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1.
The limits of resolution that can be obtained in 1H–15N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide 1H resonances. Heteronuclear decoupling of 15N from the 1H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly 2H and 15N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the 15N nuclei. The combination of these techniques results in average 1H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and 15N decoupling are described for achieving the best possible performance in these experiments. 相似文献
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Tonelli M Masterson LR Hallenga K Veglia G Markley JL 《Journal of biomolecular NMR》2007,39(3):177-185
We present a highly sensitive pulse sequence, carbonyl carbon label selective 1H–15N HSQC (CCLS-HSQC) for the detection of signals from 1H–15N units involved in 13C′–15N linkages. The CCLS-HSQC pulse sequence utilizes a modified 15N CT evolution period equal to 1/(
) (∼33 ms) to select for 13C′–15N pairs. By collecting CCLS-HSQC and HNCO data for two proteins (8 kDa ubiquitin and 20 kDa HscB) at various temperatures
(5–40°C) in order to vary correlation times, we demonstrate the superiority of the CCLS-HSQC pulse sequence for proteins with
long correlation times (i.e. higher molecular weight). We then show that the CCLS-HSQC experiment yields assignments in the
case of a 41 kDa protein incorporating pairs of 15N- and 13C′-labeled amino acids, where a TROSY 2D-HN(CO) had failed. Although the approach requires that the 1H–15N HSQC cross peaks be observable, it does not require deuteration of the protein. The method is suitable for larger proteins
and is less affected by conformational exchange than HNCO experiments, which require a longer period of transverse 15N magnetization. The method also is tolerant to the partial loss of signal from isotopic dilution (scrambling). This approach
will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large
enzymes, and partially folded or unfolded proteins. 相似文献
4.
Imino 1H–15N residual dipolar couplings (RDCs) provide additional structural information that complements standard 1H–1H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino 1H–1H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNAVal using a BEST-Jcomp-HMQC2 experiment. 1H–1H RDCs are observed between the imino protons in G–U wobble base pairs and between imino protons on neighboring base pairs
in both RNAs. These imino 1H–1H RDCs complement standard 1H–15N RDCs because the 1H–1H vectors generally point along the helical axis, roughly perpendicular to 1H–15N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the
standard experiment. The ability to measure imino 1H–1H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
5.
The heteronuclear 15N–{1H} NOE values are typically determined by taking the ratio of 15N signal intensities recorded in the presence and absence of 1H saturation prior to evolution of 15N magnetization. Since the intensity ratio of two independent experiments is taken, complete recovery of 15N magnetization during the scan repetition delay is critical to obtain reliable NOE values. Because it may not be practical
to wait for the complete recovery of magnetization at high magnetic fields, Solomon equations may be used to correct measured
NOE values. Here, based on experiments and simulations, we show that since the cross-correlation between 1H–15N dipole and 15N chemical shift anisotropy becomes significant at high fields for small or deuterated proteins, measured NOE values can not
be accurately corrected based on the Solomon equations. We also discuss ranges of rotational correlation times and proton
spin-flip rate, in which the NOE values can be corrected by the equations. 相似文献
6.
Jaroniec CP Boisbouvier J Tworowska I Nikonowicz EP Bax A 《Journal of biomolecular NMR》2005,31(3):231-241
New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond 15N1/9–13C1, 15N1/9–13C6/8, and 15N1/9–13C2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ 1H–13C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H1–C1–N1/9 (MQ-HCN-QJ) or H6/8–C6/8–N1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and 1/(2JNC) (attenuated spectrum). The different types of 15N–13C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time 15N evolution period and the offset of two frequency-selective 13C pulses. The methods are demonstrated for a uniformly 13C, 15N-enriched 24-nucleotide stem-loop RNA sequence, helix-35, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35 sample result in an estimated precision of ca. 0.07 Hz for 1DNC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of 15N1/9–13C1, 15N1/9–13C6/8, and 15N1/9–13C2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35, refined using independently measured observables, including 13C–1H, 13C–13C and 1H–1H dipolar couplings.Supplementary material to this paper is available in electronic form at
http://dx.doi.org/10.1007/s10858-005-0646-2. 相似文献
7.
We present a simple method, ARTSY, for extracting 1JNH couplings and 1H–15N RDCs from an interleaved set of two-dimensional 1H–15N TROSY-HSQC spectra, based on the principle of quantitative J correlation. The primary advantage of the ARTSY method over
other methods is the ability to measure couplings without scaling peak positions or altering the narrow line widths characteristic
of TROSY spectra. Accuracy of the method is demonstrated for the model system GB3. Application to the catalytic core domain
of HIV integrase, a 36 kDa homodimer with unfavorable spectral characteristics, demonstrates its practical utility. Precision
of the RDC measurement is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC spectrum, and is approximately
given by 30/(S/N) Hz. 相似文献
8.
