Assignment of the protonated 13C resonances of apo-neocarzinostatin by 2D heteronuclear NMR spectroscopy at natural abundance

Summary Nearly complete assignment of the protonated carbon resonances of apo-neocarzinostatin, 113-amino acid antitumor antibiotic carrier protein, has been achieved at natural ¹³C abundance using heteronuclear 2D experiments. Most of the cross peaks in the proton-carbon correlation map were identified by the combined use of HMQC, HMQC-RELAY and HMQC-NOESY spectra, using already published proton chemical shifts. However, double-DEPT and triple-quantum experiments had to be performed for the edition of CH and CH₂ side-chain groups, respectively, which were hardly visible on HMQC-type maps. The triple-quantum pulse sequence was adapted from its original scheme to be applicable to a natural abundance sample. The correlation between carbon chemical shifts and the apo-neocarzinostatin structure is discussed. In particular, ¹³C alpha secondary shifts correlate well with the backbone conformation. These shifts also yield information about the main-chain flexibility of the protein. Assignments reported herein will be used further for interpretation of carbon relaxation times in a study of the internal dynamics of apo-neocarzinostatin.     

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     

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.     

Biochemical synthesis of uniformly 13C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta

Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly ¹³C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC–MS and ¹³C-NMR. Application of the ¹³C-labeled substrates on rice leaves led to the detection of ¹³C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-¹³C₂₀] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.     

Recent progress in heteronuclear long-range NMR of complex carbohydrates: 3D H2BC and clean HMBC

The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of ¹³C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the ¹H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution.     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.     

A (13)C NMR study on the interactions of calcium chloride/potassium chloride with pyranosides in D(2)O

The (13)C NMR spectra of methyl beta-d-glucopyranoside, methyl beta-d-galactopyranoside, methyl beta-d-xylopyranoside, and methyl beta-l-arabinopyranoside were recorded in CaCl(2)/KCl+D(2)O mixtures and in D(2)O. The chemical shifts of C-1, C-3, and C-5 in the methyl beta-d-glucopyranoside and methyl beta-d-galactopyranoside decrease rapidly as molalities of CaCl(2)/KCl increase, while those of C-1, C-2, and C-3 in the methyl beta-d-xylopyranoside and methyl beta-l-arabinopyranoside decrease rapidly as molalities of CaCl(2)/KCl increase. Cations (Ca(2+)/K(+)) can weakly complex with O in OMe of the pyranosides studied. Results are discussed in terms of the stereochemistry of the pyranoside molecules and the structural properties of the ions.     

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Summary The growth factor receptor-bound protein-2 (Grb2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly ¹³C-/¹⁵N-enriched protein as well as the protein containing selectively ¹⁵N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, ¹H, ¹³C, ¹³C and ¹³CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423–438]. The core structure of the SH2 domain contains an antiparallel -sheet and two -helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.Abbreviations crk viral p47^gag-crk - EGF epidermal growth factor - GAP GTPase-activating protein - PI3K phosphatidylinositol-3-kinase - PLC- phospholipase-C-, shc, src homologous and collagen - src sarcoma family of nonreceptor tyrosine kinase     

Assignment of the backbone carbonyl resonances in 15N-labelled proteins with 13C at natural abundance by a 2D triple-resonance correlation technique

Summary A 2D NMR experiment for assignment of backbone carbon resonances in small and medium-sized ¹⁵N-labelled proteins with ¹³C at natural abundance is presented. The experiment is a two-dimensional variant of the HNCO triple-resonance experiment and is demonstrated by application to a 6 kDa protein at relatively low concentration (2 mM) and temperature (30°C). The experiment is particularly suitable for assignment of carbonyl resonances.     

