Conformationally Restricted 2-Substituted Wyosine Derivatives. 1H, 13C,and 15N NMR Study

Abstract

It was found by ¹H, ¹³C and ¹⁵N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH₃O and C₆H₅CH₂O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety.     

Variation in the export of 13C and 15N from soybean leaf: the effects of nitrogen application and sink removal

Translocation of carbon and nitrogen within a single source-sink unit, comprising a trifoliated leaf, the axillary pod and the subtending internode, and from this unit to the rest of the plant was examined in soybean (Glycine max L. cv. Akishirome) plant by feeding ¹³CO₂ and ¹⁵NO₃. The plants were grown at two levels of nitrogen in the basal medium, i.e. low-N (2 g N m^–2) and high-N (35 g N m^–2) and a treatment of depodding was imposed by removing all the pods from the plant, except the pod of the source sink unit, 13 days after flowering. The plants at high-N accumulated more biomass in its organs compared to low-N and pod removal increased the weight of the vegetative organs. When the terminal leaflet of the source-sink unit was fed with ¹³CO₂, almost all of the radioactive materials were retained inside the source-sink unit and translocation to rest of the plants was insignificant under any of the treatments imposed. Out of the¹³C exported by the terminal leaflet, less than half went into the axillary pod, as the lateral leaflets claimed equal share and very little material was deposited in the petiole. Pod removal decreased ¹³C export at high-N , but not at low-N. Similar to ¹³C, the source-sink unit retained all the ¹⁵N fed to the terminal leaflet at high-N. At low-N, the major part of ¹⁵N partitioning occurred in favour of the rest of the plant outside the source-sink unit, but removal of the competitve sinks from the rest of the plants nullified any partitioning outside the unit. Unlike the situation in ¹³C, no partitioning of ¹⁵N occurred in favour of the lateral leaflets from the terminal leaflet inside the unit. It is concluded that sink demand influences partitioning of both C and N and the translocation of carbon is different from that of nitrogen within a source-sink unit. The translocation of the N is more adjustive to a demand from other sink units compared to the C.     

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.     

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.     

1H, 15N, 13C and 13CO assignments and secondary structure determination of basic fibroblast growth factor using 3D heteronuclear NMR spectroscopy

Summary The assignments of the ¹H, ¹⁵N, ¹³CO and ¹³C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled ¹⁵N- and ¹⁵N-/¹³C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide ¹H and ¹⁵N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic ¹H and ¹³C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as ³J_H n _H coupling constants, amide exchange and ¹³C and ¹³C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined.     

Fine Roots vs. Needles: A Comparison of 13C and 15N Dynamics in a Ponderosa Pine Forest Soil

Plant allocation patterns may affect soil C and N storage due to differences in litter quality and the depth of plant C and N inputs into the soil. We studied the dynamics of dual-labeled (¹³C/¹⁵N) Pinus ponderosa needles and fine roots placed at two soil depths (O and A horizon) in a temperate conifer forest soil during 2 y. Input of C as fine roots resulted in much more C retained in soil (70.5 ± 2.2% of applied) compared with needle C (42.9 ± 1.3% of applied) after 1.5 y. Needles showed faster mass loss, rates of soil ¹³CO₂ efflux, and more ¹⁵N immobilized into microbial biomass than did fine roots. The larger proportion of labile C compounds initially present in needles (17% more needle C was water soluble than in fine roots) likely contributed to its shorter C residence time and greater degree of transformation in the soil. A double exponential decay function best described the rate of ¹³C loss, with a smaller initial pulse of C loss from fine roots (S₁k₁) and a slower decay rate of the recalcitrant C pool for fine roots (0.03 y⁻¹) compared with (0.19 y⁻¹) for needles. Soil ¹³C respiration, representing heterotrophic respiration of litter C, was much more seasonal from the O horizon than from the A. However, offsetting seasonal patterns in ¹³C dynamics in the O horizon resulted in no net effect of soil depth on total ¹³C retention in the soil after 1.5 y for either litter. Almost 90% of applied litter N was retained in the soil after 1.5 y, independent of litter quality or soil depth. Very small amounts of ¹³C or ¹⁵N (<3% of applied) moved to the horizon above or below the placement depth (i.e., O to A or A to O). Our results suggest that plant allocation belowground to fine roots results in more C retained and less N mineralized compared with allocation aboveground to needles, primarily due to litter quality differences.     

