Spectroscopic labeling of A, S/T in the 1H-15N HSQC spectrum of uniformly (15N-13C) labeled proteins

A new triple resonance two-dimensional experiment, termed (HC)NH, has been described to generate specific labels on the peaks of alanines and serines/threonines, separately, in the ¹H–¹⁵N HSQC spectrum of a protein. The performance of the pulse sequence has been demonstrated with a 151 residue protein. The method permits the investigation of local environments around those specific residues without actually having to obtain complete resonance assignments for the entire protein. With this one can envisage use of the technique for studying large protein systems from different points of view.     

Efficient measurement of (3)J(N,Cgamma) and (3)J(C',Cgamma) coupling constants of aromatic residues in (13)C, (15)N-labeled proteins

An NMR pulse sequence is proposed for the simultaneous determination of side chain chi1 torsion-angle related (3)J(N,Cgamma) and (3)J(C', Cgamma) couplings in aromatic amino acid spin systems. The method is of the quantitative J correlation type and takes advantage of attenuated (15)N and (1)H transverse relaxation by means of the TROSY principle. Unlike previously developed schemes for the measurement of either of the two coupling types, spectra contain internal reference peaks that are usually recorded in separate experiments. Therefore, the desired information is extracted from a single rather than four data sets. The new method is demonstrated with uniformly (13)C/(15)N labeled Desulfovibrio vulgaris flavodoxin, which contains 14 aromatic out of 147 total amino acid residues.     

Measurement of (1)J(NC') and (2)J(H(N))(C') couplings from spin-state-selective two-dimensional correlation spectrum.

A method for the measurement of (1)J(NC') and (2)J(H(N))(C') coupling constants from a simplified two-dimensional [(15)N, (1)H] correlation spectrum is presented. The multiplet components of the (1)J(NC') doublet in the indirect dimension and (2)J(H(N))(C') in the direct dimension are separated into two subspectra by spin-state-selective filters. Thus each subspectrum contains no more peaks than the conventional [(15)N, (1)H]-HSQC spectrum. Furthermore, the method for the measurement of (1)J(NC') and (2)J(H(N))(C') is designed to exploit destructive relaxation interference (TROSY). The results are verified against the measurements of (1)J(NC') from spin-state-selective [(13)C', (1)H] correlation spectra recorded with additional sequence described here.     

Semi-constant-time HMSQC (SCT-HMSQC-HA) for the measurement of (3)J(H(N))(H(alpha)) couplings in (15)N-labeled proteins

A simple method for accurately measuring (3)J(H(N))(H(alpha)) coupling constants in (15)N-labeled proteins is described. This semi-constant-time HMSQC-HA experiment combines the rapidity and convenience of the recently introduced CT-HMQC-HA scheme (Postingl and Otting, J. Biomol. NMR 12, 319-324 (1998)) with the high resolution and robustness of the HSQC experiment. The proposed method is demonstrated for the 76-residue human ubiquitin and Saccharopolyspora erythraea calerythrin (176 residues). Our results imply that the SCT-HMSQC-HA experiment is suitable also for proteins with less favorable NMR properties due to its good resolution and sensitivity.     

1H,13C and15N resonance assignment for barnase

The nearly complete assignment of¹H,¹³C and¹⁵N resonances for bacterial ribonuclease barnase produced byBacillus amyloliquefaciens was obtained by standard methods of heteronuclear triple-resonance nuclear magnetic resonance spectroscopy. Analysis of the secondary chemical shifts of the backbone¹H^α,¹³C^α and¹³CO nuclei reveal their strong correlation with the protein secondary structure.     

13C and 15N Abundances in the Sediment Core (VER 92/1-St-10-GC2) from Northern Lake Baikal

Abstract

Carbon and nitrogen stable isotope compositions of organic matter, TOC/TN ratio, and manganese concentration in a sediment core that was collected in northern part of Lake Baikal (VER92ST10-GC2, water depth at 922 m, about 3 m long) were investigated to elucidate the origin of the sedimentary organic matter and its associated environmental factors.

