Heteronuclear Two-dimensional NMR Research Articles

Anthocyanins with an unusual acylation pattern from stem of Allium victorialis

Two novel anthocyanins have been isolated from the stem of Allium victorialis. By means of chemical degradation and spectroscopy, especially homo- and hetero-nuclear two-dimensional NMR techniques, the structures were determined to be cyanidin 3- O-(3″,6″- O-dimalonyl-β-glucopyranoside) (76.6%) and cyanidin 3- O-(3″, O-malonyl-β-glucopyranoside) (13.8%). This is the first report of acylation of the 3-position in the sugar moiety of any anthocyanin. The stability of malonyl substitution in the 3″-position on glucose is higher than the corresponding 6″-malonylation.

Structural Elucidation of the Biological Repeating Unit of O‐specific Polysaccharide from Citrobacter Serotype O41

Sugar analysis of the O-specific polysaccharide produced by Citrobacter serotype O41 revealed the presence of a hexasaccharide repeating unit, which includes the unusual 3-amino-N-(D-3'-hydroxybutyryl)-3,6-dideoxy-D-galactosyl residue (Fuc3NAcyl). The structure of the repeating unit was determined by extensive use of homo- and heteronuclear two-dimensional NMR spectroscopy, including the application of long-range 1H-13C correlation experiments and NOE studies to establish the sequence of sugar units. Indentification of the Glc beta 1-->2Fuc3NAcyl beta 1-->6GlcNAc alpha 1-->sequence at the non-reducing terminus establishes the biological repeating unit of this O-specific polysaccharide as: -->2Glc beta 1-->Fuc3NAcyl-beta 1-->6GlcNAc alpha 1-->(Glc alpha 1-->2) 4Gal beta 1-->3GalNAc beta 1-->. This structure is similar to that found for the O-specific polysaccharide isolated from Hafnia alvei strain 1211 [Katzenellenbogen, E., Romanowska, E., Dabrowski, U. & Dabrowski, J. (1991) Eur. J. Biochem. 200, 401-407], which differs in having an acetyl substituent at O4 of the Fuc3NAcyl residue and a branch point of GalNAc alpha substituted by Glc beta at O3; these differences are responsible for the only weak serological cross-reactivity of the two strains.

NMR structure determination of tick anticoagulant peptide (TAP).

Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.

PH Titration of the histidine residues of cyclophilin and FK506 binding protein in the absence and presence of immunosuppressant ligands

Histidine residues in immunophilins, particularly His-126 of cyclophilin (CyP) and His-87 of the FK506 binding protein (FKBP), have been suggested to play important roles in ligand binding and peptidyl prolyl cis-trans isomerase (PP iase) catalysis. The charged states of the histidine residues in FKBP and CyP, which were characterized by their p K a values, have been determined in the absence and presence of the immunosuppressant ligands, ascomycin and cyclosporin A (CsA), respectively, by using a heteronuclear two-dimensional NMR method. Overall, the histidine residues in FKBP and CyP are very acidic with p K a values ranging from ≤ 2.8 to 6.5, indicating that they are predominantly uncharged at physiological pH. To our knowledge, the p K a value of ≤ 2.8 determined from this study is the lowest p K a reported for the free imidazole ring of the histidine residues in proteins. The abnormally acidic p K a 's of His-25 in FKBP and His-54 in CyP could be explained by their highly positively charged environments. His-87 of FKBP, which is located in the FK506 binding pocket, was found to exist in two forms in free FKBP with p K a values of 5.9 and 6.5 for the major and minor forms, respectively. His-126, which is part of the CsA and substrate binding site, has a p K a of 6.3 in free CyP. The p K a values of these two histidine residues in the free proteins are higher than the p K a 's obtained for the peptidyl prolyl cis-trans isomerase (PP iase) activity of these enzymes, indicating that the acid/base characters of His-87 of FKBP and His-126 of CyP are not essential in the PPiase catalysis. The hydrogen bonding of the histidine imidazole rings and the effect of hydrophobicity upon changes in p K a values are discussed.

Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR.

All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.