Time-saving methods for heteronuclear multidimensional NMR of (13C, 15N) doubly labeled proteins

Summary Heteronuclear 2D (¹³C, ¹H) and (¹⁵N, ¹H) correlation spectra of (¹³C, ¹⁵N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC heteronuclear multiple-quantum coherence spectroscopy - HSQC heteronuclear single-quantum coherence spectroscopy - HSMQC heteronuclear single- and multiple-quantum coherence spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy     

A combined HNCA/HNCO experiment for 15N labeled proteins with 13C at natural abundance

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.     

Measurements of abundances of 15N and 13C as tools in retrospective studies of N balances and water stress in forests: A discussion of preliminary results

Preliminary attempts to make retrospective studies of N balances and water stress in forest fertilization experiments by analyzing changes in the abundances of ¹⁵N and ¹³C, respectively, are discussed. Most evidence is from the Swedish Forest Optimum Nutrition Experiments, which have been running for two decades. Annual additions of N have been given either alone or in combination with other elements, notably P and K, every third year. Processes leading to loss of N, e.g. volatilization of ammonia, nitrification followed by leaching or denitrification, and denitrification alone, discriminate against the heavy isotope ¹⁵N. A correlation was found between fractional losses of added N and the change in ¹⁵N () during 19 years in current needles in a Scots pine forest, irrespective of source of N. Isotope effects were larger on urea than on ammonium nitrate plots (2 as compared to 9 ¹⁵N ()) because of ammonia volatilization and higher rates of nitrification. They developed gradually over time, which opens possibilities to analyse the development of N saturation. However, the analysis may be confounded by shifts in ¹⁵N abundance of fertilizer N. In another trial, N isotope effects could be seen in both plants and soils 10 years after the last fertilization; they were smaller in soils because of a large pretreatment memory effect, but we expect them to persist there for decades.The enzyme RuBisCo discriminates strongly against the heavy isotope ¹³C during photosynthesis, but this effect becomes less expressed as stomata close because of water stress. The supply of N may also affect the ¹³C () via effects on rates of photosynthesis, and the source of N may have an influence directly via non-RubisCo carboxylations, and indirectly via effects on water use efficiency. In a trial with Norway spruce, the effect of N fertilization on the ¹³C () of current needles was strongly correlated with production and weakly so with foliar biomass a dry year, but not a wet year. This suggested that these variations are primarily related to induced differences in the balance between supply and demand for water. Hence, studies of {au13}C abundance can disentangle the role of water as such from its effects on mineralization of N and flow of N.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Kavalactones from Piper methysticum, and their 13C NMR spectroscopic analyses

The kavalactone, 11-methoxy-5,6-dihydroyangonin, and eight previously reported analogs along with four other aromatic compounds were isolated from the root extracts of Piper methysticum (Kava Kava). Their structural elucidations were made by 1H and 13C NMR spectroscopic assignments using COSY, HMBC and HMQC experiments.     

Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.     

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.     

Hydrogen, carbon and nitrogen isotopic fractionations during chlorophyll biosynthesis in C3 higher plants

We determined hydrogen, carbon and nitrogen isotopic compositions of chlorophylls a and b isolated from leaves of five C3 higher plant species (Benthamidia japonica, Prunus japonica, Acer carpinifolium, Acer argutum and Querus mongloica), and hydrogen and carbon isotopic compositions of phytol and chlorophyllides in the chlorophylls to understand isotopic fractionations associated with chlorophyll biosynthesis in these species. Chlorophylls are depleted in D relative to ambient water by approximately 189 per thousand and enriched in (13)C relative to bulk tissue by approximately 1.6 per thousand. These data can be explained by the contribution of isotopic fractionations during phytol and chlorophyllide biosyntheses. Phytol is more depleted in both D (by approximately 308 per thousand) and (13)C (by approximately 4.3 per thousand), while chlorophyllides are less depleted in D (by approximately 44 per thousand) and enriched in (13)C (by approximately 4.8 per thousand). Such inhomogeneous distribution of isotopes in chlorophylls suggests that (1) the phytol in chlorophylls reflects strong D- and (13)C-depletions due to the isotopic fractionations during the methylerythritol phosphate pathway followed by hydrogenation, and (2) the chlorophyllides reflect D- and (13)C-enrichments in tricarboxylic acid cycle. On the other hand, chlorophylls are slightly ( approximately 1.2 per thousand) depleted in (15)N relative to the bulk tissue, indicating that net isotopic fractionation of nitrogen during chlorophyll biosynthesis is small compared with those of hydrogen and carbon.