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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
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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
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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
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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
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The carboxy-terminal pleckstrin homology (PH) domain recruits GRP1 to the plasma membrane through the specific binding to
phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3]. Here, we describe backbone and side chain assignments of the GRP1 PH domain determined by triple resonance experiments.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Kozlov G Cheng J Lievre C Banville D Gehring K Ekiel I 《Journal of biomolecular NMR》2002,24(2):169-170
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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
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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
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Lipocalin2 plays an important role in the innate immune system. In this article we report the backbone and side-chain resonance
assignments of rat lipocalin2 (rLcn2). These assignments provide a basis for determining the structure and dynamics of rLcn2.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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The heteronuclear 15N-NOE experiment was extensively tested with respect to statistical and systematic experimental error. The dependence of signal intensity in the NOE experiment and in the reference experiment on the saturation and relaxation time was experimentally investigated. The statistics of the experimental values were accessed by numerous repetitions of identical set-ups. As a model system a protein of typical size for NMR studies was chosen, i.e., a 120 amino acid residues containing fragment of the F-actin binding gelation factor (ABP-120). The fragment exhibits fast dynamics that are accessible with the 15N-NOE experiment with various amplitudes. The results of this study show that commonly used parameters are only adequate for accurate measurement of motions with moderate amplitude. Highly flexible parts require longer delay times and thus more experimental time than commonly used. On the other hand, a qualitative or semi-quantitative assessment of a protein's mobility on fast times scales can be obtained from rapidly recorded experiments with unusual short delay times. The findings of this study are of equal importance for highly accurate measurement of the 15N-NOE as well as for quick identification of mobile or even unstructured residues/parts of a protein. 相似文献
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