共查询到20条相似文献,搜索用时 15 毫秒
1.
Vladimír Sklená >r Robert D. Peterson Marita R. Rejante Juli Feigon 《Journal of biomolecular NMR》1994,4(1):117-122
Summary The advent of methods for preparing 15N- and 13C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the 13C label. It may therefore sometimes be desirable to prepare a relatively inexpensive 15N-labeled sample only. Here we show that the two-bond 1H-15N HSQC experiment can be used on 15N-labeled RNA to correlate the intranucleotide H1 and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected 1H-15N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1 and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained. 相似文献
2.
Boisbouvier J Bryce DL O'neil-Cabello E Nikonowicz EP Bax A 《Journal of biomolecular NMR》2004,30(3):287-301
New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond 2DCH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in 13C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear 1H-1H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven 1H-13C and 13C-13C couplings are measured for pyrimidines (U and C), including 1DC5H5, 1DC6H6, 2DC5H6, 2DC6H5, 1DC5C4, 1DC5C6, and 2DC4H5. For adenine, four base couplings (1DC2H2, 1DC8H8, 1DC4C5, and 1DC5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (1DC8H8, 1DC4C5, and 1DC5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide. 相似文献
3.
Sebastian Meier Bent O. Petersen Jens
. Duus Ole W. Srensen 《Carbohydrate research》2009,344(16):2274-2278
The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of 13C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the 1H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution. 相似文献
4.
The structure of the 13C,15N-labeled d(GCGAAGC) hairpin, as determined by NMR spectroscopy and refined using molecular dynamics with NOE-derived distances, torsion angles, and residual dipolar couplings (RDCs), is presented. Although the studied molecule is of small size, it is demonstrated that the incorporation of diminutive RDCs can significantly improve local structure determination of regions undefined by the conventional restraints. Very good correlation between the experimental and back-calculated small one- and two-bond 1H-13C, 1H-15N, 13C-13C and 13C-15N coupling constants has been attained. The final structures clearly show typical features of the miniloop architecture. The structure is discussed in context of the extraordinary stability of the d(GCGAAGC) hairpin, which originates from a complex interplay between the aromatic base stacking and hydrogen bonding interactions. 相似文献
5.
Wienk HL Martínez MM Yalloway GN Schmidt JM Pérez C Rüterjans H Löhr F 《Journal of biomolecular NMR》2003,25(2):133-145
A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone 1
J
NC, 1
J
NC, and 2
J
NC coupling constants are obtained for the two [15N;13C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [2H;15N;13C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J
NC values on protein backbone torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated 15N-13C residual dipolar couplings. 相似文献
6.
The solution structure of d(CGCGAATTCGCG)2 has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear 13C-1H and 15N-1H and qualitative homonuclear 1H-1H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative 1H-1H and 22 31P-1H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 31P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of 13C-1H and 15N-1H dipolar couplings. In the newly calculated structures, 31P-1H dipolar and 3Jsub
H3
P
sub couplings and 31P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of 10°, which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2-endo and C3-endo deoxyribose puckers (sugar switching). The C2-H2/H2 dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR JHH couplings, with a population of the minor C3-endo form higher for pyrimidines than for purines. 相似文献
7.
A suite of spin-state-selective excitation (S3E) NMR experiments for the measurements of small one-bond (13C-13C, 15N-13C) and two-bond (1H-13C, 1H-15N) coupling constants in 13C,15N labeled purine and pyrimidine bases is presented. The incorporation of band-selective shaped pulses, elimination of the cross talk between and sub-spectra, and accuracy and precision of the proposed approach are discussed. Merits of using S3E rather than /-half-filter are demonstrated using results obtained on isotopically labeled DNA oligonucleotides. 相似文献
8.
Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme
Eletsky A Heinz T Moreira O Kienhöfer A Hilvert D Pervushi K 《Journal of biomolecular NMR》2002,24(1):31-39
A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of 15N-15N and 1H-15N trans-hydrogen bond scalar couplings, 2h
J
NN and 1h
J
HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models. 相似文献
9.
