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1.
We show that adiabatic fast passage (AFP) pulses are robust refocusing elements of transverse 13C magnetization in multidimensional NMR experiments. A pair of identical AFP pulses can refocus selected parts or a complete 13 C chemical shift range in 13C spectra. In the constant time 13C-1H HSQC, replacement of attenuated rectangular pulses by selective AFP pulses results in a sensitivity enhancement of up to a factor of 1.8. In the 3D CBCA(CO)NH the signal-to-noise ratio is increased by a factor of up to 1.6. 相似文献
2.
Analysis of 2D [13C,1H]-HSQC spectra of biosynthetic fractionally 13C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional 13C labeling yields aromatic rings in which some of the 13C-13C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the 13C constant-time period in 2D [13C,1H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic 13C CSA and 13C-1H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution 13C constant-time spectra with good sensitivity. 相似文献
3.
Resonance assignment is necessary for the comprehensive structure determination of insoluble proteins by solid-state NMR spectroscopy. While various 2D and 3D correlation techniques involving 13C and 15N spins have been developed for this purpose, 1H chemical shift has not been exploited sufficiently. We demonstrate the combination of the regular 1H-13C heteronuclear correlation (HETCOR) experiment and a dipolar filtered HETCOR technique to obtain better resolved 1H chemical shift spectra. The dipolar filtered experiment, MELODI-HETCOR, simplifies the 1H spectra by suppressing the directly bonded C-H correlation peaks and retaining only the medium- and long-range cross peaks. We apply this MELODI-HETCOR technique to several amino acids and proteins with various isotopic labeling patterns. The enhanced 1H chemical shift resolution allows the assignment of overlapping H and H resonances in Ser, identifies the 1H chemical shift differences between neutral and cationic imidazole rings of His, and permits the assignment of residues with side chain nitrogen atoms in ubiquitin. The potential utility of this dipolar filtered HETCOR technique to resonance assignment of extensively labeled proteins is discussed. 相似文献
4.
NMR spectroscopy has great potential to provide us with information on structure and dynamics at atomic resolution of glycoproteins in solution. In larger glycoproteins, however, the detrimental fast 1H transverse relaxation renders the conventional 1H-detected NMR experiments difficult. 13C direct detection potentially offers a valuable alternative to 1H detection to overcome the fast T2 relaxation. Here, we applied 13C-detected NMR methods to observe the NMR signals of 13C-labeled glycans attached to the Fc fragment of immunoglobulin G with a molecular mass of 56 kDa. Spectral analysis revealed that a 13C-detected 13C-13C NOESY experiment is highly useful for spectral assignments of the glycans of large glycoproteins. This approach would be, in part, complementary to 13C-13C TOCSY and 1H-detection experiments. 相似文献
5.
Three improved 13C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(CaliCaro)H-TOCSY, is an HCCH-TOCSY in which all CHn moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the 13C-detected Call-TOCSY, magnetization transfer comprises all carbons—aliphatic, aromatic as well as the carbonyl carbons—making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct 13C–13C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa. 相似文献
6.
Mathias A. S. Hass Ali Yilmaz Hans E. M. Christensen Jens J. Led 《Journal of biomolecular NMR》2009,44(4):225-233
The use of 13C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues
in proteins is investigated. To illustrate the approach, measurements on three specifically 13C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for 13Cε1 nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the
chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from 15N backbone relaxation measurements. Compared to measurements of backbone nuclei, 13Cε1 dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes
of histidine side chains or their environment. Advantages and shortcomings of using the 13Cε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics
of the histidine side chains are discussed. 相似文献
7.
Bertini I Felli IC Kümmerle R Luchinat C Pierattelli R 《Journal of biomolecular NMR》2004,30(3):245-251
13C-13C NOESY experiments were performed under long mixing time conditions on reduced human superoxide dismutase (32 kDa, 15N, 13C and 70% 2H labeled). 13C-13C couplings were successfully eliminated through post-processing of in-phase-anti-phase (IPAP) data. It appears that at mixing time m of 3.0 s the spin diffusion mechanism allows the detection of 96% of the two-bond correlations involving C and C. The interpretation was confirmed by simulations. This approach broadens the range of applicability of 13C-13C NOESY spectroscopy. 相似文献
8.
13C NMR resonances of 15 simple tetrahydroisoquinolines have been assigned on the basis of chemical shift theory, 13C-1H coupling constants 相似文献
9.
A novel three-dimensional (3D) HCCH NMR experiment is introduced. It involves 13C-13C COSY or TOCSY coherence transfer plus two independent editing steps according to the number of protons attached to the individual carbons before and after the 13C-13C homonuclear mixing. This double editing leads to simplification of HCCH protein side chain spectra that otherwise are prone to spectral overlap. Another interesting feature is amino acid selectivity, i.e. that the presence of certain correlations in a doubly edited HCCH subspectrum gives a clue as to assignment to a particular subgroup of amino acids or segments thereof. Finally, the selection of two different multiplicities in the two editing steps leads to diagonal peak suppression in the 1H-1H (3D spectrum recorded with two 1H and one 13C dimension) or the 13C-13C (3D spectrum recorded with one 1H and two 13C dimensions) two-dimensional projection. The new experiment is demonstrated using a 13C,15N-labeled protein sample, chymotrypsin inhibitor 2, at 500 MHz. 相似文献
10.
Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta 总被引:2,自引:0,他引:2
A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response. NOEs between aliphatic and/or aromatic protons are first spread out into a third dimension by the 13C chemical shift of the carbon atom attached to the originating proton and subsequently into a fourth dimension by the 13C chemical shift of the carbon atom attached to the destination proton. Thus, each NOE cross peak is labeled by four chemical shifts. By this means, ambiguities in the assignment of NOEs that arise from chemical shift overlap and degeneracy are completely removed. Further, NOEs between protons with the same chemical shifts can readily be detected providing their attached carbon atoms have different 13C chemical shifts. The design of the pulse sequence requires special care to minimize the level of artifacts arising from undesired coherence transfer pathways, and in particular those associated with "diagonal" peaks which correspond to magnetization that has not been transferred from one proton to another.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully13 C/15N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue13 C-15N correlation experiments with13 C-13C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonances in solid proteins. The performance of sequences using 15N instead of13 C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the 15N and 13C resonances in human ubiquitin. 相似文献
12.
David S. Wishart Colin G. Bigam Arne Holm Robert S. Hodges Brian D. Sykes 《Journal of biomolecular NMR》1995,5(1):67-81
Summary In this study we report on the 1H, 13C and 15N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide
Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the
possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because
all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide
a complete and internally consistent set of 1H, 13C and 15N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor
effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical
shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences
arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring
for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid
substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in
determining peptide and protein chemical shifts. 相似文献
13.
Artifacts associated with the measurement of methyl 1H single quantum CPMG-based relaxation dispersion profiles are described. These artifacts arise due to the combination of cross-correlated spin relaxation effects involving intra-methyl 1H–1H dipolar interactions and imperfections in 1H refocusing pulses that are applied during CPMG intervals that quantify the effects of chemical exchange on measured transverse relaxation rates. As a result substantial errors in extracted exchange parameters can be obtained. A simple work-around is presented where the 1H chemical shift difference between the exchanging states is extracted from a combination of 13C single quantum and 13C–1H multiple quantum dispersion profiles. The approach is demonstrated with an application to a folding/unfolding reaction involving a G48M mutant Fyn SH3 domain. 相似文献
14.
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
15.
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
16.
John P. Marino Harald Schwalbe Clemens Anklin Wolfgang Bermel Donald M. Crothers Christian Griesinger 《Journal of biomolecular NMR》1995,5(1):87-92
Summary A three-dimensional 1H,13C,31P triple resonance experiment, HCP-CCH-TOCSY, is presented which provides unambiguous through-bond correlation of all 1H ribose protons on the 5′ and 3′ sides of the intervening phosphorus along the backbone bonding network in 13C-labeled RNA oligonucleotides. The correlation of the complete ribose spin system to the intervening phosphorus is obtained
by adding a C,C-TOCSY coherence transfer step to the triple resonance HCP experiment. The C,C-TOCSY transfer step, which utilizes
the large and relatively uniform 1J(C,C) coupling constant (∼40 Hz for ribose carbons), efficiently correlates the phosphorus-coupled carbons observed in the
HCP correlation experiment (i.e., C4′ and C5′ in the 5′ direction and C4′ and C3′ in the 3′ direction) to all other carbons
in the ribose spin system. Of the additional correlations observed in the HCP-CCH-TOCSY, that to the relatively well-resolved
anomeric H1′, C1′ resonance pairs provides the greatest gain in terms of facilitating assignment. The gain in spectral resolution
afforded by chemical shift labeling with the anomeric resonances should provide a more robust pathway for sequential assignment
over the intervening phosphorus in larger RNA oligonucleotides. The HCP-CCH-TOCSY experiment is demonstrated on a uniformly
13C,15N-labeled 19-nucleotide RNA stem-loop, derived from the antisense RNA I molecule found in the ColE1 plasmid replication control system. 相似文献
17.
Charles M. Stevens Mark Okon Lawrence P. McIntosh Mark Paetzel 《Biomolecular NMR assignments》2013,7(2):193-197
Herein are reported the mainchain 1H, 13C and 15N chemical shift assignments and amide 15N relaxation data for Escherichia coli DmsD, a 23.3 kDa protein responsible for the correct folding and translocation of the dimethyl sulfoxide reductase enzyme complex. In addition, the observed amide chemical shift perturbations resulting from complex formation with the reductase subunit DmsA leader peptide support a model in which the 44 residue peptide makes extensive contacts across the surface of the DmsD protein. 相似文献
18.
An understanding of side chain motions in protein is of great interest since side chains often play an important role in protein folding and intermolecular interactions. A novel method for measuring the dynamics of methyl groups in uniformly 13C-, 15N-labeled proteins has been developed by our group. The method relies on the difference in peak intensities of 13C quartet components of methyl groups, in a spectrum recording the free evolution of 13C under proton coupling in a constant-time period. Cross-correlated relaxation rates between 13C-1H dipoles can be easily measured from the intensities of the multiplet components. The degree of the methyl restrictions (S 2) can be estimated from the cross-correlated relaxation rate. The method is demonstrated on a sample of human fatty acid binding protein in the absence of fatty acid. We obtained relaxation data for 33 out of 46 residues having methyl groups in apo-IFABP. It has been found that the magnitude of the CSA tensor of spin 13C in a methyl group could be estimated from the intensities of the 13C multiplet components. 相似文献
19.
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
20.
Lindsay J. Sperling Terry L. Sasser Chad M. Rienstra 《Journal of molecular biology》2010,399(2):268-15379
We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to ∼ 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-13C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-13C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional 13C-13C spectrum. 相似文献