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1.
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
2.
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
3.
A multiple-quantum 3D HCN-CCH-TOCSY experiment is presented for the assignment of RNA ribose resonances. The experiment makes use of the chemical shift dispersion of N1 of pyrimidine and N9 of purine to distinguish the ribose spin systems. It provides an alternative approach for the assignment of ribose resonances to the currently used COSY- and TOCSY-type experiments in which either 13C or 1H is utilized to distinguish the different spin systems. Compared to the single-quantum version, the sensitivity of the multiple-quantum HCN-CCH-TOCSY experiment is enhanced on average by a factor of 2 for a 23-mer RNA aptamer complexed with neomycin. 相似文献
4.
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
5.
A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on 15N, natural abundance 13C samples. The experiment exploits the fact that transfers of magnetization from 15N to 13CO and from 15N to 13C (and back) proceed independently for samples that are not enriched in 13C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM 15N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz. 相似文献
6.
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
7.
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
8.
A methyl-detected ‘out-and-back’ NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts, SIM-HMCM(CGCBCA)CO, is described. The developed pulse-scheme serves the purpose of convenience in recording a single data set for all Ileδ1, Leuδ and Valγ (ILV) methyl positions instead of acquiring two separate spectra selective for valine or leucine/isoleucine residues. The SIM-HMCM(CGCBCA)CO experiment can be used for ILV methyl assignments in moderately sized protein systems (up to ~100 kDa) where the backbone chemical shifts of 13Cα, 13Cβ and 13CO are known from prior NMR studies and where some losses in sensitivity can be tolerated for the sake of an overall reduction in NMR acquisition time. 相似文献
9.
Christopher J. Falzone John Cavanagh Marlon Cowart Arthur G. Palmer III C. Robert Matthews Stephen J. Benkovic Peter E. Wright 《Journal of biomolecular NMR》1994,4(3):349-366
Summary By using fully 15N- and 15N/13C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific 1H and 15N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the 13C resonances in the binary folate complex. These assignments were made through a variety of three-dimensional proton-detected 15N and 13C experiments. A smaller but significant subset of side-chain 1H and 13C assignments were also determined. In this complex, only one 15N or 13C resonance was detected per 15N or 13C protein nucleus, which indicated a single conformation. Proton-detected 13C experiments were also performed with unlabeled DHFR, complexed with 13C-7/13C-9 folate to probe for multiple conformations of the substrate in its binary complex. As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7. These results are consistent with an earlier report based on 1H NMR data [Falzone, C.J. et al. (1990) Biochemistry, 29, 9667–9677] and suggest that the E. coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex. This feature of the E. coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date. 相似文献
10.
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
11.
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
12.
Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz 15N-13CO-13Cα and 15N-13Cα-13CX correlation spectra, using uniformly 13C, 15N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic 15N-13Cα-13Cβ side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively 13C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Cα and Cβ resonances indicated the presence of typical α-helical secondary structure, consistent with previous studies. 相似文献
13.
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
14.
Wayne Boucher Ernest D. Laue Sharon L. Campbell-Burk Peter J. Domaille 《Journal of biomolecular NMR》1992,2(6):631-637
Summary We recently proposed a novel 4D NMR strategy for the assignment of backbone nuclei in13C/15N-labelled proteins (Boucher et al., 1992). Intra-residue (and many sequential) assignments are obtained from a HCANNH experiment, whereas sequential assignments are based on a complementary HCA(CO)NNH experiment. We present here new constant time 4D HCANNH, HCA(CO)NNH and HNCAHA experiments that are more sensitive. Some of the data were presented at the 33rd ENC held at Asilomar, California, U.S.A., in April 1992. 相似文献
15.
