Monitoring of hypoxic metabolism in superfused plant tissues by in vivo 1H NMR

We have used a coaxial superfusion system to obtain physiologically interpretable in vivo 1H NMR spectra at 500 MHz of carrot roots, maize roots, and rice shoots in water (no 2H2O). The superfusion system was constructed from common laboratory parts, required no modification of the probe and sample loading procedure, and was inherently leak resistant. The assignment and quantitation of the in vivo 1H NMR resonances were achieved by performing two-dimensional NMR experiments in vivo, and by in vitro analysis including NMR and gas chromatography-mass spectrometry. The in vivo spectra were dominated by resonances arising from sugars, organic acids, amino acids, and ethanol. In vivo measurements of spin-lattice relaxation times and chemical shifts of beta protons of malate in carrot roots suggested that malate was located in a relatively viscous and acidic compartment. In rice shoots, the hypoxic time courses of 9 metabolites were established in vivo, and 23 in vitro. In both cases, accumulation of lactate, ethanol, Ala, and gamma-aminobutyrate as well as a decrease in Gln and Asn concentrations were observed. These findings are consistent with accelerated glycolysis and decreased tricarboxylic acid cycle activity.     

Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations

Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.     

The interaction of ammonia and xenon with the imidazole glycerol phosphate synthase from Thermotoga maritima as detected by NMR spectroscopy

The imidazole glycerol phosphate (ImGP) synthase from the hyperthermophilic bacterium Thermotoga maritima is a 1:1 complex of the glutaminase subunit HisH and the cyclase subunit HisF. It has been proposed that ammonia generated by HisH is transported through a channel to the active site of HisF, which generates intermediates of histidine (ImGP) and de novo biosynthesis of 5‐aminoimidazole‐4‐carboxamideribotide. Solution NMR spectroscopy of ammonium chloride‐titrated samples was used to study the interaction of NH₃ with amino acids inside this channel. Although numerous residues showed ¹⁵N chemical shift changes, most of these changes were caused by nonspecific ionic strength effects. However, several interactions appeared to be specific. Remarkably, the amino acid residue Thr 78—which is located in the central channel—shows a large chemical shift change upon titration with ammonium chloride. This result and the reduced catalytic activity of the Thr78Met mutant indicate a special role of this residue in ammonia channeling. To detect and further characterize internal cavities in HisF, which might for example contribute to ammonia channeling, the interaction of HisF with the noble gas xenon was analyzed by solution NMR spectroscopy using ¹H‐¹⁵N HSQC experiments. The results indicate that HisF contains three distinct internal cavities, which could be identified by xenon‐induced chemical shift changes of the neighboring amino acid residues. Two of these cavities are located at the active site at opposite ends of the substrate N′‐[(5′‐phosphoribulosyl)formimino]‐5‐aminoimidazole‐4‐carboxamide‐ribonucleotide (PRFAR) binding groove. The third cavity is located in the interior of the central β‐barrel of HisF and overlaps with the putative ammonia transport channel.     

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.     

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.     

Distribution of N within Pea, Lupin, and Soybean Nodules

The ¹⁵N abundance of some, but not all, legume root nodules is significantly elevated compared to that of the whole plant. It seems probable that differences in ¹⁵N enrichment reflect differences in the assimilatory pathway of fixed N. In that context, we have determined the distribution of naturally occurring ¹⁵N in structural fractions of nodules from soybean (Glycine max L. Merr.), yellow lupin (Lupinus luteus), and pea (Pisum sativum) nodules and in chemical components from soybean nodules and to a lesser extent, pea and lupin nodules. None of the fractions of pea nodules (cortex, bacteriod, or host plant cytoplasm) was enriched in ¹⁵N. The differences among bacteriods, cortex, and plant cytoplasm were smaller in lupin than in soybean nodules, but in both, bacteriods had the highest ¹⁵N enrichment. In soybean nodules, the ¹⁵N abundance of bacteriods and cortex was higher than plant cytoplasm, but all three fractions were more enriched in ¹⁵N than the entire plant. Plant cytoplasm from soybean nodules was fractionated into protein-rich material, nonprotein alcohol precipitable material (NA), and a low molecular weight fraction. The N of the latter was further separated into N of ureides, nucleotides and free amino acids. Most of these components were either similar to or lower in ¹⁵N abundance than the plant cytoplasm as a whole, but the NA fraction showed unusual ¹⁵N enrichment. However, the percentage of nodule N in this fraction was small. NA fractions from yellow lupin and pea nodules and from soybean leaves were not enriched in ¹⁵N. Nor was the NA fraction in ruptured bacteriods and cortical tissue of soybean nodules. Variation among soybean nodule fractions in the preponderance in protein of different amino acids was not large enough to explain the differences in ¹⁵N abundances among them. A hypothesis, consistent with all known data, concerning the mechanism leading to the observed excess ¹⁵N of lupin and soybean bacteriods is offered.     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.     