Intercalation complex of imidazoacridinone C-1311, a potential anticancer drug,with DNA helix d(CGATCG)2: stereostructural studies by 2D NMR spectroscopy

Imidazoacridinone C-1311 (Symadex®) is a powerful antitumor agent, which successfully made its way through the Phase I clinical trials and has been recommended for Phase II few a years ago. It has been shown experimentally that during the initial stage of its action C-1311 forms a relatively stable intercalation complex with DNA, yet it has shown no base-sequence specificity while binding to DNA. In this paper, the d(CGATCG)₂:C-1311 intercalation complex has been studied by means of two-dimensional NMR spectroscopy, yielding a full assignment of the resonance lines observed in ¹H NMR spectra. The observation of the intermolecular NOE contacts between C-1311 and DNA allowed locating the ligand between the guanine and adenine moieties. Formation of a symmetric complex was pointed out on the basis of the lack of a second set of the ¹H resonances. The resulting stereostructure of the complex was then improved by means of molecular dynamics, using the CHARMM force field and GROMACS software. To this end, distance restraints derived from the NOESY cross-peak volumes were applied to the atomistic model of the d(CGATCG)₂:C-1311 complex. Obtained results are in full agreement with biochemical data on the mechanism of action of C-1311, in particular with the previously postulated post-intercalation enzymatic activation of the studied drug.     

Conformation and dynamics changes of bacteriorhodopsin and its D85N mutant in the absence of 2D crystalline lattice as revealed by site-directed C NMR

¹³C NMR spectra of [3-¹³C]Ala- and [1-¹³C]Val-labeled D85N mutant of bacteriorhodopsin (bR) reconstituted in egg PC or DMPC bilayers were recorded to gain insight into their secondary structures and dynamics. They were substantially suppressed as compared with those of 2D crystals, especially at the loops and several transmembrane α_II-helices. Surprisingly, the ¹³C NMR spectra of [3-¹³C]Ala-D85N turned out to be very similar to those of [3-¹³C]Ala-bR in lipid bilayers, in spite of the presence of globular conformational and dynamics changes in the former as found from 2D crystalline preparations. No further spectral change was also noted between the ground (pH 7) and M-like state (pH 10) as far as D85N in lipid bilayers was examined, in spite of their distinct changes in the 2D crystalline state. This is mainly caused by that the resulting ¹³C NMR peaks which are sensitive to conformation and dynamics changes in the loops and several transmembrane α_II-helices of the M-like state are suppressed already by fluctuation motions in the order of 10⁴-10⁵ Hz interfered with frequencies of magic angle spinning or proton decoupling. However, ¹³C NMR signal from the cytoplasmic α-helix protruding from the membrane surface is not strongly influenced by 2D crystal or monomer. Deceptively simplified carbonyl ¹³C NMR signals of the loop and transmembrane α-helices followed by Pro residues in [1-¹³C]Val-labeled bR and D85N in 2D crystal are split into two peaks for reconstituted preparations in the absence of 2D crystalline lattice. Fortunately, ¹³C NMR spectral feature of reconstituted [1-¹³C]Val and [3-¹³C]Ala-labeled bR and D85N was recovered to yield characteristic feature of 2D crystalline form in gel-forming lipids achieved at lowered temperatures.     

Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance (13)C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the [(1)H]-(13)C NOE were determined in this study. The C alpha H relaxation measurements were compared to the previously measured (15)N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the chi(1) dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than +/-25 degrees.     

The heterogeneous nature of microbial products as shown by solid-state13C CP/MAS NMR spectroscopy

