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1.
We have developed a tool for computer-assisted assignments of protein NMR spectra from triple resonance data. The program is designed to resemble established manual assignment procedures as closely as possible. IBIS exports its results in XEASY format. Thus, using IBIS the operator has continuous visual and accounting control over the progress of the assignment procedure. IBIS achieves complete assignments for those residues that exhibit sequential triple resonance connectivities within a few hours or days. 相似文献
2.
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
3.
Summary A 4D HCCH-TOCSY experiment is described for correlating and assigning the1H and13C resonances of protein amino acid side chains that has several advantages over 3D versions of the experiment. In many cases, both the1H and13C chemical shifts can be obtained in the 4D experiment from a simple inspection of the13C(3),1H(4) planes extracted at the1H(1)/13C(2) chemical shifts. Together with the 3D and 4D triple resonance experiments, this allows sequence-specific assignments to be obtained. In addition, the increased resolution of the 4D experiment compared to its 3D counterpart allows. automation of resonance assignments. 相似文献
4.
Nathan Astrof Clay Bracken John Cavanagh Arthur G. Palmer III 《Journal of biomolecular NMR》1998,11(4):451-456
A triple resonance NMR experiment, denoted CO_H(N)CACB, correlates1HN and 13CO spins with the13C and13C spins of adjacent amino acids. Thepulse sequence is an out-and-back design that starts with1HN magnetization and transfers coherence viathe 15N spin simultaneously to the 13CO and13C spins, followed by transfer to the13C spin. Two versions of the sequence arepresented: one in which the 13CO spins are frequency labeledduring an incremented t1 evolution period prior to transfer ofmagnetization from the 13C to the13C resonances, and one in which the13CO spins are frequency labeled in a constant-time mannerduring the coherence transfer to and from the13C resonances. Because 13COand 15N chemical shifts are largely uncorrelated, thetechnique will be especially useful when degeneracy in the1HN-15N chemical shifts hindersresonance assignment. The CO_H(N)CACB experiment is demonstrated usinguniformly 13C/15N-labeled ubiquitin. 相似文献
5.
In experiments with proteins of molecular weights around 100 kDa the implementation of [15N,1H]-TROSY-elements in [15N]-constant-time triple resonance experiments yields sensitivity enhancements of one to two orders of magnitude. An additional gain of 10 to 20% may be obtained with the use of sensitivity enhancement elements. This paper describes a novel sensitivity enhancement scheme which is based on concatenation of the 13 C 15N magnetization transfer with the ST2-PT element, and which enables proper TROSY selection of the 15N multiplet components. 相似文献
6.
Improved resolution in three-dimensional constant-time triple resonance NMR spectroscopy of proteins 总被引:1,自引:0,他引:1
Arthur G. Palmer III Wayne J. Fairbrother John Cavanagh Peter E. Wright Mark Rance 《Journal of biomolecular NMR》1992,2(1):103-108
Summary Two new protocols for the three-dimensional, triple resonance, constant-time HCA(CO)N NMR experiment are presented that significantly increase the experimental resolution attainable in the C frequency dimension. Experimental verification of the new experiments is provided by spectra of the IIA domain of glucose permease fromBacillus subtilis. 相似文献
7.
Structure of the Oct-3 POU-homeodomain in solution,as determined by triple resonance heteronuclear multidimensional NMR spectroscopy.
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E. H. Morita M. Shirakawa F. Hayashi M. Imagawa Y. Kyogoku 《Protein science : a publication of the Protein Society》1995,4(4):729-739
The POU-homeodomain (POUH) forms the bipartite DNA-binding POU domain in association with the POU-specific domain. The 1H, 15N, and 13C magnetic resonances of the 67-amino acid long POUH of mouse Oct-3 have almost completely been assigned, mainly through the combined use of three-dimensional triple resonance NMR methods. Based on the distance and dihedral angle constraints derived from the NMR data, the solution structure of the POUH domain has been calculated by the ab initio simulated annealing method. The average RMS deviation for all backbone heavy atoms of the 20 best calculated structures for residues 9-53 of the total 67 amino acid residues is 0.44 A. The POUH domain consists of three alpha-helices (helix-I, 10-20; helix-II, 28-38; and helix-III, 42-53), and helices-II and -III form a helix-turn-helix motif. In comparison with other classical homeodomains, the folding of the three helices is quite similar. However, the length of helix-III is fairly short. In the complex of the Oct-1 POU domain with an octamer site (Klemm JD, et al., 1994, Cell 77:21-32), the corresponding region is involved in helix-III. The structural difference between these two cases will be discussed. 相似文献
8.
