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1.
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.
Summary Novel HCCNH TOCSY NMR experiments are presented that provide unambiguous assignment of the exchangeable imino proton resonances by intranucleotide through-bond connectivities to the (assigned) nonexchangeable purine H8 and pyrimidine H6 protons in uniformly 15N-, 13C-labeled RNA oligonucleotides. The HCCNH TOCSY experiments can be arranged as a two-dimensional experiment, correlating solely GH8/UH6 and GH1/UH3 proton resonances (HCCNH), of as three-dimensional experiments, in which additional chemical shift labeling either by GN1/UN3 (HCCNH) or by GC8/UC6 (HCCNH) chemical shifts is introduced. The utility of these experiments for the assignment of relatively large RNA oligonucleotides is demonstrated for two different RNA molecules.To whom correspondence should be addressed.  相似文献   

3.
Summary Sequence-specific backbone 1H and 15N resonance assignments have been made for 95% of the amino acids in sperm whale myoglobin, complexed with carbon monoxide (MbCO). Many assignments for side-chain resonances have also been obtained. Assignments were made by analysis of an extensive series of homonuclear 2D spectra, measured with unlabeled protein, and both 2D and 3D 1H-15N-correlated spectra obtained from uniformly 15N-labeled myoglobin. Patterns of medium-range NOE connectivities indicate the presence of eight helices in positions that are very similar to those found in the crystal structures of sperm whale myoglobin. The resonance assignments of MbCO form the basis for determination of the solution structure and for hydrogen-exchange measurements to probe the stability and folding pathways of myoglobin. They will also form a basis for assignment of the spectra of single-site mutants with altered ligand-binding properties.  相似文献   

4.
Summary Two new 3D 1H-15N-13C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the 2JNC coupling to establish the sequential assignment of backbone NH and 15N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly 15N- and 13C-labelled ribonuclease T1.  相似文献   

5.
6.
Thanks to rather large (5–9 Hz) long-range imino proton-13C J-couplings, heteronuclear correlation experiments in H2O provide unambiguous assignment of imino protons by intranucleotide through-bond connectivities to guanosine H8 and thymidine CH3 protons, or sequence-specific assignment of non-exchangeable protons when the imino protons are identified independently. This method is demonstrated in the Dickerson dodecamer [d(CGCGAATTCGCG)]2 and in a human telomeric fragment of 22 nucleotides.  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.
Summary A set of triple resonance experiments is presented, providing through-bond H2N/HN to H6 connectivities in uridines and cytidines in 13C-/15N-labeled RNAs. These connectivities provide an important link between the sequential assignment pathways for the exchangeable and nonexchangeable proton resonances in nucleic acids. Both 2D and pseudo-3D HNCCCH experiments were applied to a 30-nucleotide lead-dependent ribozyme, known as the leadzyme. The HN to H6 connectivities for three uridines in the leadzyme were identified from one 2D H(NCCC)H experiment, and the H2N to H6 connectivities were identified for seven of the eight cytidines from the combination of a 2D H(NCCC)H and a pseudo-3D H(NCC)CH experiment.  相似文献   

9.
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   

10.
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   

11.
Summary The pulse sequence of a new constant-time 3D triple-resonance experiment, ct-HA[CAN]HN, is presented. This experiment delineates exclusively scalar connectivities and uses 13C15N heteronuclear two-spin coherence to overlay the chemical shift evolution periods of the 13C and 15N nuclei, thereby providing the four resonance frequencies of the -proton, the -carbon, the amide nitrogen, and the amide proton of a given amino acid residue in three dimensions. This experiment promises to be a valid alternative to 4D experiments, providing the same information on intraresidue polypeptide backbone connectivities in 13C-15N-double-labeled proteins.Abbreviations 3D, 4D three-dimensional, four-dimensional - TPPI time-proportional phase incrementation - ct constant-time - rf radiofrequency - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - glutaredoxin(C14S) mutant E. coli glutaredoxin with the cysteine at position 14 replaced by serine  相似文献   

12.
Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated 14N amino acids that are 13C labeled at the carbonyl position to a medium for uniform deuteration and 15N labeling. This has three important benefits over conventional 15N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the 13C labeled amino acid is very sharp since its alpha position is deuterated, (2) the 13C label at the carbonyl position is less prone to scrambling than the 15N at the α-amino position, and (3) the peaks for the 1-13C labeled amino acids can be identified easily from the large intensity reduction in the 1H-15N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

