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1.
A database of peptide chemical shifts, computed at the density functional level, has been used to develop an algorithm for prediction of 15N and 13C shifts in proteins from their structure; the method is incorporated into a program called SHIFTS (version 4.0). The database was built from the calculated chemical shift patterns of 1335 peptides whose backbone torsion angles are limited to areas of the Ramachandran map around helical and sheet configurations. For each tripeptide in these regions of regular secondary structure (which constitute about 40% of residues in globular proteins) SHIFTS also consults the database for information about sidechain torsion angle effects for the residue of interest and for the preceding residue, and estimates hydrogen bonding effects through an empirical formula that is also based on density functional calculations on peptides. The program optionally searches for alternate side-chain torsion angles that could significantly improve agreement between calculated and observed shifts. The application of the program on 20 proteins shows good consistency with experimental data, with correlation coefficients of 0.92, 0.98, 0.99 and 0.90 and r.m.s. deviations of 1.94, 0.97, 1.05, and 1.08 ppm for 15N, 13C, 13C and 13C, respectively. Reference shifts fit to protein data are in good agreement with `random-coil' values derived from experimental measurements on peptides. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement. 相似文献
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Daiwen Yang Ronald A. Venters Geoffrey A. Mueller W.Y. Choy Lewis E. Kay 《Journal of biomolecular NMR》1999,14(4):333-343
HNCO-based 3D pulse schemes are presented for measuring 1HN-15N,15N-13CO, 1HN-13CO,13CO-13C and 1HN-13C dipolar couplings in 15N,13C,2-labeled proteins. The experiments are based on recently developed TROSY methodology for improving spectral resolution and sensitivity. Data sets recorded on a complex of Val, Leu, Ile (1 only) methyl protonated 15N,13C,2H-labeled maltose binding protein and -cyclodextrin as well as 15N,13C,2H-labeled human carbonic anhydrase II demonstrate that precise dipolar couplings can be obtained on proteins in the 30–40 kDa molecular weight range. These couplings will serve as powerful restraints for obtaining global folds of highly deuterated proteins. 相似文献
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A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex. 相似文献
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A 4D TROSY-based triple resonance experiment, 4D-HNCOi–1CAi, is presented which correlates intra-residue 1HN, 15N, 13 C chemical shifts with the carbonyl (13C) shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (1HN,15N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCOi–1CAi often allows resolution of the ambiguity by linking the 13C and 13C spins surrounding the (1HN,15N) pair. The experiment is demonstrated on a sample of 15N, 13C, 2 H labeled maltose binding protein in complex with -cyclodextrin that tumbles with a correlation time of 46 ns. 相似文献
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Selective diagonal-free 13C,13C-edited aliphatic–aromatic NOESY experiment with non-uniform sampling
Jan Stanek Michał Nowakowski Saurabh Saxena Katarzyna Ruszczyńska-Bartnik Andrzej Ejchart Wiktor Koźmiński 《Journal of biomolecular NMR》2013,56(3):217-226
A band-selective aromatic–aliphatic C,C-edited four-dimensional NOESY experiment is proposed here. Its key advantage is the absence of auto-correlation signals which makes it very attractive for joint use with non-uniform sampling. It is demonstrated here that the sensitivity of the experiment is not significantly affected by utilization of selective pulses (for either aromatic-13C or aliphatic-13C spins). The method was applied to the sample of E32Q mutant of human S100A1 protein, a homodimer of total molecular mass ~20 kDa. High-resolution 4D spectra were obtained from ~1.5 % of sampling points required conventionally. It is shown that superior resolution facilitates unambiguous assignment of observed aliphatic–aromatic cross-peaks. Additionally, the addition of aliphatic-13C dimension enables to resolve peaks with degenerated aliphatic 1H chemical shifts. All observed cross-peaks were validated against previously determined 3D structure of E32Q mutant of S100A1 protein (PDB 2LHL). The increased reliability of structural constraints obtained from the proposed high-resolution 4D 13C(ali),13C(aro)-edited NOESY can be exploited in the automated protocols of structure determination of proteins. 相似文献
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Weisemann R Rüterjans H Schwalbe H Schleucher J Bermel W Griesinger C 《Journal of biomolecular NMR》1994,4(2):231-240
Summary Sensitive three-dimensional NMR experiments, based on the E.COSY principle, are presented for the measurement of the 3J(HN,H) and 3J(HN,C) coupling constants in uniformly 13C- and 15N-labeled proteins. They employ gradient coherence selection in combination with the sensitivity enhancement method in HSQC-type spectra (Cavanagh et al., 1991; Palmer et al., 1991). In most cases, the two measured coupling constants unambiguously define the -angle for protein structure determination. The method is applied to uniformly 13C, 15N-labeled ribonuclease T1. 相似文献
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A [CO]HN(CA)CB-E.COSY pulse scheme is described for measurement of three-bond couplings, 3JCC, between carbonyl and aliphatic C carbons in ubiquitin, uniformly enriched with 13C and 15N. A Karplus relation, 3JCC = 1.28 cos2( - 120°) -1.02 cos( - 120°) +0.30 Hz, is obtained by correlating the 3JCC values measured for human ubiquitin with backbone angles from its crystal structure. As predicted, the new Karplus parametrization yields 3JCC values slightly larger than previously obtained by quantitative J correlation [Hu, J.-S. and Bax, A. (1997) J. Am. Chem. Soc., 119, 6360-6368], but considerably smaller than what has been reported on the basis of other E.COSY-type measurements carried out on flavodoxin. 相似文献