The sediment core was composed of mainly two parts: turbidite sections and other sections. Constant δ¹³C and δ¹⁵N values of the turbidite sections were observed (- 26.8 ±0.2 ‰ for δ¹³C and 3.2 ± 0.1 ‰ for δ¹⁵N) throughout the core. The higher δ¹³C in turbidite sections (about - 27 ‰) than that of the other sections (- 31 to - 29 ‰) was clearly observed, and δ¹⁵N was different between turbidite sections (about 3‰) and other sections (3 to 5 ‰). δ¹³C of other sections was close to that of pelagic phytoplankton, indicating that sediment other than turbidite sections is composed of autochthonous components. The variation of stable isotopes in other sections may be possibly caused by the changes in either phytoplankton growth rate or contribution ratios of terrestrial to aquatic plants for δ¹³C. Either denitrification or fluctuation of δ¹⁵N in pelagic phytoplankton can be the cause of variable δ¹⁵N in other sections.     

Determination of internucleotide (h)J(HN) couplings by the modified 2D J(NN)-correlated [(15)N, (1)H] TROSY

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J(NN)-correlated [(15)N, (1)H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147-14151, 1998) can be applied to measure (h)J(HN) in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA(Trp), since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J(NN)-correlated [(15)N, (1)H] TROSY experiment which enables direct measurement of (h)J(HN) in large nucleic acids.     

Millimetre-wave spectrum of anti-13C1 and 13C2 isotopologues of ethanol

The rotational spectra of the two monosubstituted ¹³C isotopologues of the anti conformer of ethanol have been measured between 80–800 GHz using three different spectrometers at the Cologne Laboratory Astrophysics group. The dataset was constrained for fitting with a standard Watson-S reduction Hamiltonian by rejecting transitions from high-lying states showing significant perturbation with the gauche states and by averaging some small methyl torsional splits. This treatment is compatible with the needs for a first astrophysical research for which an appropriate set of predictions is given.     

Short-term changes in delta(13)C and delta(15)N signatures of water discharged from grazed grasslands

The composition of dissolved organic matter (DOM) in a soil is the product of a variety of soil processes. Changes in the composition of DOM in water discharged from soil should, therefore, give an important insight into modifications in these soil processes. We hypothesise that these processes in soils, under different grassland management regimes, would be affected to different extents by the short-term disturbance of a storm event and that evidence of this could be detected in delta(13)C and delta(15)N signatures in drainage and surface runoff waters. During a storm event we collected discharge waters from 1 ha grassland lysimeters, with or without artificial drainage, which received contrasting fertiliser inputs, and delta(13)C and delta(15)N signatures were determined. Changes in (13)C enrichment during the storm event were clearly identifiable, as were differences between plots for (13)C and (15)N, illustrating that this technique has potential to be a useful tool for identifying and investigating short- and long-term changes in soil organic matter dynamics. Copyright 1999 John Wiley & Sons, Ltd.     

Sensitivity-enhanced MQ-HCN-CCH-TOCSY and MQ-HCN-CCH-COSY pulse schemes for (13)C/(15)N labeled RNA oligonucleotides

Sensitivity enhanced multiple-quantum 3D HCN-CCH-TOCSY and HCN-CCH-COSY experiments are presented for the ribose resonance assignment of (13)C/(15)N-labeled RNA sample. The experiments make use of the chemical shift dispersion of N1/N9 of pyrimidine/purine to distinguish the ribose spin systems. They provide a complementary approach for the assignment of ribose resonance to the currently used HCCH-COSY and HCCH-TOCSY type experiments in which either (13)C or (1)H is utilized to separate the different ribose spin systems. The pulse schemes have been demonstrated on a 23-mer (13)C/(15)N-labeled RNA aptamer complexed with neomycin and tested on a 32-mer RNA complexed with a 23-residue peptide.     

