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1.
Triple resonance HN(COCA)NH pulse sequences for correlating 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins that utilize overlapping coherence transfer periods provide increased sensitivityrelative to pulse sequences that utilize sequential coherence transfer periods. Although theoverlapping sequence elements reduce the overall duration of the pulse sequences, theprincipal benefit derives from a reduction in the number of 180° pulses. Two versions of thetechnique are presented: a 3D (H)N(COCA)NH experiment that correlates 15N(i),1H(i-1), and 15N(i-1) spins, and a 3D HN(COCA)NH experiment that correlates 1H(i), 15N(i),1H(i-1), and 15N(i-1) spins by simultaneously encoding the 1H(i) and 15N(i) chemical shiftsduring the t1 evolution period. The methods are demonstrated on a 13C/15N-enriched sampleof the protein ubiquitin and are easily adapted for application to 2H/13C/15N-enrichedproteins. 相似文献
2.
Summary A simplified scheme for sequential assignment in isotopically enriched proteins is presented. It is based on the standard triple resonance experiments HNCO, HN(CO)CA, HNCA and a modified H(N)CACO correlation, in which both of the HN connectivities to the CA/C pair of residue i and i-1 are observed. The H(N)CACO was tested on uniformly 13C/15N enriched P13 domain of mannose permease (31 kDa). 相似文献
3.
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
4.
Reimond Bernstein Christian Cieslar Alfred Ross Hartmut Oschkinat Jens Freund Tad A. Holak 《Journal of biomolecular NMR》1993,3(2):245-251
Summary An algorithm based on the technique of combinatorial minimization is used for the semi-automated assignment of multidimensional heteronuclear spectra. The program (ALFA) produces the best assignment compatible with the available input data. Even partially misleading or missing data do not seriously corrupt the final assignment. Ambiguous sequences of the possible assignment and all alternatives are indicated. The program can also use additional non-spectroscopic data to assist in the assignment procedure. For example, information from the X-ray structure of the protein and/or information about the secondary structure can be used. The assignment procedure was tested on spectra of mucous trypsin inhibitor, a protein of 107 residues. 相似文献
5.
6.
Summary A new computer-based approach is described for efficient sequence-specific assignment of uniformly 15N-labeled proteins. For this purpose three-dimensional 15N-correlated [1H, 1H]-NOESY spectra are divided up into two-dimensional 1H-1H strips which extend over the entire spectral width along one dimension and have a width of ca. 100 Hz, centered about the amide proton chemical shifts along the other dimension. A spectral correlation function enables sorting of these strips according to proximity of the corresponding residues in the amino acid sequence. Thereby, starting from a given strip in the spectrum, the probability of its corresponding to the C-terminal neighboring residue is calculated for all other strips from the similarity of their peak patterns with a pattern predicted for the sequentially adjoining residue, as manifested in the scalar product of the vectors representing the predicted and measured peak patterns. Tests with five different proteins containing both -helices and -sheets, and ranging in size from 58 to 165 amino acid residues show that the discrimination achieved between the sequentially neighboring residue and all other residues compares well with that obtained with an unguided interactive search of pairs of sequentially neighboring strips, with important savings in the time needed for complete analysis of 3D 15N-correlated [1H, 1H]-NOESY spectra. The integration of this routine into the program package XEASY ensures that remaining ambiguities can be resolved by visual inspection of the strips, combined with reference to the amino acid sequence and information on spin-system types obtained from additional NMR spectra.Abbreviations 1D, 2D, 3D, 4D
one-, two-, three-, four-dimensional
- NOE
nuclear Overhauser enhancement
- NOESY
nuclear Overhauser enhancement spectroscopy
- COSY
correlation spectroscopy
- TOCSY
total correlation spectroscopy 相似文献
7.
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
8.