Mark A. McCoy Martin J. Dellwo Diane M. Schneider Tracey M. Banks Joseph Falvo Karen J. Vavra Alan M. Mathiowetz M. Walid Qoronfleh Richard Ciccarelli Ewell R. Cook Tricia A. Pulvino Robert C. Wahl Hsin Wang 《Journal of biomolecular NMR》1997,9(1):11-24
We report here the backbone 1HN, 15N, 13C, 13CO, and 1H NMR assignmentsfor the catalytic domain of human fibroblast collagenase (HFC). Three independentassignment pathways (matching 1H, 13C, and 13CO resonances) were used to establishsequential connections. The connections using 13C resonances were obtained fromHNCOCA and HNCA experiments; 13CO connections were obtained from HNCO andHNCACO experiments. The sequential proton assignment pathway was established from a 3D(1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement withrecent X-ray structures of human fibroblast collagenase and human neutrophil collagenase aswell as similar to recent NMR structures of a highly homologous protein, stromelysin. Allthree helices were located; a five-stranded -sheet (four parallel strands, one antiparallelstrand) was also determined. -Sheet regions were identified by cross-stranddN and dNN connections and by strong intraresidue dN correlations, and were corroborated byobserving slow amide proton exchange. Chemical shift changes in a selectively 15N-labeledsample suggest that substantial structural changes occur in the active site cleft on the bindingof an inhibitor. 相似文献
9.
Two triple resonance experiments, HNN and HN(C)N, are presented which correlate HN and 15N resonances sequentially along the polypeptide chain of a doubly (13C, 15N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, C and C chemical shift dispersions. 相似文献
10.
A novel automated approach for the sequence specific NMR assignments of 1HN, 13C, 13C, 13C/1H and 15N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate 1HN and 15N chemical shifts with those of 13C, 13C and 13C/1H. The information derived from such correlations is used to create a `master_list' consisting of all possible sets of 1HN
i, 15Ni, 13C
i, 13C
i, 13Ci/1H
i, 13C
i–1, 13C
i–1 and 13Ci–1/ 1H
i–1 chemical shifts. On the basis of an extensive statistical analysis of 13C and 13C chemical shift data of proteins derived from the BioMagResBank (BMRB), it is shown that the 20 amino acid residues can be grouped into eight distinct categories, each of which is assigned a unique two-digit code. Such a code is used to tag individual sets of chemical shifts in the master_list and also to translate the protein primary sequence into an array called pps_array. The program then uses the master_list to search for neighbouring partners of a given amino acid residue along the polypeptide chain and sequentially assigns a maximum possible stretch of residues on either side. While doing so, each assigned residue is tracked in an array called assig_array, with the two-digit code assigned earlier. The assig_array is then mapped onto the pps_array for sequence specific resonance assignment. The program has been tested using experimental data on a calcium binding protein from Entamoeba histolytica (Eh-CaBP, 15 kDa) having substantial internal sequence homology and using published data on four other proteins in the molecular weight range of 18–42 kDa. In all the cases, nearly complete sequence specific resonance assignments (> 95%) are obtained. Furthermore, the reliability of the program has been tested by deleting sets of chemical shifts randomly from the master_list created for the test proteins. 相似文献
11.
Three-dimensional nuclear magnetic resonance (3D NMR) provides one of the foremost analytical tools available for the elucidation
of biomolecular structure, function and dynamics. Executing a 3D NMR experiment generally involves scanning a series of time-domain
signals S(t
3), as a function of two time variables (t
1, t
2) which need to undergo parametric incrementations throughout independent experiments. Recent years have witnessed extensive
efforts towards the acceleration of this kind of experiments. Among the different approaches that have been proposed counts
an “ultrafast” scheme, which distinguishes itself from other propositions by enabling—at least in principle—the acquisition
of the complete multidimensional NMR data set within a single transient. 2D protein NMR implementations of this single-scan
method have been demonstrated, yet its potential for 3D acquisitions has only been exemplified on model organic compounds.
This publication discusses a number of strategies that could make these spatial encoding protocols compatible with 3D biomolecular
NMR applications. These include a merging of 2D ultrafast NMR principles with temporal 2D encoding schemes, which can yield
3D HNCO spectra from peptides and proteins within ≈100 s timescales. New processing issues that facilitate the collection
of 3D NMR spectra by relying fully on spatial encoding principles are also assessed, and shown capable of delivering HNCO
spectra within 1 s timescales. Limitations and prospects of these various schemes are briefly addressed. 相似文献
12.
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
13.