Summary A time-shared [15N, 13C] half-filter technique is presented, which can be used to study proton-proton NOEs between biomolecules. The filter is demonstrated in a 2D [15N, 13C] double filtered NOESY experiment of a dimeric Mnt repressor mutant consisting of completely [15N, 13C] labeled monomer and unlabeled monomer. The benefit of this combined [15N, 13C] half-filter is that a single NMR experiment can be designed that yields all NOE interactions between labeled and unlabeled protons ((13C, 14N/12C), (15N, 14N/12C), (12C, 15N/13C) and (14N, 15N/13C)) in the protein, where conventional half-filters would require at least three separate NMR experiments to obtain these NOEs. The intermonomer NOEs of the Mnt mutant confirmed the secondary structure of the DNA-binding domain as an antiparallel ribbon, formed from an N-terminal segment contributed by each monomer. Moreover, several intersubunit NOEs were characterized in the C-terminal part of the Mnt mutant for which no structural data is available yet. 相似文献
16.
Protein-mediated cholesterol trafficking is central to maintaining cholesterol homeostasis in cells. START (Steroidogenic acute regulatory protein-related lipid transfer) domains constitute a sterol and lipid binding motif and the START domain protein StARD4 typifies a small family of mammalian sterol transport proteins. StARD4 consists of a single START domain and has been reported to act as a general cholesterol transporter in cells. However, the structural basis of cholesterol uptake and transport is not well understood and no cholesterol-bound START domain structures have been reported. We have undertaken the study of cholesterol binding and transport by StARD4 using solution state NMR spectroscopy. To this end, we report nearly complete 1H, 15N, and 13C backbone resonance assignments of an inactive but well behaved mutant (L124D) of StARD4. 相似文献
17.
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
18.
Mei Hong 《Journal of biomolecular NMR》1999,15(1):1-14
The comprehensive structure determination of isotopically labeled proteins by solid-state NMR requires sequence-specific assignment of 13C and 15 N spectra. We describe several 2D and 3D MAS correlation techniques for resonance assignment and apply them, at 7.0 Tesla, to 13C and 15N labeled ubiquitin to examine the extent of resonance assignments in the solid state. Both interresidue and intraresidue assignments of the 13C and 15N resonances are addressed. The interresidue assignment was carried out by an N(CO)CA technique, which yields Ni-Ci–1 connectivities in protein backbones via two steps of dipolar-mediated coherence transfer. The intraresidue connectivities were obtained from a new 3D NCACB technique, which utilizes the well resolved C chemical shift to distinguish the different amino acids. Additional amino acid type assignment was provided by a 13C spin diffusion experiment, which exhibits 13C spin pairs as off-diagonal intensities in the 2D spectrum. To better resolve carbons with similar chemical shifts, we also performed a dipolar-mediated INADEQUATE experiment. By cross-referencing these spectra and exploiting the selective and extensive 13 C labeling approach, we assigned 25% of the amino acids in ubiquitin sequence-specifically and 47% of the residues to the amino acid types. The sensitivity and resolution of these experiments are evaluated, especially in the context of the selective and extensive 13C labeling approach. 相似文献
19.
Oktaviani NA Otten R Dijkstra K Scheek RM Thulin E Akke M Mulder FA 《Biomolecular NMR assignments》2011,5(1):79-84
Here we present the 100% complete assignment chemical shift of non-labile 1H, 15N and 13C nuclei of Calbindin D9k P43G. The assignment includes all non-exchangeable side chain nuclei, including ones that are rarely reported, such as LysNζ
as well as the termini. NMR experiments required to achieve truly complete assignments are discussed. To the best of our knowledge
our assignments for Calbindin D9k extend beyond previous studies reaching near-completeness (Vis et al. in Biochem 33:14858–14870, 1994; Yamazaki et al. in J Am Chem Soc 116:6464–6465, 1994; Yamazaki et al. in Biochem 32:5656–5669, 1993b). 相似文献
20.
To determine the three-dimensional solution structure of the calcium bound S100P protein, the backbone and side chain resonance assignments of the S100P protein have been reported based on triple-resonance experiments using uniformly [13C, 15N]-labeled calcium bound protein. 相似文献