15N and 13C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through 13C and 15N NMR. To our knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by 13C or 15N NMR. Here we extend our 13C NMR studies to PBGS complexes with [3,3-2H2,3-13C]ALA and report 15N NMR studies of [15N]ALA bound to PBGS. As in our previous 13C NMR studies, observation of enzyme-bound 15N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pKa is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent (kexchange greater than 10(2) s-1). For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C4 of ALA and an active-site lysine. The 13C chemical shift of [3,3-2H2,3-13C]ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between [15N]ALA and hydrazine or hydroxylamine, the 15N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation; again the protons are in rapid exchange with solvent. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C4 of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.     

Amino Acid Metabolism of Lemna minor L. : IV. N-Labeling Kinetics of the Amide and Amino Groups of Glutamine and Asparagine

A serious limitation to the use of N(O,S)-heptafluorobutyryl isobutyl amino acid derivatives in the analysis of ¹⁵N-labeling kinetics of amino acids in plant tissues, is that the amides glutamine and asparagine undergo acid hydrolysis to glutamate and aspartate, respectively, during derivatization. This led us to consider an alternative procedure (G Fortier et al. [1986] J Chromatogr 361: 253-261) for derivatization of glutamine and asparagine with N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide in pyridine. Gas chromatography-mass spectrometry (electron ionization) yielded fragment ions (M-57) of mass 417 and 431 for the [¹⁴N]asparagine and [¹⁴N]glutamine derivatives, respectively, suitable for monitoring unlabeled, single-¹⁵N- and double-¹⁵N-labeled amide species from the ion clusters at mass to charge ratio (m/z) 415 to 423 for asparagine, and m/z 429 to 437 for glutamine. From separate analyses of the specific isotope abundance of the amino-N groups of asparagine and glutamine as their N-heptafluorobutyryl isobutyl derivatives, the specific amide-[¹⁵N] abundance of these amino acids was determined. We demonstrate that this approach to ¹⁵N analysis of the amides can yield unique insights as to the compartmentation of asparagine and glutamine in vivo. The ratios of unlabeled:single-¹⁵N:double-¹⁵N-labeled species are highly diagnostic of the relative sizes and turnover of metabolically active and inactive pools of the amides and their precursors. Kinetic evidence is presented to indicate that a significant proportion (approximately 10%) of the free asparagine pool may be metabolically inactive (vacuolar). If the amide group of asparagine is derived exclusively from glutamine-amide, then asparagine must be synthesized in a compartment of the cell in which both glutamine-amide and aspartate are more heavily labeled with ¹⁵N than the bulk pools of these amino acids. This compartment is presumably the chloroplast. The transaminase inhibitor aminooxyacetate is shown to markedly inhibit amino acid synthesis; several amino acid pools accumulated in the presence of aminooxyacetate and [¹⁵N]H₄⁺ are ¹⁴N-enriched and must be derived primarily from protein turnover.     

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Prospects for resonance assignments in multidimensional solid-state NMR spectra of uniformly labeled proteins

Summary The feasibility of assigning the backbone ¹⁵N and ¹³C NMR chemical shifts in multidimensional magic angle spinning NMR spectra of uniformly isotopically labeled proteins and peptides in unoriented solid samples is assessed by means of numerical simulations. The goal of these simulations is to examine how the upper limit on the size of a peptide for which unique assignments can be made depends on the spectral resolution, i.e., the NMR line widths. Sets of simulated three-dimensional chemical shift correlation spectra for artificial peptides of varying length are constructed from published liquid-state NMR chemical shift data for ubiquitin, a well-characterized soluble protein. Resonance assignments consistent with these spectra to within the assumed spectral resolution are found by a numerical search algorithm. The dependence of the number of consistent assignments on the assumed spectral resolution and on the length of the peptide is reported. If only three-dimensional chemical shift correlation data for backbone ¹⁵N and ¹³C nuclei are used, and no residue-specific chemical shift information, information from amino acid side-chain signals, and proton chemical shift information are available, a spectral resolution of 1 ppm or less is generally required for a unique assignment of backbone chemical shifts for a peptide of 30 amino acid residues.     

Uniform labeling of a recombinant antibody Fv-fragment with 15N and 13C for heteronuclear NMR spectroscopy

The expression of functional antibody fragments in Escherichia coli enables a detailed analysis by NMR spectroscopy. This is demonstrated with the uniform labeling of an Fv-fragment (25 kDa) comprising the antigen binding site of an antibody against 2-phenyloxazolone with 15N and 13C. The antigen-complexed Fv-fragment was analysed for a potential assignment by heteronuclear multi-dimensional NMR spectroscopy. For almost all backbone amides 15N/1H crosspeaks and for 80% of them TOCSY crosspeaks were observed. In a 13C-edited-HCCH-2D experiment 17 out of 18 threonine spin-systems were identified. Thus detailed assignments are possible, but some amino acid specific labeling in addition to uniform labeling will be required for complete assignments of Fv-fragments.     

