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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.
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
3.
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
4.
Leppert J Heise B Ohlenschläger O Görlach M Ramachandran R 《Journal of biomolecular NMR》2004,28(2):185-190
A reduced dimensionality magic angle spinning solid-state NMR experimental protocol for obtaining chemical shift correlation spectra of dipolar coupled nuclei in uniformly (13C, 15N) labelled biological systems is described and demonstrated. The method involves a mapping of the evolution frequencies of heteronuclear 13C-15N zero- and double-quantum coherences. In comparison to a reduced dimensionality procedure involving the simultaneous incrementation of two single-quantum chemical shift evolution periods, the approach described here could be potentially advantageous for minimising the heat dissipated in the probe by high power 1H decoupling in experiments requiring long t
1 acquisition times. 相似文献
5.
Jean-Pierre Simorre Bernhard Brutscher Michael S. Caffrey Dominique Marion 《Journal of biomolecular NMR》1994,4(3):325-333
Summary Two-dimensional versions of HNCA and HNCO experiments are described, which provide essentially the same information as the 3D sequence. A multiple-quantum coherence involving either 15N and 13C or 15N and 13CO is created. One of the two frequencies is given by the middle point between the two cross peaks (zero-and double-quantum) and the other by their separation. Quadrature detection can be performed on either nucleus, modifying only the appearence of the 2D spectrum, but not the information content. These experiments, named MQ-HNCA and MQ-HNCO, are illustrated on a (15N, 13C) doubly labelled cytochrome c2 from Rhodobacter capsulatus (116 amino acids).Abbreviations HMQC
heteronuclear multiple-quantum coherence spectroscopy
- HSQC
heteronuclear single-quantum coherence spectroscopy
- ZQ
zero quantum
- 2Q
double quantum
- MQ
multiple quantum
- TPPI
time-proportional phase increment 相似文献
6.
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
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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
7.
Triple resonance HCN and HCNCH experiments used in studies of 13C/15N labeled oligonucleotides include extended evolution periods (typically up to 100 ms) to allow coherence transfer through a complex heteronuclear spin network. Unfortunately, most of the magnetization is lost during the evolution due to fast spin–spin relaxation dominated by one-bond 1H–13C dipolar interaction. As demonstrated recently, the sensitivity of the experiments can be dramatically improved by keeping the spin system in a state of proton–carbon multiple-quantum coherence, which is not affected by the strong dipolar coupling. However, the multiple-quantum coherence is very sensitive to homonuclear as well as long-range heteronuclear interactions. Unwanted magnetization transfer due to these interactions can reduce the sensitivity back to the level of a single-quantum experiment and, for some spin moieties, even eliminate the signal completely. In the present paper we show that a modified HCN scheme that refocuses the interfering coherences improves sensitivity routinely by a factor of 1.5 to 4 over a nonselective experiment. In addition, novel multiple-quantum 2D and 3D HCNCH experiments with substantially enhanced sensitivity are presented. 相似文献
8.
Moon YH Nam SH Kang J Kim YM Lee JH Kang HK Breton V Jun WJ Park KD Kimura A Kim D 《Applied microbiology and biotechnology》2007,77(3):559-567
Two arbutin glucosides were synthesized via the acceptor reaction of a glucansucrase from Leuconostoc mesenteroides B-1299CB with arbutin and sucrose. The glucosides were purified by Bio-gel P-2 column chromatography and high-performance
liquid chromatography, and the structures were elucidated as 4-hydroxyphenyl β-isomaltoside (arbutin-G1), 4-hydroxyphenyl
β-isomaltotrioside (arbutin-G2), according to the results of 1H, 13C, heteronuclear single-quantum coherence, 1H-1H COSY, and heteronuclear multiple-bond correlation analyses. Arbutin glucoside (4-hydroxyphenyl β-isomaltoside) exhibited
slower effects on 1,1-diphenyl-2-picrylhydrazyl radical scavenging and similar effects on tyrosinase inhibition, and increased
inhibitory effect on matrix metalloproteinase-1 production induced by UVB than arbutin.
