Correlation of nucleotide base and sugar protons in a 15N-labeled HIV-1 RNA oligonucleotide by 1H-15N HSQC experiments

Summary The advent of methods for preparing ¹⁵N- and ¹³C-labeled RNA oligonucleotides holds promise for extending the size of RNA molecules that can be studies by NMR spectroscopy. A practical limitation is the expense of the ¹³C label. It may therefore sometimes be desirable to prepare a relatively inexpensive ¹⁵N-labeled sample only. Here we show that the two-bond ¹H-¹⁵N HSQC experiment can be used on ¹⁵N-labeled RNA to correlate the intranucleotide H1 and H8,H6,H5 resonances indirectly through the shared glycosidic nitrogen. The nonrefocused version of a standard HSQC experiment for 2D proton-detected ¹H-¹⁵N chemical-shift correlation is applied in order to minimize the sensitivity loss due to the relatively fast spin-spin relaxation of RNA oligonucleotides. The experiment is applied to the 30-nucleotide RNA RBE3 which contains the high-affinity binding site of the RRE (rev response element) for the Rev protein of HIV. The results indicate that this simple experiment allows a straightforward identification of the base proton resonances CH5, CH6, UH5, UH6, purine H8, and AH2 as well as the intranucleotide H1 and H8,H6,H5 connectivities. When combined with a NOESY experiment, complete sequential assignments can be obtained.     

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects

Summary In this study we report on the ¹H, ¹³C and ¹⁵N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of ¹H, ¹³C and ¹⁵N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.     

Assignment of cytosine N3 resonances in nucleic acids via intrabase three-bond coupling to amino protons

Coherences were observed between ¹⁵N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with ¹⁵N. Use of intraresidue ¹⁵ N3-amino proton couplings to assign cytosine ¹⁵ N3 signals complements the recently proposed J_NN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293–8297] method of identifying hydrogen-bonded base pairs in RNA.     

Competition between Subterranean Clover and Rygrass for uptake of 15N-labeled fertilizer

Grasses and legumes are often grown together for improving quality of forage and for better yield when soil N availability is limiting. One compatible mixture is Trifolium subterranium L., subterranean clover and Lolium multiflorum Lam, ryegrass.Experiments were conducted with plants grown in a glasshouse and plant growth chambers to determine the competitive ability of these plants for fertilizer N. Fertilizer N was enriched with ¹⁵N to measure the contribution of dinitrogen fixation and fertilizer N to the growth of clover. In pure stands, with increased fertilizer N, the legume took up similar quantities of mineral N as the grass to make up for the deficit due to less dinitrogen fixation but in mixed stands the grass by far outcompeted the legume. The growth of clover suffered due to lack of N both from less dinitrogen fixation and the inability to compete with the grass for mineral N. Increasing levels of fertilizer N reduced dinitrogen fixation by the clover. When growing with the clover the grass did not receive N from the clover. A laboratory experiment using ¹⁵N label on pure stands of the two species indicated that the grass had an inherent capability of absorbing almost twice the amount of mineral N as the legume under the same conditions even when root weight and volume was not larger for the grass. The results of this research provide insight into the often observed phenomenon that growth of clover is reduced when grown with grass in proportion to the amount of mineral N provided. This revised version was published online in June 2006 with corrections to the Cover Date.     

Specific 15N, NH correlations for residues in15 N, 13C and fractionally deuterated proteins that immediately follow methyl-containing amino acids

A triple-resonance pulse scheme is described which records¹⁵N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a¹⁵N, ¹³C and fractionally deuterated proteinsample and selects for CH₂D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone ¹⁵N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens.     

Improved sensitivity in indirect monitoring of chemical shifts of proton-heteronuclear spin pairs (1H-13C and 1H-15N) in 3D and 4D NMR spectroscopy

In three-dimensional and four-dimensional experiments on doubly labelled proteins not only heteronuclear (¹³C or ¹⁵N) but also proton (¹H) frequencies are often indirectly monitored, rather than being directly observed. In this communication we show how in these experiments by overlaying ¹H and heteronuclear evolutions one can obtain decreased apparent relaxation rates of ¹H signals, yielding improved sensitivity. The new method applies to spin pairs like ¹H-¹⁵N, as in amide groups, or ¹H-¹³C, as in methine groups of alpha or aromatic systems.     

