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1.
We present two time-shared experiments that enable the characterization of all nOes in 1H–13C-ILV methyl-labelled proteins that are otherwise uniformly deuterated and 15N enriched and possibly selectively protonated for distinct residue types. A 3D experiment simultaneously provides the spectra
of a 3D NOESY-HN-TROSY and of a 3D NOESY-HC-PEP-HSQC. Thus, nOes from any protons to methyl or amide protons are dispersed
with respect to 15N and 13C chemical shifts, respectively. The single 4D experiment presented here yields simultaneously the four 4D experiments HC-HSQC-NOESY-HC-PEP-HSQC,
HC-HSQC-NOESY-HN-TROSY, HN-HSQC-NOESY-HN-TROSY and HN-HSQC-NOESY-HC-PEP-HSQC. This allows for the unambiguous determination
of all nOes involving amide and methyl protons. The method was applied to a (1H,13C)-ILV−(1H)-FY-(U−2H,15N) sample of a 37 kDa di-domain of the E. coli enterobactin synthetase module EntF. 相似文献
2.
Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D Haro-NOESY-CH3NH and 3D Caro-NOESY-CH3NH, based on a modification of a technique for simultaneous detection of 13C-1H (of CH3) and 15N-1H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the 13C and 15N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH2 moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations. 相似文献
3.
The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-2H,15N,13C; Ile??1-{13CH3}; Leu,Val-{13CH3/12CD3}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-??YRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40?°C to improve the sensitivity of the measurements and alleviate the overlap of 1H?C15N correlations in the abundant ??-helical segments of the protein. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of 1H?C15N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D 1H?C15N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected ??out-and-back?? HMCM(CG)CBCA experiment, and (3) strong sequential HN?CHN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578?C195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments. 相似文献
4.
This paper describes a [15N,1H]/[13C,1H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide 15N–1H groups, side chain 15N–1H2 groups and aromatic 13C–1H groups in otherwise highly deuterated proteins. The 15N–1H and 13C–1H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the 1H dimension. The side-chain 15N–1H2 groups, which are suppressed in conventional [15N,1H)-TROSY, are observed with high sensitivity in the 15N–1H subspectrum. [15N,1H]/[13C,1H]-TROSY was used as the heteronuclear correlation block in a 3D [1H,1H]-NOESY-[15N,1H]/[13C,1H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain 15N–1H2 groups. 相似文献
5.
Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection 总被引:1,自引:1,他引:0
A pair of HN-methyl NOESY experiments that are based on simultaneous TROSY-type detection of amide and methyl groups is described.
The preservation of cross-peak symmetry in the simultaneous 1H–15N/13CH3 NOE spectra enables straightforward assignments of HN-methyl NOE cross-peaks in large and complex protein structures. The
pulse schemes are designed to preserve the slowly decaying components of both 1H–15N and methyl 13CH3 spin-systems in the course of indirect evolution (t
2) and acquisition period (t
3) of 3D NOESY experiments. The methodology has been tested on {U-[15N,2H]; Ileδ1-[13CH3]; Leu,Val-[13CH3,12CD3]}-labeled 82-kDa enzyme Malate Synthase G (MSG). A straightforward procedure that utilizes the symmetry of NOE cross-peaks
in the time-shared 3D NOE data sets allows unambiguous assignments of more than 300 HN-methyl interactions in MSG from a single
3D data set providing important structural restraints for derivation of the backbone global fold. 相似文献
6.
Shi P Xi Z Wang H Shi C Xiong Y Tian C 《Biochemical and biophysical research communications》2010,402(3):461-466
SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for 15N/19F-trifluoromethyl-phenylalanine (15N/19F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide (15N)1H and side-chain 19F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide (15N)1H and side-chain 19F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes. 相似文献
7.
We present a gradient selected and doubly sensitivity-enhanced DE-MQ–(H)CC
m
H
m
–TOCSY experiment for the sequence-specific assignment of methyl resonances in 13C,15N labeled proteins. The proposed experiment provides improved sensitivity and artifact suppression relative to the phase-cycled experiments. One part of the 13Cchemical shift evolution takes place under heteronuclear multiple quantum coherence, whereas the other part occurs under 13C single quantum coherence in a semi-constant time fashion. The feasibility of the experiment was assessed using 15N,13C labeled Mus musculus coactosin (16 kDa), having a rotational correlation time of 14.5 ns at 15 °C in D2O. A 16-h experiment on 600 MHz 1H yielded good quality data and enabled the assignment of 70 out of 72 methyl groups in coactosin. As well as being an improved approach for methyl resonance assignment, this experiment can also be highly valuable for the rapid assignment of methyl resonances in SAR by NMR studies. 相似文献
8.
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
9.
The 1H, 13C, and 15N backbone resonance assignments have been made for the Src homology 2 (SH2) domain of the human molecular adapter protein Grb14. The assignments, along with the majority of the non-aromatic side-chain 1H and 13C resonances are reported. The SH2 domain has been complexed with a phosphotyrosine-containing peptide (pY766) corresponding to the putative binding site in the fibroblast growth factor receptor (FGFR1). Chemical shift changes upon binding are also reported. 相似文献
10.
