenyl as sole source of carbon along with 0.01 % yeast extract, formation of chloride ions was measured. 16S rRNA (1,489 bases) nucleotide sequence of isolated strain was compared with those of closely related Bacillus type strains and confirmed that the strain belongs to the genus Bacillus. Strain IITR-54T differs from all other species of Bacillus by at least 2.1 % at the 16S rRNA level, and the moderately related species are Bacillus oceanisediminis (97.9 %) followed by Bacillus infantis (97.7 %), Bacillus firmus (97.4 %), Bacillus drentensis (97.3 %), Bacillus circulans (97.2 %), Bacillus soli (97.1 %), Bacillus horneckiae (97.1 %), Bacillus pocheonensis (97.1 %) and Bacillus bataviensis (97.1 %), respectively. The cell wall peptidoglycan contained meso-diaminopimelic acid and the major isoprenoid quinone was MK-7. Major fatty acids are iso-C15:0 (32.4 %) and anteiso-C15:0 (27.4 %). Predominant polar lipids are diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The results of physiological and biochemical tests allowed the genotypic and phenotypic distinctiveness of strain IITR-54T with its phylogenetic relatives and suggest that the strain IITR-54T should be recognized as a novel species, for which the name Bacillus mesophilum sp. nov. is proposed. The type strain is IITR-54T (= MTCC 11060T = JCM 19208T).  相似文献   

16.
17.
Dendrocalamus sinicus, which is the largest bamboo species in the world, has broad prospects in the fields of bioenergy and biorefinery application. In this study, dewaxed D. sinicus samples were sequentially treated with 80 % ethanol containing 0.025 M HCl, 80 % ethanol containing 0.5 % NaOH, and aqueous alkaline solutions (containing 2.0, 5.0, and 8.0 % NaOH, respectively) at 75 °C for 4 h, in which 9.63, 8.71, 21.83, 21.09, and 13.09 % of the original lignin were isolated, respectively. The lignin fractions obtained were comparatively characterized by chemical composition, molecular weights, and structural features by wet chemical and instrumental analysis methods. It was found that the bamboo lignin fractions isolated by ethanol had lower weight-average molecular weights (1,360–1,380 g?mol?1) and contained much higher amounts of associated hemicelluloses, while the lignin fractions isolated by aqueous alkaline solutions had higher weight-average molecular weights (5,300–6,040 g?mol?1) and contained lower amounts of associated hemicelluloses. Spectroscopy analyses indicated that the bamboo lignin was a typical grass lignin, consisting of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. A small percentage of the lignin side-chain was found to be acetylated at the γ-carbon, predominantly at syringyl units. The major interunit linkages present in the bamboo lignin obtained were β-O-4′ aryl ether linkages, together with lower amounts of β-β′, β-5′, and β-1′ linkages.  相似文献   

18.
A haloalkaliphilic, thermophilic Bacillus strain (T14), isolated from a shallow hydrothermal vent of Panarea Island (Italy), produced a new exopolysaccharide (EPS). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T14 was highly related (99 % similarity) to Bacillus licheniformis DSM 13T and Bacillus sonorensis DSM 13779T. Further DNA–DNA hybridization analysis revealed 79.40 % similarity with B. licheniformis DSM 13T and 39.12 % with B. sonorensis DSM 13779T. Sucrose (5 %) was the most efficient carbon source for growth and EPS production. The highest EPS production (366 mg l?1) was yielded in fermenter culture at 300 rpm after 48 h of incubation. The purified fraction EPS1 contained fructose/fucose/glucose/galactosamine/mannose in a relative proportion of 1.0:0.75:0.28:tr:tr and possessed a molecular weight of 1,000 kDa displaying a trisaccharide unit constituted by sugars with a β-manno-pyranosidic configuration. Screening for biological activity showed anti-cytotoxic effect of EPS1 against Avarol in brine shrimp test, indicating a potential use in the development of novel drugs.  相似文献   

19.
We have analyzed the relaxation properties of all 31P nuclei in an RNA cUUCGg tetraloop model hairpin at proton magnetic field strengths of 300, 600 and 900 MHz in solution. Significant H, P dipolar contributions to R 1 and R 2 relaxation are observed in a protonated RNA sample at 600 MHz. These contributions can be suppressed using a perdeuterated RNA sample. In order to interpret the 31P relaxation data (R 1, R 2), we measured the 31P chemical shift anisotropy (CSA) by solid-state NMR spectroscopy under various salt and hydration conditions. A value of 178.5 ppm for the 31P CSA in the static state (S 2 = 1) could be determined. In order to obtain information about fast time scale dynamics we performed a modelfree analysis on the basis of our relaxation data. The results show that subnanosecond dynamics detected around the phosphodiester backbone are more pronounced than the dynamics detected for the ribofuranosyl and nucleobase moieties of the individual nucleotides (Duchardt and Schwalbe, J Biomol NMR 32:295–308, 2005; Ferner et al., Nucleic Acids Res 36:1928–1940, 2008). Furthermore, the dynamics of the individual phosphate groups seem to be correlated to the 5′ neighbouring nucleobases.  相似文献   

20.
Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 Å, an all atom RMSD value of 1.2 Å to the crystal structure for well-defined regions, and RMSD value of 1.1 Å to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with 15N- and 13C-edited NOESY data obtained with a perdeuterated 15N, 13C-enriched 13CH3 methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR.  相似文献   

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