M. L. Remerowski T. Domke A. Groenewegen H. A. M. Pepermans C. W. Hilbers F. J. M. van de Ven 《Journal of biomolecular NMR》1994,4(2):257-278
Summary
1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the -protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020–9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C and H frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C chemical shifts and TOCSY ladders. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D 15N NOESY-HMQC data and C secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427–445] shows no major differences.Supplementary material available from F.J.M. van de Ven: the source code (PASCAL) for the computer program described in this paper. 相似文献
9.
Summary A new protocol is described for obtaining intraresidual and sequential correlations between carbonyl carbons and amide 1H and 15N resonances of amino acids. Frequency labeling of 13CO spins occurs during a period required for the 13C-15N polarization transfer, leading to an optimized transfer efficiency. In a four-dimensional version of the experiment, 13C chemical shifts are used to improve the dispersion of signals. The resonance frequencies of all backbone nuclei can be detected in a 3D variant in which cross peaks are split along two frequency axes. This pulse scheme is the equivalent of a five-dimensional experiment. The novel pulse sequences are applied to flavodoxin from Desulfovibrio vulgaris. 相似文献
10.
Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins 总被引:1,自引:0,他引:1
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
11.
Thomas Szyperski Bogdan Banecki Daniel Braun Ralf W. Glaser 《Journal of biomolecular NMR》1998,11(4):387-405
We recently introduced a new line of reduced-dimensionality experiments making constructive use of axial peak magnetization, which has so far been suppressed as an undesirable artifact in multidimensional NMR spectra [Szyperski, T., Braun, D., Banecki, B. and Wüthrich, K. (1996) J. Am. Chem. Soc., 118, 8146–8147]. The peaks arising from the axial magnetization are located at the center of the doublets resulting from projection. Here we describe the use of such projected four-dimensional (4D) triple resonance experiments for the efficient sequential resonance assignment of 15N/13C-labeled proteins. A 3D
/
/(CO)NHN experiment is recorded either in conjunction with 3D HNN<
> or with the newly presented 3D HNN
scheme. The first combination yields sequential assignments based on the measurement of13 C chemical shifts and provides a complete 1H, 13C and 15N resonance assignment of polypeptide backbone and CHn
moieties. When employing the second combination, 13C=O chemical shifts are not measured, but the sequential assignment relies on both 13C and1 H chemical shifts. The assignment is performed in a semi-automatic fashion using the program XEASY in conjunction with the newly implemented program SPSCAN. This program package offers routines for the facile mutual interconversion of single-quantum and zero/double-quantum frequencies detected in conventional and reduced-dimensionality spectra, respectively. In particular, SPSCAN comprises a peak picking routine tailored to cope with the distinct peak patterns of projected NMR experiments performed with simultaneous acquisition of central peaks. Data were acquired at 13 °C for the N-terminal 63-residue polypeptide fragment of the 434 repressor. Analysis of these spectra, which are representative for proteins of about 15 kDa when working at commonly used temperatures around 30 °C , demonstrates the efficiency of our approach for the assignment of medium-sized15 N/13C doubly labeled proteins. 相似文献
12.
Robin T. Clowes Wayne Boucher Colin H. Hardman Peter J. Domaille Ernest D. Laue 《Journal of biomolecular NMR》1993,3(3):349-354
Summary We recently proposed a novel four-dimensional (4D) NMR strategy for the assignment of backbone nuclei in spectra of 13C/15N-labelled proteins (Boucher et al. (1992) J. Am. Chem. Soc., 114, 2262–2264 and J. Biomol. NMR, 2, 631–637). In this paper we extend this approach with a new constant time 4D HCC(CO)NNH experiment that also correlates the chemical shifts of the aliphatic sidechain (1H and 13C) and backbone (1H, 13C and 15N) nuclei. It separates the sidechain resonances, which may heavily overlap in spectra of proteins with large numbers of similar residues, according to the backbone nitrogen and amide proton chemical shifts. When used in conjunction with a 4D HCANNH or HNCAHA experiment it allows, in principle, complete assignment of aliphatic sidechain and backbone resonances with just two 4D NMR experiments. 相似文献
13.