A novel way of amino acid-specific assignment in 1H-15N HSQC spectra with a wheat germ cell-free protein synthesis system

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones.In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.     

Multidimensional1H and15N NMR investigation of glutamine-binding protein ofEscherichia coli

Summary Specific and uniform¹⁵N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His¹⁵⁶, Trp³² and Trp.²²⁰ residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport ofl-glutamine across the cytoplasmic membrane ofEscherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H₂ of His¹⁵⁶ refines the earlier, model where this particular proton formas an intermolecular hydrogen bond to the -carbonyl ofl-glutamine, while assignments of both Trp³² and Trp²²⁰ show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8–9 amino acid residues at a time. This paper illustrates the usefulness of combining¹⁵N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25 000.     

A suite of Mathematica notebooks for the analysis of protein main chain 15N NMR relaxation data

A suite of Mathematica notebooks has been designed to ease the analysis of protein main chain ¹⁵N NMR relaxation data collected at a single magnetic field strength. Individual notebooks were developed to perform the following tasks: nonlinear fitting of ¹⁵N-T ₁ and -T ₂ relaxation decays to a two parameter exponential decay, calculation of the principal components of the inertia tensor from protein structural coordinates, nonlinear optimization of the principal components and orientation of the axially symmetric rotational diffusion tensor, model-free analysis of ¹⁵N-T ₁, -T ₂, and {¹H}–¹⁵N NOE data, and reduced spectral density analysis of the relaxation data. The principle features of the notebooks include use of a minimal number of input files, integrated notebook data management, ease of use, cross-platform compatibility, automatic visualization of results and generation of high-quality graphics, and output of analyses in text format.L. Spyracopoulos is an AHFMR Medical Research Senior Scholar     

Triple resonance experiments for the simultaneous correlation of H6/H5 and exchangeable protons of pyrimidine nucleotides in 13C,15N-labeled RNA applicable to larger RNA molecules

Triple-resonance two-dimensional H6/H5(C4N)H and C6/C5(C4N)H experiments are described that provide through-bond H6/H5 or C6/C5 to imino/amino correlations in pyrimidine bases in ¹³C,¹⁵N-labeled RNA. The experiments simultaneously transfer H6/H5 magnetization by an INEPT step to the C6/C5 nuclei and by homonuclear CC- and heteronuclear CN-TOCSY steps via the intervening C4 nucleus to the N3/N4 nuclei and then by a reverse INEPT step to the imino/amino hydrogens. The sensitivity of these experiments is high as demonstrated using a 30-nucleotide pyrimidine rich RNA at a concentration of 0.9 mM at temperatures of 10°C and 25°C. This indicates the general applicability of the experiments and the possibility to obtain correlations for imino resonances in non-canonical regions of the target RNA.     

Measurement of 15N-1H coupling constants in uniformly 15N-labeled proteins: Application to the photoactive yellow protein

A modified HNHB experiment is presented that allows thedetermination of J(NH) coupling constants directly from the ratio ofcross-peak to diagonal-peak intensities. The experiment was applied to thephotoactive yellow protein (PYP) and yielded the magnitude of 117³J(NH) coupling constants. In addition, 29³J(NH^(i–1)) coupling constantscould be measured, providing information about the backbone angle .These data, in conjunction with the magnitudes of the³J(H^NH) coupling constantsobtained from the HNHA spectrum, effectively discriminate the twopossibilities for the stereospecific assignment of theH resonances in glycine residues. For all eight glycineresidues in PYP that were not subject to conformational averaging and hadnon-degenerate H resonance frequencies, the J-couplingdata, together with limited NOE data, yielded the stereospecific assignmentof the H resonances for these residues. In addition,reliable and precise , dihedral constraints were also derived forthese residues from the J-coupling data.     

Forests losing large quantities of nitrogen have elevated 15N:14N ratios

Summary Urea (U) and ammonium nitrate (AN) had been applied to a Scots pine (Pinus sylvestris L.) forest in northern Sweden for 18 consecutive years at four doses resulting in total N applications ranging from 0 to 1980 kg ha^–1. The ¹⁵N abundance ( ¹⁵N) of the grass Deschampsia flexuosa (L.) Trin. increased linearly (from –0.7 to 11.0) with application rate in the case of U. The response to AN was in the same direction but smaller. While others have shown that the initial response of nitrogen-limited systems to additions of N is a change of ¹⁵N abundance towards that of added N, this study shows that further and excessive additions leads to a retention of ¹⁵N. Monitoring ¹⁵N abundance over time in dose-response trials of this type thus opens new possibilities to estimate critical loads of N and the point of nitrogen saturation.     