Triple resonance experiments for aligned sample solid-state NMR of (13)C and (15)N labeled proteins

Initial steps in the development of a suite of triple-resonance (1)H/(13)C/(15)N solid-state NMR experiments applicable to aligned samples of (13)C and (15)N labeled proteins are described. The experiments take advantage of the opportunities for (13)C detection without the need for homonuclear (13)C/(13)C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are approximately 20% randomly labeled with (13)C in all backbone and side chain carbon sites and approximately 100% uniformly (15)N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are (13)C labeled at only the alpha-carbon and (15)N labeled at the amide nitrogen of a few residues. The requirement for homonuclear (13)C/(13)C decoupling while detecting (13)C signals is avoided in the first case because of the low probability of any two (13)C nuclei being bonded to each other; in the second case, the labeled (13)C(alpha) sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the (13)C chemical shift and (1)H-(13)C and (15)N-(13)C heteronuclear dipolar coupling frequencies associated with the (13)C(alpha) and (13)C' backbone sites, which provide orientation constraints complementary to those derived from the (15)N labeled amide backbone sites. (13)C/(13)C spin-exchange experiments identify proximate carbon sites. The ability to measure (13)C-(15)N dipolar coupling frequencies and correlate (13)C and (15)N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the (13)C chemical shift and (1)H-(13)C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.     

Dual 13C, 15N labelling of terrestrial slugs (Deroceras reticulatum)

A simple, rapid and cost-effective laboratory method is described for labelling terrestrial slugs simultaneously with 13C and 15N. Slugs (Deroceras reticulatum) were provided with a mixture of [U-13C6]glucose, 15N-enriched lettuce powder, and wheat bran. Assimilation efficiencies for 13C (24.2%) and 15N (27.4%) were not affected by feeding regimes offering ad libitum or restricted access to unlabelled food during the labelling period. Body tissue was significantly more highly enriched in 13C but significantly less in 15N than cutaneous mucus after 15 days.     

Intensity of N and P signals of a two-dimensional two-particle NQR spectrum (J=1)

We calculate the intensities and phases of the N and P peaks of a 2D NMR spectrum under two-frequency excitation during preparation and mixing by means of simultaneous radio-frequency pulses with repetition frequencies close to those of adjacent transitions of the nuclear species studied. The 2D spectrum is shown to contain up to four cross-peaks, in addition to the two diagonal peaks.Kaliningrad State University. Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 6, pp. 63–65, June, 1995.     

Isolation of NO(3)(-)-N as 1-phenylazo-2-naphthol (Sudan-1) for measurement of delta(15)N

The conversion of nitrate (NO(3)(-)) to 1-phenylazo-2-naphthol (Sudan-1) has been examined as a method for natural abundance measurement of delta(15)N of NO(3)(-). The reaction results in dilution of NO(3)(-)-N with only one reagent-derived N and the product is readily concentrated from dilute samples by reverse phase chromatography. There is systematic isotopic fractionation during the reaction, but this can be allowed for by analysing known NO(3)(-) standards along with each sample set. Sudan-1 prepared from surface water samples containing approximately 50 &mgr;g NO(3)(-)-N can be analysed by automated continuous flow isotope ratio mass spectrometry with a precision of 0.2 per thousand (one standard deviation) and the accuracy is not affected by interference from other nitrogenous species in the sample or reagents. Copyright 1999 John Wiley & Sons, Ltd.     

Evaluation of 15N Methods for the measurement of Denitrification in Soils

The precision of the ¹⁵N-emission and that of the ¹⁵N-balance methods was evaluated and both methods were compared in a denitrification experiment. ¹⁵N-analysis was performed with an isotope ratio mass spectrometer which was coupled to an elemental analyzer. The measuring sensitivity in soil and gas analysis was tested by analyzing ¹⁵N-standards. The detection limit for gas samples with two different procedures of ¹⁵N-gas analysis was δ¹⁵N = (4.5 ± 1.0)‰ and (0.5 ± 0.05)‰, respectively. The error in measurement was 19% and 12% respectively. ¹⁵N-analysis of a ¹⁵N-labelled soil (4.15 ppm ¹⁵N) resulted in a CV of 1.32%. The measurements had to be calibrated with soil standards because the ¹⁵N-values showed a continuous downward fluctuation in a range of 10–20% within several days, when only acetanilid was used for calibration. Mean ¹⁵N-losses which were determined with both methods during the denitrification experiment were in good agreement. The precision of the ¹⁵N-emission method was adequate in all variants of the experiment. The precision of the ¹⁵N-balance method however was unsatisfactory at low ¹⁵N-losses (0.2–2% of added ¹⁵N).     