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An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins (15N, 13Cα, 13CO, 1Hα) and 13Cβ nuclei arising from 1H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond 13Cα isotope shifts, 2ΔCα(NiD) and 3ΔCα(Ni+1D), and three-bond 13Cβ isotope shifts, 3ΔCβ(NiD), are useful reporters of the local geometry of the protein backbone. 相似文献
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The ζ-COP is one subunit of the COP I coatomer, which mediates the protein trafficking from the cis-Golgi complex to the endoplasmic
reticulum and also functions in the intra-Golgi trafficking. The NMR assignments of the ζ-COP are essential for its solution
structure determination. 相似文献
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Summary Relaxation times of 13C carbons of uniformly 13C/15N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T1, T2 and the heteronuclear NOE of 13C in uniformly 13C/15N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T1 and the heteronuclear NOE were similar to those of the corresponding 15N experiments published previously. The determination of T2 for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T2 evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T2 evolution period were simulated in order to optimize the bandwidth for which reliable T2 relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual 1H–13C cross peaks, in a series of (1H, 13C)-ct-HSQC spectra with a modified CPMG sequence as well as a T1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm. 相似文献
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Although a significant number of proteins include bound metals as part of their structure, the identification of amino acid
residues coordinated to non-paramagnetic metals by NMR remains a challenge. Metal ligands can stabilize the native structure
and/or play critical catalytic roles in the underlying biochemistry. An atom’s chemical shift is exquisitely sensitive to
its electronic environment. Chemical shift data can provide valuable insights into structural features, including metal ligation.
In this study, we demonstrate that overlapped 13Cβ chemical shift distributions of Zn-ligated and non-metal-ligated cysteine residues are largely resolved by the inclusion
of the corresponding 13Cα chemical shift information, together with secondary structural information. We demonstrate this with a bivariate distribution
plot, and statistically with a multivariate analysis of variance (MANOVA) and hierarchical logistic regression analysis. Using
287 13Cα/13Cβ shift pairs from 79 proteins with known three-dimensional structures, including 86 13Cα and13Cβ shifts for 43 Zn-ligated cysteine residues, along with corresponding oxidation state and secondary structure information,
we have built a logistic regression model that distinguishes between oxidized cystines, reduced (non-metal ligated) cysteines,
and Zn-ligated cysteines. Classifying cysteines/cystines with a statisical model incorporating all three phenomena resulted
in a predictor of Zn ligation with a recall, precision and F-measure of 83.7%, and an accuracy of 95.1%. This model was applied in the analysis of Bacillus subtilis IscU, a protein involved in iron–sulfur cluster assembly. The model predicts that all three cysteines of IscU are metal ligands.
We confirmed these results by (i) examining the effect of metal chelation on the NMR spectrum of IscU, and (ii) inductively
coupled plasma mass spectrometry analysis. To gain further insight into the frequency of occurrence of non-cysteine Zn ligands,
we analyzed the Protein Data Bank and found that 78% of the Zn ligands are histidine and cysteine (with nearly identical frequencies),
and 18% are acidic residues aspartate and glutamate.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
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A triple resonance pulse scheme is presented for recording 13C-1H one-bond dipolar couplings in 15N, 13C labeled proteins. HNCO correlation maps are generated where the carbonyl chemical shift is modulated by the 13C-1H coupling, with the two doublet components separated into individual data sets. The experiment makes use of recently described methodology whereby the protein of interest is dissolved in a dilute solution of bicelles which orient above a critical temperature, thus permitting measurement of significant couplings (Tjandra and Bax, 1997a). An application to the protein ubiquitin is described. 相似文献
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Molecular size has limited solution NMR analyses of proteins. We report 13C–13C NOESY experiments on a 480 kDa protein, the multi-subunit ferritin nanocage with gated pores. By exploiting 13C-resonance-specific chemical shifts and spin diffusion effects, we identified 75% of the amino acids, with intraresidue C–C
connectivities between nuclei separated by 1–4 bonds. These results show the potential of 13C–13C NOESY for solution studies of molecular assemblies >100 kDa. 相似文献
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Human regenerating (Reg) genes belong to the C-type lectin superfamily and express secretory proteins in various tissues. Reg Iα, also named lithostathine, has multiple roles in numerous biological events such as cytokines, anti-apoptotic factors and the calcium carbonate crystals inhibitor. Under physiological pH, Reg Iα becomes largely insoluble after a self-proteolysis process, and the N-terminally truncated form readily polymerizes into fibrils, which leads to neurodegenerative diseases. Reg Iα may form protofibril via lateral hydrophobic interactions with a native-like conformation. The structural basis from the native to fibril form, as well as the carbohydrate binding sites on Reg Iα, remain unknown. Here we present the NMR backbone and side-chain assignments of Reg Iα for use in further NMR investigations. 相似文献