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1.
Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy. 总被引:1,自引:1,他引:0 下载免费PDF全文
G. J. Kroon J. Grtzinger K. Dijkstra R. M. Scheek G. T. Robillard 《Protein science : a publication of the Protein Society》1993,2(8):1331-1341
This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray. 相似文献
2.
Rasmus H. Fogh Dick Schipper Rolf Boelens Robert Kaptein 《Journal of biomolecular NMR》1995,5(3):259-270
Summary The 1H, 13C and 15N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CHn carbons, all amide (NH and NH2) nitrogens and 99.2% of the amide and CHn protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D
two-/three-/four-dimensional
- HSQC
heteronuclear single-quantum coherence
- HMQC
heteronuclear multiple-quantum coherence
- COSY
correlation spectroscopy
- TOCSY
total correlation spectroscopy
- NOE
nuclear Overhauser enhancement (connectivity)
- NOESY
2D NOE spectroscopy
Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667]. 相似文献
3.
N A van Nuland A A van Dijk K Dijkstra F H van Hoesel R M Scheek G T Robillard 《European journal of biochemistry》1992,203(3):483-491
We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with 15N and 13C to overcome spectral overlap. Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three-dimensional heteronuclear 1H NOE 1H-15N multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation 1H-15N multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on 15N-enriched HPr and an additional three-dimensional triple-resonance 1HN-15N-13C alpha correlation spectroscopy (HNCA) experiment on 13C, 15N-enriched HPr. Many of the sequential backbone 1H assignments, as derived from two-dimensional NMR studies [Klevit, R.E., Drobny, G.P. & Waygood, E.B. (1986) Biochemistry 25, 7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact. 相似文献
4.
Summary Heteronuclear 2D (13C, 1H) and (15N, 1H) correlation spectra of (13C, 15N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC
heteronuclear multiple-quantum coherence spectroscopy
- HSQC
heteronuclear single-quantum coherence spectroscopy
- HSMQC
heteronuclear single- and multiple-quantum coherence spectroscopy
- NOESY
nuclear Overhauser enhancement spectroscopy 相似文献
5.
We report the 13C NMR data for 20 compounds bearing a substituent (alkyl, alkenyl, alkynyl, alkylamide, spiro-γ-lactone, phenyl, benzyl, naphthyl, etc.) at the 17α-position of estradiol. The carbon assignments were done using 1D and 2D NMR experiments (distortionless enhancement by polarization transfer, homonuclear correlated spectroscopy, heteronuclear shift correlation, and heteronuclear shift correlation via long-range couplings). Only the chemical shifts of carbons 12–18, which surround the substitution site, were affected by the addition of a substituent. The magnitude of the effects (shielding or deshielding) was influenced by the 17α-substituent. The individual effects at these carbons were sufficiently distinctive to identify specific centers and should be valuable for signal assignment of a variety of 17α-derivatives of estradiol. In addition to carbon-skeleton assignment, we also report the carbon-substituent assignments. 相似文献
6.
Cytotoxic biotransformed products from cinobufagin by Mucor spinosus and Aspergillus Niger 总被引:2,自引:0,他引:2
Cinobufagin (1) was one of important cardenolidal steroids and major components of Chan'Su, a famous traditional Chinese medicine. Bioconversion of cinobufagin by the fungi of Mucor spinosus and Aspergillus niger were investigated. Nine bioconversion products were obtained from M. spinosus and seven products from A. niger. Their structures were elucidated by high-resolution fast atom bombardment mass spectroscopy (HR-FAB-MS), extensive NMR techniques, including (1)H NMR, (13)C NMR, DEPT, (1)H-(1)H correlation spectroscopy (COSY), two-dimensional nuclear Overhauser effect correlation spectroscopy (NOESY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC). The in vitro cytotoxic activities against human hepatoma cells (HepG2, SMMC-7221 and BEL-7402) and human leukemia cells (K562, HL-60 and HEL) of all bioconversion products were determined by the MTT method, and their structure-activity relationships (SAR) were discussed. 相似文献
7.
Kerstin Nordstrand Hannes Ponstingl Arne Holmgren Gottfried Otting 《European biophysics journal : EBJ》1996,24(3):179-184
Virtually complete sequence specific 1H and 15N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F1-decoupled ROESY-15N-HSQC and 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC using a single uniformly 15N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-15N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without 13C/15N double labelling. Chemical shifts, 3J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC
heteronuclear single quantum coherence
- NMR
nuclear magnetic resonance
- NOE
nuclear Overhauser effect
- NOESY
two-dimensional NOE spectroscopy
- ROE
nuclear Overhauser effect in the rotating frame
- ROESY
two-dimensional ROE spectroscopy
- TOCSY
total correlation spectroscopy
- TPPI
time proportional phase incrementation
Correspondence to: G. Otting 相似文献
8.
Siddhartha Sarma Russell J. DiGate Debra L. Banville R. D. Guiles 《Journal of biomolecular NMR》1996,8(2):171-183
Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain 13C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b
5. The assignment of backbone 13C resonances was used to confirm previous 1H and 15N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone 13C chemical shifts, the solution secondary structure of rat cytochrome b
5 has been determined. The striking similarity of backbone 13C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the 13C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE
delays alternating with nutation for tailored excitation
- DEAE
diethylaminoethyl
- DQF-COSY
2D double-quantum-filtered correlation spectroscopy
- EDTA
ethylenediaminetetraacetic acid
- HCCH-TOCSY
3D proton-correlated carbon TOCSY experiment
- HMQC
2D heteronuclear multiple-quantum correlation spectroscopy
- HNCA
3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons
- HNCO
3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons
- HNCOCA
3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons
- HOHAHA
2D homonuclear Hartmann-Hahn spectroscopy
- HOHAHA-HMQC
3D HOHAHA relayed HMQC
- HSQC
2D heteronuclear single-quantum correlation spectroscopy
- IPTG
isopropyl thiogalactoside
- NOESY
2D nuclear Overhauser enhancement spectroscopy
- NOESY-HSQC
3D NOESY relayed HSQC
- TOCSY
2D total correlation spectroscopy
- TPPI
time-proportional phase incrementation
- TSP
trimethyl silyl propionate 相似文献
9.
The aerial parts of Teucrium oliverianum yielded two neo-clerodane diterpenoids, teucrolin F and G, together with the known teucrolin E. The previously proposed structure for teucrolin E was revised so that it contains a tetrahydrofuran ring instead of an oxetane ring. This was based on analysis of the NMR spectroscopic data of its diacetate, including its NOE spectra. In addition, the structural assignments of the new diterpenoids were based on 1H and 13C NMR spectroscopic studies, mainly 2D NMR experiments, including homonuclear and heteronuclear correlations. 相似文献
10.
The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and 13C, 1H, and 13C/1H heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6'-O-methylcarrabiose 2,4'-disulfate (the repeating unit of 6'-O-methylated iota-carrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported. 相似文献