Backbone NMR assignments and H/D exchange studies on the ferric azide- and cyanide-inhibited forms of Pseudomonas aeruginosa heme oxygenase

The 198 amino acid long heme oxygenase from Pseudomonas aeruginosa (pa-HO) was studied by multinuclear and multidimensional NMR spectroscopy in its paramagnetic cyanide-inhibited (pa-HO-CN) and azide-inhibited (pa-HO-N3) forms. Nearly complete backbone assignments (>93%) of all non-proline residues have been obtained, with the majority of the nonassigned residues corresponding to the first 10 amino terminal residues. Resonances strongly affected by heme iron paramagnetism were assigned with the aid of selective amino acid labeling and experiments tailored to detect fast relaxing signals, whereas the rest of the polypeptide was assigned using conventional three-dimensional NMR experiments. Amide chemical shift assignments were used to monitor the rate of exchange of backbone protons in hydrogen-deuterium exchange experiments. The polypeptide in the pa-HO-N3 complex was found to be significantly less prone to exchange than the polypeptide in pa-HO-CN, which we interpret to indicate that pa-HO-N3 is conformationally less flexible than pa-HO-CN. The differences in protection factors extend to regions of the protein remote from the heme iron and distal ligand. Mapping the differences in protection factors into the X-ray crystal structure of pa-HO [Friedman, J., Lad, L., Li, H., Wilks, A. Poulos, T. L. (2004) Biochemistry 43, 5239-5345] suggests that the distinct chemical properties imparted by the coordination of azide or cyanide to the heme iron [Zeng, Y. Caignan, G. A., Bunce, R. A., Rodríguez, J. C., Wilks, A., Rivera, M. (2005) J. Am. Chem. Soc. 127, 9794-9807] are transmitted to the polypeptide by a network of structural water molecules extending from the active site to the surface of the enzyme. Finally, while the 1H amide resonance of Gly125 was too broad to detect, the corresponding 15N resonance exhibits a large downfield shift, large line width, steep temperature dependence, and a larger than usual upfield deuterium isotope effect. These properties indicate unpaired spin delocalization from the heme iron into the Gly 15N atom via formation of a hydrogen bond between the coordinated azide nitrogen and the Gly125 N-H.     

N6-(1-carboxyethyl)lysine formation by Streptococcus lactis. Purification, synthesis, and stereochemical structure

During growth in an arginine-deficient (chemically defined) medium, cells of Streptococcus lactis K1 formed significant amounts of a previously undetected ninhydrin-positive compound. This intracellular compound did not cochromatograph with any of a wide range of amino acids or amino acid analogs tested. However, by two-dimensional thin layer chromatography, the unknown compound migrated close to the recently discovered N5-(1-carboxyethyl)ornithine (Thompson, J., Curtis, M. A., and Miller, S. P. F. (1986) J. Bacteriol. 167, 522-529; Miller, S. P. F., and Thompson, J. (1987) J. Biol. Chem. 262, 16109-16115). The purified compound behaved as a neutral amino acid and eluted between valine and methionine in the amino acid analyzer. The results of 1H NMR spectroscopy suggested the presence of a lysine backbone and a coupled methyl-methine unit in the molecule, and 13C NMR showed that there were nine carbon atoms, of which two (C-1 and C-7) were carboxyl carbons. The simplest structure compatible with the physicochemical data was that of an alkylated derivative of lysine. The identity of this new amino acid, N6-(1-carboxyethyl)lysine, was confirmed by chemical synthesis. In vivo labeling experiments conducted using L[U-14C]lysine and [epsilon-15N]lysine showed that exogenous lysine served as the precursor of intracellular N6-(1-carboxyethyl)lysine and that the epsilon-amino N atom was conserved during biosynthesis of the lysine derivative. Of the two possible diastereomers (2S,8S or 2S,8R) of N6-(1-carboxyethyl)lysine, comparative 13C NMR spectroscopy established that the amino acid produced by S. lactis K1 was exclusively of the 2S,8S configuration.     

Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.     

Intracellular pH regulation in maize root tips exposed to ammonium at high external pH

Ammonium-induced changes in the cytoplasmic and vacuolar pH values of excised maize (Zea mays L.) root tips, measured by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy, were correlated with the ammonium content of the tissue, determined by 14N NMR. Calculations based on these measurements indicated that the pH changes observed during exposure to 10 mM ammonium for 1 h at pH 9.0, and in the recovery following the removal of the external ammonium supply, were largely determined by the influx and efflux of the weak base NH3. Carboxylate synthesis, detected by both in vivo 13C NMR and the incorporation of [14C]bicarbonate, was stimulated by the ammonium-induced alkalinization of the root tips, but the contribution that this proton-generating process made to pH regulation during and after the ammonium treatment was quantitatively insignificant. Similarly, ammonium assimilation, which was shown to occur via the proton-generating glutamine synthetase/glutamate synthase pathway using in vivo 15N NMR, was also quantitatively insignificant in comparison with the large changes in ammonium content that occurred during the ammonium treatment and subsequent recovery. The results are discussed in relation to several recent studies in which ammonium was used to perturb intracellular pH values, and it is argued (i) that a new method for probing the subcellular compartmentation of amino acids, based on an ammonium-induced alkalinization of the cytoplasm may be difficult to implement in dense heterogeneous tissues; and (ii) that observations on the apparently proton-consuming effect of ammonium assimilation in rice root hairs may actually reflect unusually rapid assimilation.     

Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria.

The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.