13.
Methods are described to correlate aromatic 1H 2/13C 2 or 1H 1/15N 1 with aliphatic 13C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.  相似文献   

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   

15.
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   

16.
Summary The assignments of1H–15N magnetic resonances of the -cro repressor are presented. Individual15N-amino acids were incorporated into the protein, or it was uniformly labeled with15N. For the13C–15N double-labeling experiments,13C-amino acids were incorporated into the uniformly15N-labeled protein. All the amide1H–15N resonances could be assigned with such specific labeling, and sequential connectivities obtained by two-dimensional (2D)1H–15N reverse correlation spectroscopies and three-dimensional (3D)1H/15N NOESY-HMQC spectroscopy. Conventional 2D1H–1H correlation spectroscopies were applied to the assignment of the side-chain protons. Some of the1H resonance assignments are inconsistent with those previously reported [Weber, P.L., Wemmer, D.E. and Reid, B.R. (1985)Biochemistry,24, 4553–4562]. The sequential NOE connectivities and H-D exchange rates indicate several elements of the secondary structure, including -helices consisting of residues 8–15, 19–25 and 28–37, and three extended strands consisting of residues 4–7, 39–45 and 49–55. Based on several long-range NOEs, the three extended strands could be combined to form an antiparallel -sheet. The amide proton resonances of the C-terminal residues except Ala66 (residues 60–65) were hardly observed at neutral pH, indicating that the arm is flexible. The identified secondary structure elements in solution show good agreement with those in the crystal structure of the cro protein [Anderson, W.F., Ohlendorf, D.H., Takeda, Y. and Matthews, B.W. (1981)Nature,290, 754–758].  相似文献   

17.
利用自编的脉冲程序,采用预饱和和自旋锁定对水峰进行双重抑制的方法,得到了 15N标记蛋白GAL4(62)的2D 1H-15N HSQC、HSQC-NOESY、HSQC-TOCSY谱,并对这几个谱在蛋白质 1H谱的归属中所起的作用进行了讨论.  相似文献   

18.
Summary For methine sites the relaxation rate of 13C-1H two-spin coherence is generally slower than the relaxation rate of the individual 13C and 1H single spin coherences. The slower decay of two-spin coherence can be used to increase the sensitivity and resolution in heteronuclear experiments, particularly those that require correlation of H and C chemical shifts. To avoid dephasing of the two-spin coherence caused by 1H-1H J-couplings, the 1H spin is locked by the application of a weak rf field, resulting in a spin-locked multiple quantum coherence. For a sample of calcium-free calmodulin, use of the multiple quantum approach yields significant signal enhancement over the conventional constant-time 2D HSQC experiment. The approach is applicable to many multidimensional NMR experiments, as demonstrated for a 3D 13C-separated ROESY CT-HMQC spectrum.  相似文献   

19.
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   

20.
Summary Sequence-specific assignments for the 1H and 15N backbone resonances of cellular retinoic acid-binding protein (CRABP), with and without the bound ligand, have been obtained. Most of the side-chain resonances of both apo- and holo-CRABP have also been assigned. The assignments have been obtained using two-dimensional homonuclear and heteronuclear NMR data, and three-dimensional 1H-15N TOCSY-HMQC and NOESY-HMQC experiments. The secondary structure, deduced from nuclear Overhauser effects, amide H/D exchange rates and H chemical shifts, is analogous in both forms of the protein and is completely consistent with a model of CRABP that had been constructed by homology with the crystal structure of myelin P2 protein [Zhang et al. (1992) Protein Struct. Funct. Genet., 13, 87–99]. This model comprises two five-stranded -sheets that form a sandwich or -clam structure, and a short N-terminal helix-turn-helix motif that closes the binding cavity between the two sheets. Comparison of the data obtained for apo- and holo-CRABP indicates that a region around the C-terminus of the second helix is much more flexible in the apo-protein. Our data provide experimental evidence for the hypothesis that the ligand-binding mechanism of CRABP, and of other homologous proteins that bind hydrophobic ligands in the cytoplasm, involves opening of a portal to allow entry of the ligand into the cavity.  相似文献   

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