共查询到20条相似文献,搜索用时 15 毫秒
1.
Recent studies have indicated that the relaxation rate of the 1H-13C multiple-quantum coherence is much slower than that of the 1H-13C single-quantum coherence for non-aromatic methine sites in 13 C labeled proteins and in nucleic acids at the slow tumbling limit. Several heteronuclear experiments have been designed to use a multiple-quantum coherence transfer scheme instead of the single-quantum transfer method, thereby increasing the sensitivity and resolution of the spectra. Here, we report a constant time, gradient and sensitivity enhanced HMQC experiment (CT-g/s-HMQC) and demonstrate that it has a significant sensitivity enhancement over constant time HMQC and constant time gradient and sensitivity enhanced HSQC experiments (CT-g/s-HSQC) when applied to a 13C and 15 N labeled calmodulin sample in D2O. We also apply this approach to 3D NOESY-HMQC and doubly sensitivity enhanced TOCSY-HMQC experiments, and demonstrate that they are more sensitive than their HSQC counterparts. 相似文献
2.
Jonathan H. Davis 《Journal of biomolecular NMR》1995,5(4):433-437
Summary 2D 15N-1H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T2 relaxation during the refocusing delays. However, we show here that, when high 15N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH2 geminal peaks and their truncation artefacts at a modest cost in sensitivity. 相似文献
3.
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
4.
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
5.
In this study, a set of nuclear magnetic resonance experiments, some of them commonly used in the study of 13C-labeled proteins and/or nucleic acids, is applied for the structure determination of uniformly 13C-enriched carbohydrates. Two model substances were employed: one compound of low molecular weight [(UL-13C)-sucrose, 342 Da] and one compound of medium molecular weight (13C-enriched O-antigenic polysaccharide isolated from Escherichia coli O142, ~10 kDa). The first step in this approach involves the assignment of the carbon resonances in each monosaccharide spin system using the anomeric carbon signal as the starting point. The 13C resonances are traced using 13C–13C correlations from homonuclear experiments, such as (H)CC–CT–COSY, (H)CC–NOESY, CC–CT–TOCSY and/or virtually decoupled (H)CC–TOCSY. Based on the assignment of the 13C resonances, the 1H chemical shifts are derived in a straightforward manner using one-bond 1H–13C correlations from heteronuclear experiments (HC–CT–HSQC). In order to avoid the 1 J CC splitting of the 13C resonances and to improve the resolution, either constant-time (CT) in the indirect dimension or virtual decoupling in the direct dimension were used. The monosaccharide sequence and linkage positions in oligosaccharides were determined using either 13C or 1H detected experiments, namely CC–CT–COSY, band-selective (H)CC–TOCSY, HC–CT–HSQC–NOESY or long-range HC–CT–HSQC. However, due to the short T2 relaxation time associated with larger polysaccharides, the sequential information in the O-antigen polysaccharide from E. coli O142 could only be elucidated using the 1H-detected experiments. Exchanging protons of hydroxyl groups and N-acetyl amides in the 13C-enriched polysaccharide were assigned by using HC–H2BC spectra. The assignment of the N-acetyl groups with 15N at natural abundance was completed by using HN–SOFAST–HMQC, HNCA, HNCO and 13C-detected (H)CACO spectra. 相似文献
6.
Dan Gibson Yonit Kasherman Dina Kowarski Inna Freikman 《Journal of biological inorganic chemistry》2006,11(2):179-188
In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl2(13CH3NH2)2] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear
single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release
of 13CH3NH2 but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds
are formed and Pt–NH3 bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C]
HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best
method for characterizing the different species in solution. However, the intensity of the peaks in the 15NH3–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity,
and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary
techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum
complexes with sulfur-containing ligands. 相似文献
7.
Kanelis V Donaldson L Muhandiram DR Rotin D Forman-Kay JD Kay LE 《Journal of biomolecular NMR》2000,16(3):253-259
Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) 13C and variable time (VT) 15N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/JCC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2HzNz state, then prior to the VT chemical shift labelling period is converted into 2HzNy coherence. The VT -15N mode eliminates the effect of 1
J
N,CO and 1,2
J
N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH2 groups, thus removing any potential overlap with the CH3 signals. The CT-13CH3,VT-15N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH3 and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH3 and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin. 相似文献
8.
Yanpeng Hou Shugeng Cao Peggy J. Brodie James S. Miller Chris Birkinshaw Mamisoa N. Andrianjafy Rabodo Andriantsiferana Vincent E. Rasamison Karen TenDyke Yongchun Shen Edward M. Suh David G.I. Kingston 《Phytochemistry》2010,71(5-6):669-674
Fractionation of an ethanol extract of a Madagascar collection of the leaves and fruit of Cassipourea lanceolata Tul. led to the isolation of three euphane triterpenoids 1–3. The 1H and 13C NMR spectra of all compounds were fully assigned using a combination of 2D NMR experiments, including COSY, TOCSY, HSQC (HMQC), HMBC and ROESY sequences. The three compounds showed weak antiproliferative activities against the A2780 human ovarian cancer cell line, with IC50 values of 25, 25 and 32 μM, respectively. 相似文献
9.
Patrik Andersson Kerstin Nordstrand Maria Sunnerhagen Edvards Liepinsh Ivars Turovskis Gottfried Otting 《Journal of biomolecular NMR》1998,11(4):445-450
Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,1 H-detected 15N-1H or13 C-1H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra. 相似文献
10.
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
11.