We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides. 相似文献
9.
James S. McKenzie Adrian J. Charlton James A. Donarski Alan D. MacNicoll Julie C. Wilson 《Metabolomics : Official journal of the Metabolomic Society》2010,6(4):574-582
A modified Lorentzian distribution function is used to model peaks in two-dimensional (2D) 1H–13C heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra. The model fit is used to determine
accurate chemical shifts from genuine signals in complex metabolite mixtures such as blood. The algorithm can be used to extract
features from a set of spectra from different samples for exploratory metabolomics. First a reference spectrum is created
in which the peak intensities are given by the median value over all samples at each point in the 2D spectra so that 1H–13C correlations in any spectra are accounted for. The mathematical model provides a footprint for each peak in the reference
spectrum, which can be used to bin the 1H–13C correlations in each HSQC spectrum. The binned intensities are then used as variables in multivariate analyses and those
found to be discriminatory are rapidly identified by cross referencing the chemical shifts of the bins with a database of
13C and 1H chemical shift correlations from known metabolites. 相似文献
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NMR relaxation of arginine (Arg) 15Nε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal 15N–15N scalar couplings on measurements of transverse relaxation rates (R 2 ) for Arg side-chain 15Nε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal 15N–15N couplings ( 2 J NN ) of the 15Nε nuclei and found that the magnitudes of the 2 J NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all 15N nuclei, suggested that the two 2 J NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg 15Nε R 2 measurements. However, our experimental data show that the expected modulation via two 2 J NN couplings vanishes during the 15N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R 2 relaxation rates for Arg side-chain 15Nε nuclei despite the presence of two 2 J NN couplings. Although the so-called recoupling conditions may cause overestimate of R 2 rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings. 相似文献
12.
A TROSY-based NMR experiment is described for simultaneous measurement of the 15N longitudinal relaxation rate constant R1 and the {1H}–15N nuclear Overhauser enhancement. The experiment is based on the observation that the TROSY mixing pulse sequence element symmetrically exchanges 1H and 15N magnetizations. The accuracy of the proposed technique is validated by comparison to independent measurements of both relaxation parameters for the protein ubiquitin. The simultaneous experiment is approximately 20–33% shorter than conventional sequential measurements. 相似文献
13.
Luke W. Arbogast Frank Delaglio Joel R. Tolman John P. Marino 《Journal of biomolecular NMR》2018,72(3-4):149-161
While the use of 1H–13C methyl correlated NMR spectroscopy at natural isotopic abundance has been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies, spectral interference from aliphatic excipients remains a significant obstacle to its widespread application. These signals can cause large baseline artifacts, obscure protein resonances, and cause dynamic range suppression of weak peaks in non-uniform sampling applications, thus hampering both traditional peak-based spectral analyses as well as emerging chemometric methods of analysis. Here we detail modifications to the 2D 1H–13C gradient-selected HSQC experiment that make use of selective pulsing techniques for targeted removal of interfering excipient signals in spectra of the NISTmAb prepared in several different formulations. This approach is demonstrated to selectively reduce interfering excipient signals while still yielding 2D spectra with only modest losses in protein signal. Furthermore, it is shown that spectral modeling based on the SMILE algorithm can be used to simulate and subtract any residual excipient signals and their attendant artifacts from the resulting 2D NMR spectra. 相似文献
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Martin D. Gelenter Tuo Wang Shu-Yu Liao Hugh O’Neill Mei Hong 《Journal of biomolecular NMR》2017,68(4):257-270