Summary A new 3D1H–15N–13C triple resonance experiment is presented that provides in-phase absorptive cross peaks between amide protons and -protons of the same and the sequentially preceding residue. The experiment yields similar connectivities as those described previously by Montelione and Wagner (1990a) [J. Magn. Reson.,87, 183–188] and Kay et al. (1991) [J. Magn. Reson.,91, 84–92]. However, the pulse sequence was designed to minimize the time that transverse coherence of the13C nucleus is present, since this nucleus has the shortest transverse relaxation time of all the nuclei involved in these experiments. This is achieved by using a coherence transfer pathway from1HN to15N,13C,1H and back to the1HN. In the sequence described, transverse13C coherence is present only for a length of ca. I1J(C-H). This reduces loss of signal due to transverse relaxation. We tested the technique on uniformly15N- and13C-enriched T4 lysozyme. 相似文献
14.
Gerard J. Kleywegt Rolf Boelens Michel Cox Miguel Llinás Robert Kaptein 《Journal of biomolecular NMR》1991,1(1):23-47
Summary A suite of computer programs (CLAIRE) is described which can be of assistance in the process of assigning 2D1H NMR spectra of proteins. The programs embody a software implementation of the sequential assignment approach first developed by Wüthrich and co-workers (K. Wüthrich. G. Wider, G. Wagner and W. Braun (1982)J. Mol. Biol.
155, 311). After data-abstraction (peakpicking), the software can be used to detect patterns (spin systems), to find cross peaks between patterns in 2D NOE data sets and to generate assignments that are consistent with all available data and which satisfy a number of constraints imposed by the user. An interactive graphics program calledCONPAT is used to control the entire assignment process as well as to provide the essential feedback from the experimental NMR spectra. The algorithms are described in detail and the approach is demonstrated on a set of spectra from the mistletoe protein phoratoxin B, a homolog of crambin. The results obtained compare well with those reported earlier based entirely on a manual assignment process. 相似文献
15.
Victoria A. Higman Jeremy Flinders Matthias Hiller Stefan Jehle Stefan Markovic Sebastian Fiedler Barth-Jan van Rossum Hartmut Oschkinat 《Journal of biomolecular NMR》2009,44(4):245-260
In recent years, solid-state magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) has been growing into
an important technique to study the structure of membrane proteins, amyloid fibrils and other protein preparations which do
not form crystals or are insoluble. Currently, a key bottleneck is the assignment process due to the absence of the resolving
power of proton chemical shifts. Particularly for large proteins (approximately >150 residues) it is difficult to obtain a
full set of resonance assignments. In order to address this problem, we present an assignment method based upon samples prepared
using [1,3-13C]- and [2-13C]-glycerol as the sole carbon source in the bacterial growth medium (so-called selectively and extensively labelled protein).
Such samples give rise to higher quality spectra than uniformly [13C]-labelled protein samples, and have previously been used to obtain long-range restraints for use in structure calculations.
Our method exploits the characteristic cross-peak patterns observed for the different amino acid types in 13C-13C correlation and 3D NCACX and NCOCX spectra. An in-depth analysis of the patterns and how they can be used to aid assignment
is presented, using spectra of the chicken α-spectrin SH3 domain (62 residues), αB-crystallin (175 residues) and outer membrane
protein G (OmpG, 281 residues) as examples. Using this procedure, over 90% of the Cα, Cβ, C′ and N resonances in the core
domain of αB-crystallin and around 73% in the flanking domains could be assigned (excluding 24 residues at the extreme termini
of the protein).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
Ingmar Sethson Ulf Edlund Tadeusz A. Holak Alfred Ross Bengt-Harald Jonsson 《Journal of biomolecular NMR》1996,8(4):417-428
Summary The backbone NMR resonances of human carbonic anhydase I (HCA I) have been assigned. This protein is one of the largest monomeric proteins assigned so far. The assignment was enabled by a combination of 3D triple-resonance experiments and extensive use of amino acid-specific 15N-labeling. The obtained resonance assignment has been used to evaluate the secondary structure elements present in solution. The solution structure appears to be very similar to the crystal structure, although some differences can be observed. Proton-deuteron exchange experiments have shown that the assignments provide probes that can be used in future folding studies of HCA I.The chemical shift data have been deposited in the BioMagResBank in Madison, WI, U.S.A. 相似文献
17.