H2-forming N5, N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum catalyzes a stereoselective hydride transfer as determined by two-dimensional NMR spectroscopy.

5,6,7,8-Tetrahydromethanopterin is a coenzyme playing a key role in the energy metabolism of methanogenic archaea. In Methanobacterium thermoautotrophicum, the reduction of N5, N10-methenyl-5,6,7,8-tetrahydromethanopterin at C(14a) with H2 to N5, N10-methylene-5,6,7,8-tetrahydromethanopterin can be catalyzed by H2-forming methylenetetrahydromethanopterin dehydrogenase, a new hydrogenase present in most methanogenic archaea, which is unique because it does not contain nickel or iron/sulfur clusters. In this work, the stereochemistry of this enzymatic hydride-transfer reaction is elucidated by means of a series of heteronuclear two-dimensional NMR experiments. It is found that the hydride from H2 is transferred by the enzyme into the rel-(pro-R) position of the C(14a) methylene group of the reaction product N5, N10-methylene-5,6,7,8-tetrahydromethanopterin. NMR experiments are described that show that the hydrogen nucleus of the hydride transferred to the oxidized coenzyme partially originates from water. The stereochemical course of this reaction is the same as that for direct hydride transfer. It is demonstrated that the diastereotopic atoms at C(14a) of the reaction product epimerize in an uncatalyzed reaction under the conditions of operation of the enzyme (k = 0.01 s-1 at 58 degree C and pH 6.5).(ABSTRACT TRUNCATED AT 250 WORDS)

1H and 15N Nuclear Magnetic Resonance Assignments, Secondary Structure in Solution, and Solvent Exchange Properties of Azurin from Alcaligenes denitrificans

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) = 14,000, 129 residues) from Alcaligenes denitrificans have been obtained at pH 5.5 and 32 degrees C using homo- and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Comparison of the resonance assignments for the backbone protons with those of Pseudomonas aeruginosa azurin, which is 68% homologous in its amino acid sequence and has a very similar three-dimensional structure, showed a high similarity in chemical shift positions. After adjustment for random coil contributions the mean difference in NH chemical shifts is 0.00 ppm (root mean square width = 0.30 ppm), whereas for C alpha protons the mean difference is 0.09 ppm (root mean square width = 0.23 ppm). Characteristic NOE connectivities and 3JHN alpha values were used to determine the secondary structure of azurin in solution. Two beta-sheets, one helix, and nine tight and four helical turns were identified, and some long-range NOE contacts were found that connect the helix with the beta-sheets. The secondary structure obtained is in agreement with the structure derived from X-ray diffraction data [Baker, E. N. (1988) J. Mol. Biol. 203, 1071-1095]. Studies of the hydration of the protein in the vicinity of the copper ligand residue His117 revealed that the solvent-exposed N epsilon 2 of His117 is in slow exchange with the bulk solvent. However, no evidence was obtained for the presence of a long-lived water molecule at the position corresponding to a well-defined water molecule observed in the crystal structures of A. denitrificans and Ps. aeruginosa azurin.

Fine structure of 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (m-THPC): a 1H, 13C and 15N NMR study

The 1H, 13C and natural abundance 15N NMR spectra of tetrakis(m-hydroxyphenyl)chlorin (m-THPC) are assigned through the complementary analysis of the data from one-dimensional, and both homo- and hetero-nuclear two-dimensional NMR experiments. The analysis of the 1H and 13C spectra showed that the symmetry of the structure includes an effective mirror plane, σv, and the 1H–15N coupling constant of 99 Hz (measured from the ‘inverse-mode’1H–15N 2D experiment) showed that the central imino protons undergo slow intramolecular tautomeric exchange. The single 15N chemical shift (δ 111.8 relative to 15NH4+) of the proton-bearing nitrogens is characteristic of a ‘pyrrole’ type, and the long range coupling 1H chemical shift correlation (2D) spectrum showed that, on the NMR time scale, the imino hydrogens are localised on five-membered rings that are not in the σv plane. Taken together these data prove that the fine structure of m-THPC is correctly represented with the imino hydrogens placed on opposite rings which are not reduced, as shown in structure 4.