Homoionic Na-, Ca-, and Al-clays were prepared from the <2 m fractions of Georgia kaolinite and Wyoming bentonite and mixed with sand to give artificial soils with 5, and 25% clay. The artificial soils were inoculated with microbes from a natural soil before incubation. Unlabelled and uniformly¹³C-labelled (99.9% atom) glucose were incorporated into the artificial soils to study the effects of clay types, exchangeable cations and clay contents on the mineralization of glucose-carbon and glucose-derived organic materials. Chemical transformation of glucose-carbon upon incorporation into microbial products and metabolites, was followed using solid-state¹³C CP/MAS NMR spectroscopy.There was a significant influence of exchangeable cations on the mineralization of glucose-carbon over a period of 33 days. At 25% clay content, mineralization of glucose-carbon was highest in Ca-soils and lowest in Al-soils. The influence of exchangeable cations on mineralization of glucose-carbon was more pronounced in soils with bentonite clay than those with kaolinite clay. Statistical analysis of data showed no overall effect of clay type on mineralization of glucose-carbon. However, the interactions of clay type with clay content and clay type with clay content and exchangeable cations were highly significant. At 25% clay content, the mineralization of glucose-carbon was significantly lower in Na- and Al-soils with Wyoming bentonite compared with Na- and Al-soils with Georgia kaolinite. For Ca-soils this difference was not significant. Due to the increased osmotic tension induced by the added glucose, mineralization of glucose-carbon was slower in soils with 5% clay than soils with 25% clay.Despite the differences in the chemical and physical characteristics of soils with Ca-, Na- and Al-clays, the chemical composition of organic materials synthesised in these soils were similar in nature. Assuming CP/MAS is quantitative, incorporation of uniformly¹³C-labelled glucose (99.9% atom) in these soils resulted in distribution of carbon in alkyl (24–25%), O-alkyl (56–63%), carbonyl (11–15%) and small amounts of aromatic and olefinic carbon (2–4%). However, as decomposition proceeded, the chemistry of synthesised material showed some changes with time. In the Ca- and Na-soils, the proportions of alkyl and carbonyl carbon decreased and that of O-alkyl carbon increased with time of incubation. However, the opposite trend was found for the Al-soil.Proton-spin relaxation editing (PSRE) subspectra clearly showed heterogeneity within the microbial products. Subspectra of the slowly-relaxing (long T₁(H)) domains were dominated by alkyl carbon in long- and short-chain structures. The signals due to N-alkyl (55 ppm) and carbonyl carbon were also strong in these subspectra. These subspectra were very similar to those obtained for microbial and fungal materials and were probably microbial tissues attached to clay surfaces by polysaccharide extracellular mucilage. Subspectra of fast-relaxing (short T₁(H)) domains comprised mostly O-alkyl and carbonyl carbon and were probably microbial metabolites released as neutral and acidic sugars into the extracellular environment, and strongly sorbed by clay surfaces.     

Noninvasive evaluation of liver metabolism by 2H and 13C NMR isotopomer analysis of human urine

Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.     

Study of hydration of cross-linked high amylose starch by solid state 13C NMR spectroscopy

Starch is subjected to chemical treatments such as cross-linking or hydroxypropylation to meet the material requirements for food uses or controlled release in the pharmaceutical industries. In this work, two types of cross-linking formulations have been employed for the preparation of high amylose starch for use as an excipient for sustained drug release. The structural differences and chain dynamics of the modified starches in the dry and hydrated states have been compared by the use of variable contact time cross polarization-magic angle spinning solid state (13)C NMR spectroscopy.     

Crystal structure and solid state 13C NMR analysis of nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-gluco- and D-galactopyranosides

The X-ray diffraction analysis of o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside (1), m-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, p-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside and o-nitrophenyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside was performed. It was found that except in the case of 1, all other crystals have one molecule in the independent part of the crystal unit cell. The results support the opinion that the nitro group does not conjugate effectively with the phenyl ring. In the 13C CP MAS spectrum of 1 the signals are split, confirming the presence of two independent molecules. Similarly, the 13C CP MAS NMR spectrum of p-nitrophenyl-2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside indicated the presence of two non-equivalent molecules in the crystal unit. One of these molecules has more conformational freedom enabling rotation of the phenyl ring.     