Young Hwan Moon and Seung Hee Nam contributed equally to this work. 相似文献
9.
Summary The simultaneous acquisition of a 4D gradient-enhanced and sensitivity-enhanced [13C,15N]/[15N,15N]-separated NOESY is presented for the 74-residue [13C,15N]-labeled N-terminal SH3 domain of mGrb2 complexed with a peptide gragment from mSOS-2 in 90% H2O. The method readily accommodates different 13C and 15N spectral widths, but requires that the same number of increments be collected for both 13C and 15N in the simultaneous dimension (F2). For purposes of display and analysis, the two 4D spectra can be deconvolved during the processing stage by the appropriate linear combination of separately stored FIDs. Compared to collecting each of these two 4D data sets separately, the presented method is a factor (2)1/2 more efficient in sensitivity per unit acquisition time. The interleaved nature of this method may also lead to improved peak registration between the two 4D spectra. 相似文献
10.
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
11.
Summary Novel strategies for sensitivity enhancement in heteronuclear multidimensional spectra are introduced and evaluated theoretically and experimentally. It is shown that in 3D sequences employing several Coherence Order Selective Coherence Transfer (COS-CT) steps, enhancement factors of up to 2 can be achieved. This sensitivity enhancement is compatible with the use of heteronuclear gradient echoes, yielding spectra with excellent water suppression. HNCO and HCCH-TOCSY pulse sequences are proposed and experimentally tested. These experiments employ recently developed coherence order selective pulse sequence elements, e.g., COS-INEPT and planar TOCSY for antiphase to in-phase transfers 2F-S2S- or in-phaseaCOS-CT for in-phase transfer F-S-, and the well-known isotropic TOCSY mixing sequences for homo- and heteronuclear in-phase transfer.This work has been presented in part at the 35th ENC, April 10–15, 1994, Asilomar, CA, U.S.A. 相似文献
12.
Emmanuel Maes Doina Florea Florence Delplace Jerome Lemoine Yves Plancke Gerard Strecker 《Glycoconjugate journal》1997,14(1):127-146
The carbohydrate chains of the mucins which constitute the jelly coat surrounding the eggs of Rana temporaria were released by alkaline borohydride treatment. Neutral and acidic oligosaccharide-alditols were purified by ion-exchange chromatography and HPLC. From the structural analysis, based upon 1H and 13C-NMR spectroscopy in combination with MALDI-TOF, the following glycan units are proposed. Abbreviations: MALDI-TOF, matrix assisted laser desorption ionization - time of flight; HPLC, high performance liquid chromatography; COSY, correlation spectroscopy; HSQC, heteronuclear single-quantum coherence spectroscopy; HMQC, heteronuclear multiple-quantum coherence spectroscopy; ROESY, rotating-frame overhauser enhancement spectroscopy; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; GalNAc-ol, N-acetylgalactosaminitol; GlcA, glucuronic acid 相似文献
13.
Summary NMR (nuclear magnetic resonance) spectroscopy was used to identify metabolic solutes in one normal and two habituated sugarbeet cell lines (Beta vulgaris L.altissima) obtained from the same mother strain. This technique was applied to investigate the intracellular naturally occurring13C isotopes (1.1% of total natural carbon) in living sugarbeet suspension cells and perchloric cell extracts. A combination of1H,13C, double-quantum filter correlation spectroscopy, heteronuclear multiple-bond correlation, and heteronuclear multiple-quantum coherence spectra from perchloric cell extracts enabled us to identify the main compounds in the different extract solutions. This was verified by spiking the solutions with small amounts of reference compounds to exclude the influence exerted by pH on the chemical shifts of the different compounds in the1H and13C spectra. The comparison of the three sugarbeet cell lines' NMR spectra showed the presence of sucrose, glucose, and fructose in the three strains. On the other hand, it revealed a strong discrepancy between metabolic solutes. Spectra from the habituated lines showed the presence of glutamine. Some amino acids such as alanine or valine, and unidentified signals corresponding to aromatic rings were only characterized in the habituated nonorganogenic cells. On the basis of these13C NMR data we assumed that the discrepancy between the different sugarbeet cell lines could be due to an increase in the metabolic activity of the habituated cell lines in relation to their autonomous growth.Abbreviations DQF-COSY
double-quantum filter correlation spectroscopy
- HO
habituated organogenous
- HNO
habituated nonorganogenous
- HMBC
heteronuclear multiple-bond correlation
- HMQC
heteronuclear multiple-quantum coherence
- N
normal
- NMR
nuclear magnetic resonance
- TSP
sodium tetradeutero-3-(trimethylsilyl)-propionate 相似文献
14.