Summary A 3D1H–15N–13C triple resonance experiment is presented that contains exclusively cross peaks between the1HN and15N nuclei of one residue with the H of the preceding residue. The pulse sequence, designed to minimize the time coherence, is transverse on nuclei with short T2 values. The experiment consists of coherence transfers via one-bond couplings from the HN via N, CO, C to the H and back to the HN for detection; it is called HN(COCA)HA. The experiment was tested on uniformly15N- and13C-enriched T4 lysozyme. 相似文献
18.
Jonathan A. Lukin Andrew P. Gove Sarosh N. Talukdar Chien Ho 《Journal of biomolecular NMR》1997,9(2):151-166
We present a computer algorithm for the automated assignment of polypeptide backbone and13C resonances of a protein of known primary sequence. Input to the algorithm consistsof cross peaks from several 3D NMR experiments: HNCA, HN(CA)CO, HN(CA)HA,HNCACB, COCAH, HCA(CO)N, HNCO, HN(CO)CA, HN(COCA)HA, and CBCA(CO)NH.Data from these experiments performed on glutamine-binding protein are analyzed statisticallyusing Bayes' theorem to yield objective probability scoring functions for matching chemicalshifts. Such scoring is used in the first stage of the algorithm to combine cross peaks fromthe first five experiments to form intraresidue segments of chemical shifts{Ni,HiN,Ci,Ci,Ci}, while the latter five are combined into interresiduesegments {Ci,Ci,Ci,Ni+1,HNi+1}. Given a tentative assignment of segments,the second stage of the procedure calculates probability scores based on the likelihood ofmatching the chemical shifts of each segment with (i) overlapping segments; and (ii) chemicalshift distributions of the underlying amino acid type (and secondary structure, if known). Thisjoint probability is maximized by rearranging segments using a simulated annealing program,optimized for efficiency. The automated assignment program was tested using CBCANH andCBCA(CO)NH cross peaks of the two previously assigned proteins, calmodulin and CheA.The agreement between the results of our method and the published assignments wasexcellent. Our algorithm was also applied to the observed cross peaks of glutamine-bindingprotein of Escherichia coli, yielding an assignment in excellent agreement with that obtainedby time-consuming, manual methods. The chemical shift assignment procedure described hereshould be most useful for NMR studies of large proteins, which are now feasible with the useof pulsed-field gradients and random partial deuteration of samples. 相似文献
19.
Kanelis V Donaldson L Muhandiram DR Rotin D Forman-Kay JD Kay LE 《Journal of biomolecular NMR》2000,16(3):253-259
Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) 13C and variable time (VT) 15N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/JCC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2HzNz state, then prior to the VT chemical shift labelling period is converted into 2HzNy coherence. The VT -15N mode eliminates the effect of 1
J
N,CO and 1,2
J
N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH2 groups, thus removing any potential overlap with the CH3 signals. The CT-13CH3,VT-15N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH3 and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH3 and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin. 相似文献
20.
Andy C. Wang Stephan Grzesiek Rolf Tschudin Patricia J. Lodi Ad Bax 《Journal of biomolecular NMR》1995,5(4):376-382
Summary It is demonstrated that sequential resonance assignment of the backbone 1H and 15N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate 1H with the intraresidue 15N and with the 15N resonance of the next residue. The experiments are preferably conducted in D2O, where very high resolution in the 15N dimension can be achieved by using 2H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in 13C and 15N. Complete backbone and 13C/1H resonance assignments are presented. 相似文献