Construction of a DNA four-way junction: Design and NMR spectroscopy

In 1964 Holliday postulated the formation of cruciform structures (four-way junctions) in duplex DNA as intermediate in genetic recombination. Since then, many biochemical and biophysical investigations were directed at solving questions concerning structural details of stable four-way junctions. Thus far, NMR spectroscopy played a minor part in these investigations on account of the minimum size of the molecule (expressed as the number of nucleotide residues) that was thought necessary to produce a stable cruciform structure. Indeed, the smallest four-way junction studied thus far by NMR methods was built from four separate DNA strands, each containing 16 nucleotides, a total of 64. Obviously, with such a large structure one runs into assignment problems. We considered the possibility of constructing a stable four-way junction from a single strand of DNA. The underlying idea was to make use of our detailed knowledge of the building principles of stable minihairpin loops. These loops, containing only two nucleotides to bridge the gap between antiparallel strands, are maximally stable in DNA sequences like 5-d(-C-TT-G-), where C and G form a normal Watson-Crick base pair and the two T residues cross the minor groove to form the minihairpin loop. Three of such miniloops could in principle cap three arms of the cruciform. The fourth arm would have an open end. The problem to be solved is to find the minimum length that is required to insure stability of the three closed arms and of the fourth open arm. We were successful with a structure that has three short stems (four base pairs each) and an open-end stem consisting of eight base pairs, a total of 46 residues. NMR experiments, carried out on this molecule in the presence of Mg²⁺, showed details of folding which have never been observed before.     

A13C NMR study of the hinge region of a mouse monoclonal antibody

Summary A¹³C NMR study is reported of the hinge region of an intact mouse monoclonal antibody with a molecular weight of 150 K. Cys, Ile, and Pro analogs of the antibody labeled with¹³C at the carbonyl carbon were prepared by growing hybridoma cells in the serum-free media. Resonance assignments have been performed as described previously [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I. and Arata, Y. (1991)Biochemistry,30, 270–278]. The spectral data obtained show that¹³C NMR can give detailed information about the structure of the hinge region of the intact antibody molecule. Prospects for the future role of¹³C NMR in the structural analyses of larger proteins are briefly discussed.Dedicated to the memory of Professor V.F. Bystrov     

Metabolic profiling by 13C-NMR spectroscopy: [1,2-13C2]glucose reveals a heterogeneous metabolism in human leukemia T cells

Metabolic profiling is defined as the simultaneous assessment of substrate fluxes within and among the different pathways of metabolite synthesis and energy production under various physiological conditions. The use of stable-isotope tracers and the analysis of the distribution of labeled carbons in various intermediates, by both mass spectrometry and NMR spectroscopy, allow the role of several metabolic processes in cell growth and death to be defined. In the present paper we describe the metabolic profiling of Jurkat cells by isotopomer analysis using (13)C-NMR spectroscopy and [1,2-(13)C(2)]glucose as the stable-isotope tracer. The isotopomer analysis of the lactate, alanine, glutamate, proline, serine, glycine, malate and ribose-5-phosphate moiety of nucleotides has allowed original integrated information regarding the pentose phosphate pathway, TCA cycle, and amino acid metabolism in proliferating human leukemia T cells to be obtained. In particular, the contribution of the glucose-6-phosphate dehydrogenase and transketolase activities to phosphoribosyl-pyrophosphate synthesis was evaluated directly by the determination of isotopomers of the [1'-(13)C], [4',5'-(13)C(2)]ribosyl moiety of nucleotides. Furthermore, the relative contribution of the glycolysis and pentose cycle to lactate production was estimated via analysis of lactate isotopomers. Interestingly, pyruvate carboxylase and pyruvate dehydrogenase flux ratios measured by glutamate isotopomers and the production of isotopomers of several metabolites showed that the metabolic processes described could not take place simultaneously in the same macrocompartments (cells). Results revealed a heterogeneous metabolism in an asynchronous cell population that may be interpreted on the basis of different metabolic phenotypes of subpopulations in relation to different cell cycle phases.