Incorporation of atmospheric 15NO2-nitrogen into free amino acids by Norway spruce Picea abies (L.) Karst.

During spring and autumn 1991, potted 6-yearold spruce trees (Picea abies (L.) Karst.) were fumigated with 60 nl·1^{–1 15}NO₂ for 4 days under controlled conditions in constant light. Current and previous flush needles, the bark and the fine roots were analysed for total ¹⁵N content and incorporation of ¹⁵N into the -amino nitrogen of free amino acids. In addition, in vitro nitrate reductase activity and stomatal conductance of the needles were measured. Nitrate reductase activity was significantly higher in the needles of fumigated trees compared to control trees exposed to filtered air. With an average of 9.1% ¹⁵N, free glutamate was the pool with the most label. Taking into account the time-course of the labelling of this pool, this figure can be taken as an estimate of the minimum contribution of NO₂ to the N nutrition of the needles. ¹⁵N-labelled amino acids were also detected in the bark and the roots, indicating export from the needles.     

Simultaneous CT-13C and VT-15N chemical shift labelling: Application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH

Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) ¹³C and variable time (VT) ¹⁵N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J_CC=28.6 ms). The magnetisation originating from NH protons is initially stored in the 2H_zN_z state, then prior to the VT chemical shift labelling period is converted into 2H_zN_y coherence. The VT -¹⁵N mode eliminates the effect of ¹ J _N,CO and ^1,2 J _N,CA coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH₂ groups, thus removing any potential overlap with the CH₃ signals. The CT-¹³CH₃,VT-¹⁵N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH₃NH and 3D ¹³C,¹⁵N HSQC-NOESY-CH₃NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH₃ and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH₃ and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

Below-ground nitrogen transfer between different grassland species: Direct quantification by 15N leaf feeding compared with indirect dilution of soil 15N

Nitrogen (N) transfer from one species to another is important for the N cycling in low-input grassland. In the present work, estimates obtained by an indirect ¹⁵N dilution technique were compared with estimates obtained by a direct ¹⁵N leaf feeding technique over two complete growing seasons in red clover-ryegrass and white clover-ryegrass mixtures under field conditions. The direct technique confirmed that N transfer between clovers and ryegrass is a bi-directional process. The transfer of N from both clovers to ryegrass occurred within 25 days upon the first labelling event. A very high N transfer occurred from white clover to the associated ryegrass, 4.5 and 7.5 g m⁻² in the 1st and 2nd production year, respectively. The corresponding values for transfer from red clover to the associated ryegrass were 1.7 and 3.6 g m⁻². Quantified relatively to the total above-ground N content of white clover- ryegrass and red clover-ryegrass mixtures, the N transfer exceeded 50% and 10%, respectively, in three out of seven harvests. The N transfer from ¹⁵N labelled grass to associated clovers constituted a relatively constant proportion of approx. 8% of the above-ground N content of the mixtures. Estimates based on the soil ¹⁵N dilution technique generally underestimated the net N transfer by more than 50% compared to the direct ¹⁵N labelling technique. Furthermore, the indirect ¹⁵N dilution technique estimated only marginal differences between red and white clover in the quantities of N transferred, whereas the direct ¹⁵N labelling technique showed the N transfer from white clover to the associated ryegrass to be significantly higher than that involving red clover. It is concluded that N transfer is a much more dynamic and quantitatively important process in grassland than previously recognised. This revised version was published online in June 2006 with corrections to the Cover Date.     

The relative orientation of the fibronectin 6F11F2 module pair: A 15N NMR relaxation study

The structure of a pair of modules (⁶F1¹F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from ¹H-¹H NOE and ³ J _HHN data [Bocquier et al. (1999) Structure, 7, 1451–1460] and a weak module–module interface, comprising Leu19 and Leu28, in ⁶F1, and Tyr68 in ²F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of ¹⁵N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and ³ J _HHN data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where ¹⁵N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443–449].