Van Melckebeke H Pardi A Boisbouvier J Simorre JP Brutscher B 《Journal of biomolecular NMR》2005,32(4):263-271
New base-type-edited transverse-relaxation optimized CT-HCN(C) experiments are presented that yield intra-base and sugar-to-base
correlations for 13C−15N labeled RNA. High spectral resolution in the 13C and 15N dimensions is achieved by constant time (CT) frequency editing. A spectral editing filter applied during the CT 15N labeling period separates the correlation peaks arising from G/U and A/C nucleotide bases. This provides the increased spectral
resolution required to unambiguously connect the 1H and 13C resonances in sugar and base moieties of RNA nucleotides. In addition, the experiment allows base type identification for
each residue, and therefore presents an attractive spectroscopic alternative to nucleotide-specific isotope labeling. Application
to a 33-nucleotide RNA aptamer demonstrates the performance of the new pulse scheme.
Electronic supplementary material Electronic supplementary material is available for this article at
and accessible for authorised users. 相似文献
11.
Two novel 3D 13C-detected experiments, hNcocaNCO and hnCOcaNCO, are proposed to facilitate the resonance assignment of intrinsically disordered proteins. The experiments correlate the 15N and 13C′ chemical shifts of two consecutive amide moieties without involving other nuclei, thus taking advantage of the good dispersion shown by the 15N–13C′ correlations, even for proteins that lack a well defined tertiary structure. The new pulse sequences were successfully tested using Nupr1, an intrinsically disordered protein of 93 residues. 相似文献
12.
van Rossum BJ Castellani F Pauli J Rehbein K Hollander J de Groot HJ Oschkinat H 《Journal of biomolecular NMR》2003,25(3):217-223
In this paper, we present a strategy for the 1HN resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the -spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue HN-N-C correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D (1H-15N) and 3D (1H-15N-13C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone 15N resonances and as an aid in the amide proton assignment. 相似文献
13.
D.R. Muhandiram Philip E. Johnson Daiwen Yang Ouwen Zhang Lawrence P. McIntosh Lewis E. Kay 《Journal of biomolecular NMR》1997,10(3):283-288
A triple-resonance pulse scheme is described which records15N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a15N, 13C and fractionally deuterated proteinsample and selects for CH2D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone 15N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens. 相似文献
14.
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
15.
We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone 1Hα and 13Cα to those of 13C′ in the preceding residue. By applying 2H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO,
HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone 13C′ and amide 15N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for 13C/15N-labeled GB1 dissolved in 2H2O. The quality of the GB1 structure determined in 2H2O was similar to that determined in H2O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures
in 2H2O or H2O at high pH values. 相似文献
16.
A general method for assigning NMR spectra of denatured proteins using 3D HC(CO)NH-TOCSY triple resonance experiments 总被引:11,自引:4,他引:11
Timothy M. Logan Edward T. Olejniczak Robert X. Xu Stephen W. Fesik 《Journal of biomolecular NMR》1993,3(2):225-231
Summary A general approach for assigning the resonances of uniformly 15N- and 13C-labeled proteins in their unfolded state is presented. The assignment approach takes advantage of the spectral dispersion of the amide nitrogen chemical shifts in denatured proteins by correlating side chain and backbone carbon and proton frequencies with the amide resonances of the same and adiacent residues. The 1H resonances of the individual amino acid spin systems are correlated with their intraresidue amide in a 3D 15N-edited 1H, 1H-TOCSY-HSQC experiment, which allows the spin systems to be assigned to amino acid type. The spin systems are then linked to the adjacent i-1 spin system using the 3D H(C)(CO)NH-TOCSY experiment. Complete 13C assignments are obtained from the 3D (H)C(CO)NH-TOCSY experiment. Unlike other methods for assigning denatured proteins, this approach does not require previous knowledge of the native state assignments or specific interconversion rates between the native and denatured forms. The strategy is demonstrated by assigning the 1H, 13C, and 15N resonances of the FK506 binding protein denatured in 6.3 M urea. 相似文献
17.
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
18.
Sunil Prasannan R. Bryn Fenwick Louise J. Campbell Katrina A. Evetts Daniel Nietlispach Darerca Owen Helen R. Mott 《Biomolecular NMR assignments》2007,1(1):147-149
We report 1H, 15N and 13C resonance assignments for the small G protein RalB in its active conformation. Backbone amide dynamics parameters for a
majority of residues have also been obtained. The BMRB accession code is 15320.
An erratum to this article can be found at 相似文献
19.
Kevin H. Gardner Robert Konrat Michael K. Rosen Lewis E. Kay 《Journal of biomolecular NMR》1996,8(3):351-356
Summary A biosynthetic strategy has recently been developed for the production of 15N, 13C, 2H-labeled proteins using 1H3C-pyruvate as the sole carbon source and D2O as the solvent. The methyl groups of Ala, Val, Leu and Ile (2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a 15N, 13C, 2H, 1H3C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range. 相似文献
20.
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