Differences between the electronic environments of reduced and oxidized Escherichia coli DsbA inferred from heteronuclear magnetic resonance spectroscopy. 总被引:1,自引:1,他引:0
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J. Couprie M. L. Remerowski A. Bailleul M. Couron N. Gilles E. Qumneur N. Jamin 《Protein science : a publication of the Protein Society》1998,7(10):2065-2080
DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two redox states was undertaken. We report here the backbone 1HN, 15N, 13C(alpha) 13CO, 1H(alpha), and 13Cbeta NMR assignments for both oxidized and reduced Escherichia coli DsbA (189 residues). Ninety-nine percent of the frequencies were assigned using a combination of triple (1H-13C-15N) and double resonance (1H-15N or 1H-13C) experiments. Secondary structures were established using the CSI (Chemical Shift Index) method, NOE connectivity patterns, 3(J)H(N)H(alpha) and amide proton exchange data. Comparison of chemical shifts for both forms reveals four regions of the protein, which undergo some changes in the electronic environment. These regions are around the active site (residues 26 to 43), around His60 and Pro 151, and also around Gln97. Both the number and the amplitude of observed chemical shift variations are more substantial in DsbA than in E. coli thioredoxin. Large 13C(alpha) chemical shift variations for residues of the active site and residues Phe28, Tyr34, Phe36, Ile42, Ser43, and Lys98 suggest that the backbone conformation of these residues is affected upon reduction. 相似文献
14.
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
15.
A new method for backbone resonance assignment suitable for large proteins with the natural 1H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, 13C-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the 13C antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly 15N,13C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric proteins. 相似文献
16.
We propose a (3, 2)D CT-HCCH-COSY experiment to rapidly collect the data and provide significant dispersion in the spectral region containing (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues. This enables one to carry out chemical shift based editing and grouping of all the (13)C-(1)H cross peaks of CH(3) groups belonging to Ala, Ile, Leu, Met, Thr and Val residues in fractionally (10%) (13)C-labelled proteins, which in turn aids in the sequence-specific resonance assignments in general and side-chain resonance assignments in particular, in any given protein. Further, we demonstrate the utility of this experiment for stereospecific assignments of the pro-R and pro-S methyl groups belonging to the Leu and Val residues in fractionally (10%) (13)C-labelled proteins. The proposed experiment opens up a wide range of applications in resonance assignment strategies and structure determination of proteins. 相似文献
17.
Daniel A. Fox Linda Columbus 《Protein science : a publication of the Protein Society》2013,22(8):1133-1140
Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β‐barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight‐stranded β‐barrel with long extracellular loops (~63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β‐barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β‐barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 β‐barrel region (97% of the resonances corresponding to the β‐barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full‐length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β‐barrel membrane proteins. 相似文献
18.
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
19.
Summary A generally applicable method for the automated classification of 2D NMR peaks has been developed, based on a Bayesian approach coupled to a multivariate linear discriminant analysis of the data. The method can separate true NMR signals from noise signals, solvent stripes and artefact signals. The analysis relies on the assumption that the different signal classes have different distributions of specific properties such as line shapes, line widths and intensities. As to be expected, the correlation network of the distributions of the selected properties affects the choice of the discriminant function and the final selection of signal properties. The classification rule for the signal classes was deduced from Bayes's theorem. The method was successfully tested on a NOESY spectrum of HPr protein from Staphylococcus aureus. The calculated probabilities for the different signal class memberships are realistic and reliable, with a high efficiency of discrimination between peaks that are true NOE signals and those that are not. 相似文献
20.
Diane Zimmerman Casimir Kulikowski Lingze Wang Barbara Lyons Gaetano T. Montelione 《Journal of biomolecular NMR》1994,4(2):241-256
Summary We have developed an automated approach for determining the sequential order of amino acid spin systems in small proteins. A key step in this procedure is the analysis of multidimensional HCC(CO)NH-TOCSY spectra that provide connections from the aliphatic resonances of residue i to the amide resonances of residue i+1. These data, combined with information about the amino acid spin systems, provide sufficient constraints to assign most proton and nitrogen resonances of small proteins. Constraint propagation methods progressively narrow the set of possible assignments of amino acid spin systems to sequence-specific positions in the process of NMR data analysis. The constraint satisfaction paradigm provides a framework in which the necessary constraint-based reasoning can be expressed, while an object-oriented representation structures and facilitates the extensive list processing and indexing involved in matching. A prototype expert system, AUTOASSIGN, provides correct and nearly complete resonance assignments with one real and 31 simulated 3D NMR data sets for a 72-amino acid domain, derived from the Protein A of Staphylococcus aureus, and with 31 simulated NMR data sets for the 50-amino acid human type- transforming growth factor. 相似文献