Vladimír Sklenář Robert D. Peterson Marita R. Rejante Juli Feigon 《Journal of biomolecular NMR》1993,3(6):721-727
Summary New 2D and 3D 1H-13C-15N triple resonance experiments are presented which allow unambiguous assignments of intranucleotide H1'-H8(H6) connectivities in 13C-and 15N-labeled RNA oligonucleotides. Two slightly different experiments employing double INEPT forward and back coherence transfers are optimized to obtain the H1'-C1'-N9/N1 and H8/H6-C8/C6-N9/N1 connectivities, respectively. The correlation of H1' protons to glycosidic nitrogens N9/N1 is obtained in a nonselective fashion. To correlate H8/H6 with their respective glycosidic nitrogens, selective 13C-refocusing and 15N-inversion pulses are applied to optimize the magnetization transfers along the desired pathway. The approach employs the heteronuclear one-bond spin-spin interactions and allows the 2D 1H-15N and 3D1H-13C-15N chemical shift correlation of nuclei along and adjacent to the glycosidic bond. Since the intranucleotide correlations obtained are based exclusively on through-bond scalar interactions, these experiments resolve the ambiguity of intra-and internucleotide H1'-H8(H6) assignments obtained from the 2D NOESY spectra. These experiments are applied to a 30-base RNA oligonucleotide which contains the binding site for Rev protein from HIV. 相似文献
10.
Direct measurement of 1H-1H dipolar couplings in proteins: A complement to traditional NOE measurements 总被引:1,自引:0,他引:1
Tian F Fowler CA Zartler ER Jenney FA Adams MW Prestegard JH 《Journal of biomolecular NMR》2000,18(1):23-31
An intensity-based constant-time COSY (CT-COSY) method is described for measuring 1H-1H residual dipolar couplings of proteins in weakly aligned media. For small proteins, the overall sensitivity of this experiment is comparable to the NOESY experiment. In cases where the 1H-1H distances are defined by secondary structure, such as 1H-1HN and 1HN-1HN sequential distances in -helices and -sheets, these measurements provide useful orientational constraints for protein structure determination. This experiment can also be used to provide distance information similar to that obtained from NOE connectivities once the angular dependence is removed. Because the measurements are direct and non-coherent processes, such as spin diffusion, do not enter, the measurements can be more reliable. The 1/r
3 distance dependence of directly observed dipolar couplings, as compared with the 1/r
6 distance dependence of NOEs, also can provide longer range distance information at favorable angles. A simple 3D, 15N resolved version of the pulse sequence extends the method to provide the improved resolution required for application to larger biomolecules. 相似文献
11.
NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings 总被引:1,自引:0,他引:1
A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar 1H-1H coupling constants with respect to the sign of one-bond 1H-X coupling constants in linear three-spin systems X-1H-1H, where X = 13C or 15N. In contrast to the original JHH-TOCSY experiments, the signs of J
HH couplings may be determined for CH2-CH2 moieties and for uniformly 13C/15N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum. 相似文献
12.
Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination. However, partial orientation of protein samples also causes severe 1 H line broadening resulting from residual 1 H-1H dipolar couplings. In this communication we show that band-selective 1H homonuclear decoupling during data acquisition is an efficient way to suppress residual 1H-1H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment. As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond 1 HN-15N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles. 相似文献
13.
Permi P 《Journal of biomolecular NMR》2002,22(1):27-35
Recently, a set of selective 1D experiments with spin-state-selective excitation for CH spin systems was introduced by Parella and Belloc (J. Magn. Reson.,
148, 78–87 (2001)). We have expanded and generalized this concept further, and demonstrated that a very simple experiment utilizing spin-state-selective filtering can be used for simultaneous measurement of heteronuclear 1
J
NH (or 1
J
CH) and geminal 2
J
HH couplings from two-dimensional 15N-1H (or 13C-1H) correlation spectrum. The experiment has very high sensitivity owing to the preservation of equivalent coherence transfer pathways analogous to the sensitivity and gradient enhanced HSQC experiment. However, overall length of the pulse sequence is 1/(2J) shorter than the gradient selected SE-HSQC experiment. Furthermore, the spin-state-selection can be utilized between NH and NH2 (or CH and CH2) moieties by changing the phase of only one pulse. The pulse scheme will be useful for the measurement of scalar and residual dipolar couplings in wide variety of samples, due to its high sensitivity and artifact suppression efficiency. The method is tested on NH2 and CH2 moieties in 15N- and 15N/13C–labeled ubiquitin samples. 相似文献
14.
Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.COSY-type methods designed to measure both the sign and magnitude of long-range 1H-19F dipolar couplings in selectively fluorine labeled RNA oligonucleotides oriented in solution by a liquid crystalline medium. The techniques for measuring 1H-19F dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at the 2-hydroxyl position of three ribose sugars. Experimentally measured 1H-19F dipolar couplings for the 2-deoxy-2-fluoro-sugars located in the helical region of the RNA hairpin were found to be in excellent agreement with values predicted using canonical A-form helical geometry, demonstrating that these couplings can provide accurate restraints for the refinement of RNA structures determined by NMR. 相似文献
15.