Site-specific determination of molecular motion and water accessibility by indirect detection of 2H NMR spectra has advantages over dipolar-coupling based techniques due to the large quadrupolar couplings and the ensuing high angular resolution. Recently, a Rotor Echo Short Pulse IRrAdiaTION mediated cross polarization (RESPIRATIONCP) technique was developed, which allowed efficient transfer of 2H magnetization to 13C at moderate 2H radiofrequency field strengths available on most commercial MAS probes. In this work, we investigate the 2H–13C magnetization transfer characteristics of one-bond perdeuterated CD n spin systems and two-bond H/D exchanged C–(O)–D and C–(N)–D spin systems in carbohydrates and proteins. Our results show that multi-bond, broadband 2H–13C polarization transfer can be achieved using 2H radiofrequency fields of ~50 kHz, relatively short contact times of 1.3–1.7 ms, and with sufficiently high sensitivity to enable 2D 2H–13C correlation experiments with undistorted 2H spectra in the indirect dimension. To demonstrate the utility of this 2H–13C technique for studying molecular motion, we show 2H–13C correlation spectra of perdeuterated bacterial cellulose, whose surface glucan chains exhibit a motionally averaged C6 2H quadrupolar coupling that indicates fast trans-gauche isomerization about the C5–C6 bond. In comparison, the interior chains in the microfibril core are fully immobilized. Application of the 2H–13C correlation experiment to H/D exchanged Arabidopsis primary cell walls show that the O–D quadrupolar spectra of the highest polysaccharide peaks can be fit to a two-component model, in which 74% of the spectral intensity, assigned to cellulose, has a near-rigid-limit coupling, while 26% of the intensity, assigned to matrix polysaccharides, has a weakened coupling of 50 kHz. The latter O–D quadrupolar order parameter of 0.22 is significantly smaller than previously reported C–D dipolar order parameters of 0.46–0.55 for pectins, suggesting that additional motions exist at the C–O bonds in the wall polysaccharides. 2H–13C polarization transfer profiles are also compared between statistically deuterated and H/D exchanged GB1. 相似文献
16.
V. A. Belyaev M. M. Dubrovin D. A. Kozlov A. A. Terent’ev A. E. Trenin G. V. Sholin 《Plasma Physics Reports》2010,36(11):964-968
D2− ions produced in collisions of D− ions with relative energies of 2.5–9.2 eV were detected for the first time. It is shown that the effective cross section
for this reaction is no less than 1.5 × 10−14 cm2. Along with the theoretically predicted short-lived state of negative molecular deuterium ions, a state existing for more
than 1 μs was observed. 相似文献
17.
The ability of an ecosystem to retain anthropogenic nitrogen (N) deposition is dependent upon plant and soil sinks for N,
the strengths of which may be altered by chronic atmospheric N deposition. Sugar maple (Acer saccharum Marsh.), the dominant overstory tree in northern hardwood forests of the Lake States region, has a limited capacity to take
up and assimilate NO3−. However, it is uncertain whether long-term exposure to NO3− deposition might induce NO3− uptake by this ecologically important overstory tree. Here, we investigate whether 10 years of experimental NO3− deposition (30 kg N ha−1 y−1) could induce NO3− uptake and assimilation in overstory sugar maple (approximately 90 years old), which would enable this species to function
as a direct sink for atmospheric NO3− deposition. Kinetic parameters for NH4+ and NO3− uptake in fine roots, as well as leaf and root NO3− reductase activity, were measured under conditions of ambient and experimental NO3− deposition in four sugar maple-dominated stands spanning the geographic distribution of northern hardwood forests in the
Upper Lake States. Chronic NO3− deposition did not alter the V
max or K
m for NO3− and NH4+ uptake nor did it influence NO3− reductase activity in leaves and fine roots. Moreover, the mean V
max for NH4+ uptake (5.15 μmol 15N g−1 h−1) was eight times greater than the V
max for NO3− uptake (0.63 μmol 15N g−1 h−1), indicating a much greater physiological capacity for NH4+ uptake in this species. Additionally, NO3− reductase activity was lower than most values for woody plants previously reported in the literature, further indicating
a low physiological potential for NO3− assimilation in sugar maple. Our results demonstrate that chronic NO3− deposition has not induced the physiological capacity for NO3− uptake and assimilation by sugar maple, making this dominant species an unlikely direct sink for anthropogenic NO3− deposition. 相似文献
18.
A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr2+ and Cl– and concentrations of stable isotope 18O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of 15N and 18O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in 15N and 18O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems. 相似文献
19.
Weingarth M Masuda Y Takegoshi K Bodenhausen G Tekely P 《Journal of biomolecular NMR》2011,50(2):129-136
Sensitive 2D solid-state 13C–13C correlation spectra of amyloid β fibrils have been recorded at very fast spinning frequencies and very high magnetic fields. It is demonstrated that PARIS-xy recoupling using moderate rf amplitudes can provide structural information by promoting efficient magnetization transfer even under such challenging experimental conditions. Furthermore, it has been shown both experimentally and by numerical simulations that the method is not very sensitive to dipolar truncation effects and can reveal direct transfer across distances of about 3.5–4Å. 相似文献