Ouwen Zhang Lewis E. Kay J. Paul Olivier Julie D. Forman-Kay 《Journal of biomolecular NMR》1994,4(6):845-858
Summary The backbone 1H and 15N resonances of the N-terminal SH3 domain of the Drosophila signaling adapter protein, drk, have been assigned. This domain is in slow exchange on the NMR timescale between folded and predominantly unfolded states. Data were collected on both states simultaneously, on samples of the SH3 in near physiological buffer exhibiting an approximately 1:1 ratio of the two states. NMR methods which exploit the chemical shift dispersion of the 15N resonances of unfolded states and pulsed field gradient water suppression approaches for avoiding saturation and dephasing of amide protons which rapidly exchange with solvent were utilized for the assignment.Abbreviations 2D, 3D
two-, three-dimensional
- drkN SH3
N-terminal SH3 domain of Drosophila drk
- HSQC
heteronuclear single-quantum spectroscopy
- NOE
nuclear Overhauser enhancement
- SH3
Src homology domain 3
- TOCSY
total correlation spectroscopy 相似文献
15.
Summary New pulse sequences are introduced and discussed that allow for simultaneous acquisition of 15N,1H-and 13C,1H-HSQC correlations for fully 13C,15N-labeled biomacromolecules in combination with hetero-nuclear gradient echoes and sensitivity enhancement. The pulse sequence
experimentally found to be optimal can be used as a building block, especially in time-consuming multidimensional NMR experiments.
Due to the excellent solvent suppression obtained by employing heteronuclear gradient echoes, which allows detection of resonances
under the water resonance, it would be possible to record two sensitivity-enhanced 4D experiments simultaneously on one sample
dissolved in H2O, e.g. a 4D 13C,1H-HSQC-NOESY-15N, 1H/13C,1H-HSQC. 相似文献
16.
Summary Sequence-specific 1H and 15N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly 15N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping 1HN chemical shifts were resolved by a three-dimensional 1H-15N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, 3JNH
coupling constants, and 1HN exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI
chemical shift index
- DQF-COSY
double-quantum-filtered correlation spectroscopy
- DIPSI
decoupling in the presence of scalar interactions
- FMN
flavin mononucleotide
- GARP
globally optimized alternating phase rectangular pulse
- HMQC
heteronuclear multiple-quantum coherence
- HSQC
heteronuclear single-quantum coherence
- NOE
nuclear Overhauser effect
- NOESY
nuclear Overhauser enhancement spectroscopy
- TOCSY
total correlation spectroscopy
- TPPI
time-proportional phase increments
- TSP
3-(trimethylsilyl)propionic-2,2,3,3-d
4 acid, sodium salt 相似文献
17.