Arafa I. Hamed Milena Masullo Mohamed G. Sheded Usama A. Mahalel Moatz M. Tawfik Angela Perrone Sonia Piacente 《Phytochemistry letters》2011,4(3):353-356
Continuing our investigations on medicinal plants of the Egyptian desert, two new triterpene glycoside derivatives, along with three known compounds have been isolated from the roots of Salsola imbricata, a shrub widely growing in Egypt. Their structures have been established as 3-O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucuronopyranosyl-akebonic acid 28-O-β-d-glucopyranoside and 3-O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucuronopyranosyl-29-hydroxyoleanolic acid 28-O-β-d-glucopyranoside on the basis of spectroscopic methods including 1D- (1H, 13C) and 2D-NMR (DQF-COSY, HSQC, HMBC) experiments as well as mass spectrometry analysis. 相似文献
12.
Wiesława Bylka 《Acta Physiologiae Plantarum》2004,26(4):393-398
A new acetylated flavonol glycoside: patuletin 3-O-[5′″-O-feruloyl-β-D-apiofuransyl (1′″→2′′)-β-D-glucopyranoside] (2), together with a known patuletin 3-O-β-D-glucopyranoside (1) were isolated from the aerial part of Artiplex littoralis L. (Chenopodiacease). Their structures were elcidated by acid hydrolysis and spectroscopic methods including UV, 1H, 13C NMR and ESI-MS for both compounds, additionally 2D-NMR, HSQC, HMBC experiments were performed for 2. 相似文献
13.
Shivanand M. Pudakalakatti Abhinav Dubey Garima Jaipuria U. Shubhashree Satish Kumar Adiga Detlef Moskau Hanudatta S. Atreya 《Journal of biomolecular NMR》2014,58(3):165-173
We present a new method for rapid NMR data acquisition and assignments applicable to unlabeled (12C) or 13C-labeled biomolecules/organic molecules in general and metabolomics in particular. The method involves the acquisition of three two dimensional (2D) NMR spectra simultaneously using a dual receiver system. The three spectra, namely: (1) G-matrix Fourier transform (GFT) (3,2)D [13C, 1H] HSQC–TOCSY, (2) 2D 1H–1H TOCSY and (3) 2D 13C–1H HETCOR are acquired in a single experiment and provide mutually complementary information to completely assign individual metabolites in a mixture. The GFT (3,2)D [13C, 1H] HSQC–TOCSY provides 3D correlations in a reduced dimensionality manner facilitating high resolution and unambiguous assignments. The experiments were applied for complete 1H and 13C assignments of a mixture of 21 unlabeled metabolites corresponding to a medium used in assisted reproductive technology. Taken together, the experiments provide time gain of order of magnitudes compared to the conventional data acquisition methods and can be combined with other fast NMR techniques such as non-uniform sampling and covariance spectroscopy. This provides new avenues for using multiple receivers and projection NMR techniques for high-throughput approaches in metabolomics. 相似文献
14.
Ronald A. Venters Chih-Chin Huang Bennett T. Farmer II Ronald Trolard Leonard D. Spicer Carol A. Fierke 《Journal of biomolecular NMR》1995,5(4):339-344
Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties. 相似文献
15.
Simultaneous data acquisition in time-sharing (TS) multi-dimensional NMR experiments has been shown an effective means to reduce experimental time, and thus to accelerate structure determination of proteins. This has been accomplished by spin evolution time-sharing of the X and Y heteronuclei, such as 15N and 13C, in one of the time dimensions. In this work, we report a new 3D TS experiment, which allows simultaneous 13C and 15N spin labeling coherence in both t
1 and t
2 dimensions to give four NOESY spectra in a single 3D experiment. These spectra represent total NOE correlations between 1HN and 1HC resonances. This strategy of double time-sharing (2TS) results in an overall four-fold reduction in experimental time compared with its conventional counterpart. This 3D 2TS CN-CN-H HSQC-NOESY-HSQC pulse sequence also demonstrates improvements in water suppression, 15N spectral resolution and sensitivity, which were developed based on 2D TS CN-H HSQC and 3D TS H-CN-H NOESY-HSQC experiments. Combining the 3D TS and the 3D 2TS NOESY experiments, NOE assignment ambiguities and errors are considerably reduced. These results will be useful for rapid protein structure determination to complement the effort of discerning the functions of diverse genomic proteins. 相似文献
16.
《Phytochemistry》2002,60(8)
Two apiose-containing kaempferol triosides, together with nine known flavonoids were isolated from the leaves of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic methods including UV, LSI MS, FAB MS, CI MS, 1H, 13C and 2D-NMR, DEPT, HMQC and HMBC experiments. The two new compounds were identified as kaempferol 3-O-β-
-apiofuranoside 7-O-α-
-rhamnosyl-(1′→6)-O-β-
-galactopyranoside and kaempferol 3-O-β-
-apiofuranoside 7-O-α-
-rhamnosyl-(1→ 6)-O-β-
(2-O-E-caffeoylgalactopyranoside). 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.
Bychowska A Theilacker C Czerwicka M Marszewska K Huebner J Holst O Stepnowski P Kaczyński Z 《Carbohydrate research》2011,(17):2816-2819
Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using 1H, 13C, and 31P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC–MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-d-galactose, glycerol (Gro), and phosphate, and has the structure shown below:→6)-α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→2)-Gro-(3→P→ 相似文献
19.
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
20.
The Lys residues in the 75-residue Ca2+-binding protein calbindin D9k were reductively methylated with13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca2+- and apo-forms of the protein, the13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H,13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca2+-calbindin D9k and apo-calbindin D9k were obtained by combiningpH titration experiments and (1H,13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca2+- and apo-forms of calbindin D9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca2+-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of thepKa values of the Lys residues. 相似文献