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1.
Summary The 1H NMR signals of the heme methyl, propionate and related chemical groups of cytochrome c
3 from Desulfovibrio vulgaris Miyazaki F ( D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. ( Biochemistry, 29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val 18. All the results contradicted the heme assignments for D.v. MF cytochrome c
3 made on the basis of electron spin resonance (Gayda et al. (1987) FEBS Lett., 217 57–61). Based on these assignments, the interaction of cytochrome c
3 with D.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochrome c
3 was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochrome c
3 molecules per monomer of ferredoxin I and 10 8 M –2 (at 53 mM ionic strength and 25°C), respectively. 相似文献
2.
The complete sequence-specific assignments of resonances in the 1H-NMR spectrum of huwentoxin-I from the Chinese bird spider, Selenocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D 2O and in H 2O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification of d αN connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification of d NN and d αN connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin. 相似文献
3.
The orphan nuclear receptor Nurr1 has been implicated in a number of conditions including Parkinson’s disease and Schizophrenia. As such, it is of interest to study its interactions with other proteins, possibly mediated by small molecules, considering possible use as a drug target. We produced 2H, 15N, 13C labelled-Nurr1 to generate the backbone amide NH, carbonyl C′, C alpha and C beta assignments. About 84.0% of residues could be assigned. Most of the 37 missing assignments fall in 3 regions of the protein. Two of these surround a putative ligand-binding region of Nurr1, suggesting that this region of the protein is flexible, despite the ligand-binding pocket being filled with hydrophobic side-chains from residues surrounding the ligand binding pocket. 相似文献
4.
Recently published chemical shifts for haem 13C nuclei in bovine ferricytochrome b
55 (Lee KB, Kweon J, Park H (1995) Assignment of hyperfine-shifted heme carbon resonances in ferricytochrome b
5. FEBS Lett. 367:77–80) are analysed in terms of haem molecular orbitals with perturbed D 4h symmetry. Since a crystal structure of this protein is available, together with extensive 1H assignments both in the oxidised and reduced forms, the paramagnetic shifts can be separated into dipolar and Fermi contact contributions by using an empirical magnetic susceptibility tensor. The results are compared with the orientation of the tensor and the geometry of the haem ligands. This comparison casts doubt on one of the 13C assignments and demonstrates that the asymmetry of the haem electronic structure is dominated by the influence of both of the His ligands. The 13C chemical shifts of two haem methyl groups in the minor form of the protein, in which the haem is approximately rotated by 180° about its 5CH15CH axis, are also evaluated. 相似文献
5.
Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP 3Rs) and a variety of voltage-gated Ca 2+ channels in the brain. We report complete NMR chemical shift assignments of Ca 2+-free CaBP1 (residues 1–167, BMRB no. 15197). 相似文献
6.
Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP 3Rs) and a variety of voltage-gated Ca 2+ channels in the brain. We report complete NMR chemical shift assignments of Ca 2+-bound CaBP1 (residues 1–167, BMRB no. 15623). 相似文献
7.
Ketopantoate reductase is an essential enzyme for pantothenate (vitamin B 5) synthesis and a potential antibiotic target. Here we report the 15N and 1HN, 13C′, 13C α and 13C β chemical shift assignments of the 34 kDa ketopantoate reductase in its apo state. 相似文献
8.
Abstract A high-field 1H and 31P-NMR study of the oligomer d[CpGp ApTpCpG] 2 was carried out in H 22O and water signal suppression was employed in all 1H NMR acquisitions. Particular attention was given to imino proton and 31P assignments. Two dimensional 31P- 1H shift correlation contours were particularly useful in 31P assignments and confirming previous 1H assignments. Titrimetric addition of aliquots of the anticancer agent mitoxantrone resulted in selective and progressive chemical shifts with critical changes at stoichiometrics of 1:1 and 2:1 drug to DNA ratios. The results indicate ultimate intercalative binding of the drug at both C.G termini of the oligomer in accord with the previously determined C.G preference and with non-nearest neighbor intercalation. 相似文献
9.
Monoclonal and polyclonal antibodies directed against peptides of F 1-ATPase or F 1F 0-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti- monoclonal antibodies has shown asymmetry of in the complex as already shown for . In addition, the involvement of with respect to nucleotide site cooperativity has been detected. Finally, the formation of F 1F 0-antibody complexes of various masses, seems to exclude the functional rotation of F 1 around F 0 during catalysis.Abbreviations IF 1
natural protein inhibitor of the ATPase-ATP synthase
- OSCP
oligomycin sensitivity-conferring protein
- DCCD
dicyclohexylcarbodiimide
- SDS-PAGE
sodium dodecylsulfate polyacrylamide gel electrophoreses
- F 1
F 1-ATPase, coupling factor F 1 of ATPase
- F 1F 0
F 1F 0-ATP synthase, ATPase-ATP synthase complex 相似文献
10.
Calcium-binding protein 1 (CaBP1) regulates inositol 1,4,5-trisphosphate receptors (InsP 3Rs) and a variety of voltage-gated Ca 2+ channels in the brain. We report complete NMR chemical shift assignments of the Ca 2+-saturated form of CaBP1 with Ca 2+ bound at EF1, EF3 and EF4 (residues 1–167, BMRB no. 16862). 相似文献
11.