S. S. Wijmenga H. A. Heus H. A. E. Leeuw H. Hoppe M. van der Graaf C. W. Hilbers 《Journal of biomolecular NMR》1995,5(1):82-86
Summary A new 1H−13C−31P triple resonance experiment is described which allows unambigous sequential backbone assignment in 13C-labeled oligonucleotides via through-bond coherence transfer from 31P via 13C to 1H. The approach employs INEPT to transfer coherence from 31P to 13C and homonuclear TOCSY to transfer the 13C coherence through the ribose ring, followed by 13C to 1H J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route
involves transfer of 31Pi coherence via C4′i and C4′i-1, because of the relatively large J′PC4 couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4′i and C4′i-1 coherences are subsequently transferred to, amongst others, H1′i and H1′i-1, respectively, leading to a 2D 1H−31P spectrum which allows a sequential assignment in the 31P−1H1′ region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on
a 13C-labeled RNA hairpin with the sequence 5′(GGGC-CAAA-GCCU)3′. 相似文献
18.
Weontae Lee Matthew Revington Neil A. Farrow Asao Nakamura Naoko Utsunomiya-Tate Yoko Miyake Masatsune Kainosho Cheryl H. Arrowsmith 《Journal of biomolecular NMR》1995,5(4):367-375
Summary [ul-13C/15N]-l-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (l-Trp) were unambiguously identified from gradient-enhanced 15N–1H HSQC, 13C–1H HSQC, 13C-and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4±0.52 s–1 at 45°C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or breathing of the protein and/or DNA.Abbreviations NOESY
nuclear Overhauser enhancement spectroscopy
- HSQC
heteronuclear single-quantum coherence
- PFG
pulsed field gradient
-
l-Trp
l-tryptophan 相似文献
19.
Summary A simple constant-time 3D heteronuclear NMR pulse sequence has been developed to quantitatively determine the heteronuclear three-bond couplings 3J(HN,C) and 3J(H,C) in uniformly 13C-enriched proteins. The protocols for measuring accurate coupling constants are based on 1H,13C-heteronuclear relayed E.COSY [Schmidt, J.M., Ernst, R.R., Aimoto, S. and Kainosho, M. (1995) J. Biomol. NMR, 6, 95–105] in combination with numerical least-squares spectrum evaluation. Accurate coupling constants are extracted from 2D spectrum projections using 2D multiplet simulation. Confidence intervals for the obtained three-bond coupling constants are calculated from F-statistics. The three-bond couplings are relevant to the determination of and X
1 dihedral-angle conformations in the amino acid backbone and side chain. The methods are demonstrated on the recombinant 13C, 15N-doubly enriched 147-amino acid protein Desulfovibrio vulgaris flavodoxin with bound flavin mononucleotide in its oxidized form. In total, 109 3J(HN,C) and 100 3J(H,C) coupling constants are obtained from a single spectrum.Abbreviations ANOVA
analysis of variances
- COSY
correlated spectroscopy
- E.COSY
exclusive correlation spectroscopy
- FMN
flavin mononucleotide
- HMQC
heteronuclear multiple-quantum coherence
- HSQC
heteronuclear single-quantum coherence 相似文献
20.
A new strategy is demonstrated that simultaneously enhances sensitivity and resolution in three- or higher-dimensional heteronuclear
multiple quantum NMR experiments. The approach, referred to as mixed-time parallel evolution (MT-PARE), utilizes evolution
of chemical shifts of the spins participating in the multiple quantum coherence in parallel, thereby reducing signal losses
relative to sequential evolution. The signal in a given PARE dimension, t
1, is of a non-decaying constant-time nature for a duration that depends on the length of t
2, and vice versa, prior to the onset of conventional exponential decay. Line shape simulations for the 1H–15N PARE indicate that this strategy significantly enhances both sensitivity and resolution in the indirect 1H dimension, and that the unusual signal decay profile results in acceptable line shapes. Incorporation of the MT-PARE approach
into a 3D HMQC-NOESY experiment for measurement of HN–HN NOEs in KcsA in SDS micelles at 50°C was found to increase the experimental sensitivity by a factor of 1.7±0.3 with a concomitant
resolution increase in the indirectly detected 1H dimension. The method is also demonstrated for a situation in which homonuclear 13C–13C decoupling is required while measuring weak H3′–2′OH NOEs in an RNA oligomer.
Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献