Removal of t(1) noise from metabolomic 2D (1)H-(13)C HSQC NMR spectra by Correlated Trace Denoising

The presence of t(1) noise artefacts in 2D phase-cycled Heteronuclear Single Quantum Coherence (HSQC) spectra constrains the use of this experiment despite its superior sensitivity. This paper proposes a new processing algorithm, working in the frequency-domain, for reducing t(1) noise. The algorithm has been developed for use in contexts, such as metabolomic studies, where existing denoising techniques cannot always be applied. Two test cases are presented that show the algorithm to be effective in improving the SNR of peaks embedded within t(1) noise by a factor of more than 2, while retaining the intensity and shape of genuine peaks.     

Linewidth-resolved 15N HSQC, a simple 3D method to measure 15N relaxation times from T1 and T2 linewidths

A three-dimensional approach for measuring 15N relaxation times is described. Instead of selecting particular values for the relaxation period, in the proposed method the relaxation period is incremented periodically in order to create a 3D spectrum. This additional frequency domain of the transformed spectrum contains the relaxation time information in the T1 and T2 linewidths, and thus the longitudinal and transverse 15N relaxation times can be measured without determination of 2D cross peak volumes/intensities and subsequent curve fitting procedures.     

Sensitivity-enhanced E.COSY-type HSQC experiments for accurate measurements of one-bond 15N-1H(N) and 15N-13C' and two-bond 13C'-1H(N) residual dipolar couplings in proteins

Novel E.COSY-type HSQC experiments are presented for the accurate measurement of one-bond 15N-1H(N) and 15N-13C(') and two-bond 13C(')-1H(N) residual dipolar couplings in proteins. Compared with existing experiments, the (delta,J)-E.COSY experiments described here are composed of fewer pulses and the resulting spectra exhibit 1.4 times the sensitivity of coupled HSQC spectra. Since residual dipolar couplings play increasingly important roles in structural NMR, the proposed methods should find wide spread application for structure determination of proteins and other biological macromolecules.     

Improved measurement of (3)J(H(alpha)(i),N(i+1)) coupling constants in H(2)O dissolved proteins.

A modification to the recently proposed alpha/beta-HN(CO)CA-J TROSY pulse sequence (P. Permi et al., J. Magn. Reson. 146, 255-259 (2000)) makes it possible to determine (3)J(H(alpha)(i), N(i+1)) coupling constants from a single E.COSY-type cross-peak pattern rather than from two (1)H(alpha) spin-state-edited subspectra. Advantages are increased (15)N resolution, critical to extracting accurate (1)H(alpha)-(15)N coupling constants, and minimized differential relaxation due to nested (13)C(alpha) and (15)N evolution periods. Application of the improved pulse sequence to Desulfovibrio vulgaris flavodoxin results in (3)J(H(alpha)(i), N(i+1)) values being systematically larger than those obtained with the original scheme. Parametrization of the coupling dependence on the protein backbone torsion angle psi yields the Karplus relation (3)J(H(alpha)(i), N(i+1))=-1.00 cos(2)(psi-120 degrees )+0.65 cos(psi-120 degrees )-0.15 Hz, with a residual root-mean-square difference of 0.13 Hz between measured and back-calculated coupling constants. The curve compares with data derived from ubiquitin (A. C. Wang and A. Bax, J. Am. Chem. Soc. 117, 1810-1813 (1995)), although spanning a slightly larger range of J values in flavodoxin. The orientation of the Ala39/Ser40 peptide link, forming a type-II beta-turn in flavodoxin, is twisted against X-ray-derived torsions by approximately 10 degrees in the NMR structure as evident from the analysis of straight phi- and psi-related (3)J coupling constants. The remaining deviation of some experimental values from the prediction is likely to be due to strong hydrogen bonding, substituent effects, or the additional dependence on the adjacent torsions straight phi.