The analytical and preparative scale optical resolution of 1-cyclohexyl-1-phenylethyl hydroperoxide and 1,2,3,4-tetrahydro-1-napthyl hydroperoxide has been achieved by chiral HPLC on a cellulose tris(3,5-dimethylphenyl carbamate) stationary phase coated on silica gel. The method has been used to obtain several hundred milligrams of highly enriched enantiomers (%ee >98) which were characterized by [α] D and circular dichroism spectra, respectively. Configurational assignments were achieved for 1,2,3,4-tetrahydro-1-naphthyl hydroperoxide enantiomers. © 1995 Wiley-Liss, Inc. 相似文献
12.
Phosphatase of regenerating liver-1 (PRL-1) is a novel target for potentially treating cancer metastases. Although its specific
biochemical role in these processes has yet to be delineated, considerable evidence suggests the phosphatase activity of PRL-1
is required for promoting cancer and metastasis. PRL-1 belongs to the protein tyrosine phosphatase (PTPase) family and functions
using the CX 5R consensus active site motif. Like other PTPases, PRL-1 is inhibited by oxidation at its active site Cys, however, disulfide
bond formation occurs unusually readily in wild-type PRL-1. Chemical shift assignments are available for oxidized wild type,
but numerous, substantial changes are observed in the spectra upon reduction. Because the reduced form is active, we sought
to identify a stable mutant that would resist oxidation and be useful for facilitating drug screening and development using
NMR-based assays. We present here NMR assignments for a full-length, reduced and active form of PRL-1, PRL-1-C170S-C171S,
that is well suited for this purpose. 相似文献
13.
Summary Specific and uniform 15N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His 156, Trp 32 and Trp. 220 residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport of l-glutamine across the cytoplasmic membrane of Escherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H 2 of His 156 refines the earlier, model where this particular proton formas an intermolecular hydrogen bond to the -carbonyl of l-glutamine, while assignments of both Trp 32 and Trp 220 show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8–9 amino acid residues at a time. This paper illustrates the usefulness of combining 15N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25 000. 相似文献
14.
The carboxy-terminal pleckstrin homology (PH) domain recruits GRP1 to the plasma membrane through the specific binding to
phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3]. Here, we describe backbone and side chain assignments of the GRP1 PH domain determined by triple resonance experiments.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
Two-dimensional 1H-nmr methods are described to obtain information on the sidechain conformation of valyl residues of the lac repressor headpiece and to assign the resonances of their methyl groups stereospecifically. The spin–spin coupling constants ( Jαβ) between C αand C β protons are obtained from two-dimensional correlated spectroscopy experiments. Large values for J αβ(10–12 Hz) corresponding to trans orientations for these protons ( g+ conformation) are found for all valyl residues in α-helical segments. For these valyl residues, the distance between one methyl group (γ 1)and the valyl amide proton is much shorter than for the other methyl group, so that stereospecific resonance assignments follow from relative intensities of the corresponding cross peaks in a two-dimensional nuclear Overhauser enhancement spectrum. Thus, streospecific assignments could be made for the methyl groups of Val 9, 20, 23, and 38 (of a total of eight valyl residues). 相似文献
16.
Lipocalin-type prostaglandin D synthase (L-PGDS) acts as both a PGD 2-synthesizing enzyme and an extracellular transporter for small lipophilic molecules. Here we report the backbone and side-chain resonance assignments of uniformly 15N, 13C labeled rat L-PGDS. 相似文献
17.
The complete sequence-specific assignments of resonances in the 1H-NMR spectrum of huwentoxin-I from the Chinese bird spider, Selenocosmia huwena, is described. A combination of two-dimensional NMR experiments including 2D-COSY, 2D-NOESY, and 2D-TOCSY has been employed on samples of the toxin dissolved in D 2O and in H 2O for assignment purposes. Protons belonging to spin systems for each of the 33 amino acids were identified. The sequence-specific assignments were facilitated by the identification of d
N connectivities on the fingerprint regions of the COSY and NOESY spectra and were supported by the identification of d
NN and d
N connectivities in the TOCSY and NOESY spectra. These studies provide a basis for the determination of the solution-phase conformation of this toxin.Abbreviations HWTX-I
huwentoxin-I
- 2D
two-dimensional
- COSY
2D homonuclear correlation spectroscopy
- NOE
nuclear Overhauser enhancement
- NOESY
2D nuclear Overhauser enhancement spectroscopy
- TOCSY
2D total correlation spectroscopy
- TPPI
time-proportional phase incrementation
- TSP
sodium 3-(trimethyl-silyl)propionate-d4
- EDTA
ethylenediaminetetraacetic acid 相似文献
18.
Living organisms rely on the F oF 1 ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the F o motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F 1 ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single F oF 1 molecules embedded in lipid bilayer nanodiscs, we now report the observation of F o-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with F o stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation. 相似文献
19.
Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites. 相似文献
20.
[ 2H, 3H]Gibberellin A 4 (GA 4) or [ 2H, 3H] GA 9 were applied to the shoot tips of seedlings of elongated internode (ein), a tall mutant of rapid cycling Brassica rapa. Following [ 2H]GA 9 application, [ 2H]GA 51, [ 2H]GA 20 and [ 2H]GA 4 were identified as products by GC-MS, while [ 2H]GA 34 and [ 2H]GA 1 were formed from [ 2H]GA 4. Other isotopically labelled products, including abundant putative conjugates, were also produced, but were not identified. Thus, in B. rapa, GA 1 biosynthesis involves the convergence of at least two metabolic pathways; it can be formed via GA 4 or GA 20, the latter of which can originate from GA 9 or from GA 19. 相似文献
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