A novel NMR pulse sequence has been developed that correlates the H2 resonances with the C2 and the N1 (N3) resonances in
adenine nucleobases of 13C, 15N labeled oligonucleotides. The pulse scheme of the new 3D-HNHC experiment is composed of a 2J-15N-HSQC and a 1J-13C-HSQC and utilizes large 2J(H2, N1(N3)) and 1J(H2, C2) couplings. The experiment was applied to a medium-size 13C, 15N-labeled 36mer RNA. It is useful to resolve assignment ambiguities occurring especially in larger RNA molecules due to resonance
overlap in the 1H-dimension. Therefore, the missing link in correlating the imino H3 resonances of the uracils across the AU base pair to
the H8 resonances of the adenines via the novel pulse sequence and the TROSY relayed HCCH-COSY (Simon et al. in J Biomol NMR
20:173–176 2001) is provided.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
In three-dimensional and four-dimensional experiments on doubly labelled proteins not only heteronuclear (13C or 15N) but also proton (1H) frequencies are often indirectly monitored, rather than being directly observed. In this communication we show how in these experiments by overlaying 1H and heteronuclear evolutions one can obtain decreased apparent relaxation rates of 1H signals, yielding improved sensitivity. The new method applies to spin pairs like 1H-15N, as in amide groups, or 1H-13C, as in methine groups of alpha or aromatic systems. 相似文献
17.
A simple spectral editing procedure is described that generates separate subspectra for the methyl 13C-1H3 multiplet components of 1H-13C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields 1H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of 1H-13C J and dipolar couplings. The editing procedure is illustrated for a Ca2+-calmodulin sample in isotropic and liquid crystalline phases. 相似文献
18.
T. Niimi G. Kawai M. Takayanagi T. Noguchi N. Hayashi T. Kohno Y. Muto K. Watanabe T. Miyazawa S. Yokoyama 《Biochimie》1993,75(12)
Imino 15N and 1H resonances of Escherichia coli tRNAlIle were observed in the absence and presence of E coli isoleucyl-tRNA synthetase. Upon complex formation of tRNAlIle with isoleucyl-tRNA synthetase, some imino 15N-1H resonances disappeared, and some others were significantly broadened and/or shifted in the 1H chemical shift, while the others were observed at the same 15H-1H chemical shifts. It was indicated that the binding of tRNAlIle with IleRS affect the following four regions: the anticodon stem, the junction of the acceptor and T stems, the middle of the D stem, and the region where the tertiary base pair connects the T, D, and extra loops. This result is consistent with those of chemical footprinting and site-directed mutagenesis studies. Taken together, these three independent results reveal the recognition mechanism of tRNAlIle by IleRS: IleRS recognizes all the identity determinants distributed throughout the tRNAlIle molecule, which induces changes in the secondary and tertiary structures of tRNAlIle. 相似文献
19.
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
20.
We report the determination of two- and three-bond 1H-15N spin–spin couplings in the nmr spectra of a polypeptide. The 1H- and 15N-nmr spectra of 99.2% 15N-enriched alumichrome have been studied at 360 MHz and 10.1 MHz, repectively. While some 2J and 3J coupling are of the order of 5 Hz, most splitting resulting from the heteronuclear interaction are ?2 Hz, which introduces strigent requirements of spectral resolution. In the 1H spectra these requirements were met by digital deconvolution with a sine bell routine combined with positive exponential filtering. Although the 15N spectra clearly exhibit features of fine structure, mainly because of the intrinsic higher nmir sensitivity of protons, observation of 1H-15N spin–spin couplings was found to be more practical in the 1H than in the 15N spectra. We find that the alumichrome data do not satisfy a simple cyclic relationship linking the heteronuclear couplings to the crystallographic ψ dihedral angles. It is suggested that a formal treatment of the ψ-related interresidue 1H-15N coupling might have to take into account a more complex dependence of the intervening 3J on the overall local electronic structure, which is dependent on ?,ψ, and ω simultaneoulsy. In contrast, our analysis indicates that χ1 can be readily determined from the measurement of the corresponding heteronuclear 3J coupling in the 1Hβ or in the amide 15N resonances. Karplus relationships are proposed that relate this heteronuclear 3J to the corresponding dihedral angle θ and which, on average, yield 相似文献