Backbone dynamics of ribonuclease T1 and its complex with 2?GMP studied by two-dimensional heteronuclear NMR spectroscopy

The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2'GMP have been studied by (15)N longitudinal and transverse relaxation experiments, combined with {(1)H, (15)H} NOE measurements. The intensity decay of individual amide cross peaks in a series of ((1)H, (15)N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972-8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359-375) was fitted to a single exponential by using a simplex algorithm in order to obtain (15)N T(1) and T(2) relaxation times. These experimentally obtained values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559; 4559-4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.

Homonuclear and heteronuclear NMR studies of oxidized Desulfovibrio vulgaris flavodoxin. Sequential assignments and identification of secondary structure elements.

Recombinant Desulfovibrio vulgaris flavodoxin (molecular mass 16.3 kDa) was produced in Escherichia coli. The oxidized protein has been investigated with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Sequence-specific assignment of all backbone and most of the side chain 1H and 15N resonances has been obtained. The secondary structure has been inferred from the pattern of sequential, medium-, and long-range NOEs, together with information about slowly exchanging amide hydrogens and HN-H alpha spin-spin coupling constants. In solution, flavodoxin consists of a five-stranded parallel beta-sheet and four alpha-helices. Residues 3-9, 32-36, 52-58, 87-96, and 123-128 are involved in the beta-sheet whereas the a-helical regions comprise residues 13-28, 69-76, 104-114, and 134-148. Several proton resonances of the bound flavin mononucleotide cofactor have been assigned. NOE contacts between the prosthetic group and the apoprotein have been detected.

Isotope-edited nuclear magnetic resonance study of Fv fragment of anti-dansyl mouse monoclonal antibody: recognition of the dansyl hapten

An isotope-edited proton nuclear magnetic resonance study is reported of Fv, which is the smallest antigen recognition unit composed of VH and VL domains. Fv has been obtained by clostripain digestion of a short-chain anti-dansyl mouse IgG2a monoclonal antibody [Igarashi, T., Sato, M., Katsube, Y., Takio, K., Tanaka, T., Nakanishi, M., & Arata, Y. (1990) Biochemistry 29, 5727-5733]. A variety of stable-isotope-labeled anti-dansyl Fv analogues have been prepared. The aromatic proton resonances for all Tyr residues of the Fv fragment have been assigned in the absence and presence of epsilon-dansyl-L-lysine by means of isotope-edited homonuclear and heteronuclear two-dimensional NMR experiments. On the basis of the established assignments, it has been concluded that the dansyl ring is bound through Tyr-96H and Tyr-104H to both ends of H3, the third hypervariable region of the heavy chain. We also suggest that the antigen binding results in the formation of a hydrophobic core comprising the dansyl ring and the aromatic rings of Tyr-96H and Tyr-104H.

High-field NMR study of the tautomers resulting from the condensation of 2-methylindole and phenalenone in the presence ofp-toluenesulphonic acid

As phenalene is an alternant hydrocarbon it is oxidized very easily to phenalenone, which condenses with various nitrogen nucleophiles in the presence of p-toluenesulphonic acid. 2-Methylindolylphenalene is obtained with 2- methylindole, together with other products. This product is a mixture of five tautomers in equilibrium, and these were analysed by homo- and heteronuclear two-dimensional high-field NMR (500 MHz) spectroscopy.

Sequential 1H and 15N NMR assignments and secondary structure of a recombinant anti-digoxin antibody VL domain.

A uniformly 15N-labeled recombinant light-chain variable (VL) domain from the anti-digoxin antibody 26-10 has been investigated by heteronuclear two-dimensional (2D) and three-dimensional (3D) NMR spectroscopy. Complementary homonuclear 2D NMR studies of the unlabeled VL domain were also performed. Sequence-specific assignments for 97% of the main-chain and 70% of the side-chain proton resonances have been obtained. Patterns of nuclear Overhauser effects observed in 2D NOESY, 3D NOESY-HSQC, and 3D NOESY-TOCSY-HSQC spectra afford a detailed characterization of the VL domain secondary structure in solution. The observed secondary structure--a nine-stranded antiparallel beta-barrel--corresponds to that observed crystallographically for VL domains involved in quaternary associations. The locations of slowly exchanging amide protons have been discerned from a 2D TOCSY spectrum recorded after dissolving the protein in 2H2O. Strands B, C, E, and F are found to be particularly stable. The possible consequences of these results for domain-domain interactions are discussed.

Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis.

Solution studies of staphylococcal nuclease H124L. 2. Proton, carbon-13, and nitrogen-15 chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3',5'-bisphosphate-calcium ternary complex

Accurate 1H, 15N, and 13C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone 1H and 15N assignments, obtained by analysis of three-dimensional 1H-15N HMQC-NOESY data [Wang, J., Mooberry, E.S., Walkenhorst, W.F., & Markley, J. L. (1992) Biochemistry (preceding paper in this issue)], were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear 1H NMR studies were at natural isotopic abundance or labeled randomly with 2H (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with 15N (to an isotope level greater than 95%) or uniformly with 13C (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single 15N- or 13C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L.pdTp.Ca2+ ternary complex [Wang, J., LeMaster, D. M., & Markley, J.L. (1990) Biochemistry 29, 88-101; Wang, J., Hinck, A.P., Loh, S. N., & Markley, J.L. (1990) Biochemistry 29, 102-113; Wang, J., Hinck, A.P., Loh, S.N., & Markley, J.L. (1990) Biochemistry 29, 4242-4253]. The results reveal that the binding of pdTp and Ca2+ induces large shifts in the resonances of several amino acid segments. These chemical shift changes are interpreted in terms of changes in backbone torsion angles that accompany the binding of pdTp and Ca2+; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds.

How does RNase H recognize a DNA.RNA hybrid?

The mechanism of RNase H substrate recognition is proposed from a model of a chemically modified DNA.RNA hybrid Escherichia coli RNase H complex. Site-directed mutagenesis of the enzyme and substrate titration observed by heteronuclear two-dimensional NMR spectra have been carried out. A model complex has been built, based on free structures of the enzyme and the substrate independently determined by x-ray crystallography and NMR distance geometry, respectively. In addition to steric and electrostatic complementarities between the molecular surfaces of the enzyme and the minor groove of the hybrid in the model, putative hydrogen bonds between the polar groups in the enzyme and 2'-oxygens of the RNA strand of the hybrid fix the hybrid close to the active site of the enzyme. The enzymatic activities of the mutant proteins and the changes in NMR spectra during the course of substrate titration are consistent with the present model. Moreover, the specific cleavage of the RNA strand in DNA.RNA hybrids can be explained as well as cleavage modes in modified heteroduplexes. A mechanism of enzymatic action is proposed.

Cyanidin 3-[6-( p-coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of sambucus canadensis

From the fruits of Sambucus canadensis four anthocyanin glycosides have been isolated by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The structure of the novel, major (69.8%) pigment, cyanidin 3- O-[6- O-( E- p-coumaroyl-2- O-(β- d-xylopyranosyl)-β- d-glucopyranoside]-5- O-β- d-glucopyranoside, was determined by means of chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques. The other anthocyanins were identified as cyanidin 3-sambubioside-5-glucoside (22.7%), cyanidin 3-sambubioside (2.3 %) and cyanidin 3-glucoside (2.1 %).

Histidine-121 of staphylococcal nuclease. Correction of the H.delta.2 1H NMR assignment and reinterpretation of the role this residue plays in conformational heterogeneity of the protein

Heteronuclear two-dimensional NMR studies of wild-type staphylococcal nuclease containing histidine residues uniformly labeled with carbon-13 (26% isotope) have led to full analysis of the aromatic parts of the histidine {sup 1}H and {sup 13}C spin systems. The {sup 1}H{sup {delta}2} and {sup 13}C{sup {delta}2} resonances of His{sup 121} were found to be split as the result of the N{sub al} {r equilibrium} N{sub a2} conformational equilibrium described previously and attributed to cis-trans isomerism about the Lys{sup 116}-Pro{sup 117} peptide bond (N{sub al}, cis; N{sub a2}, trans). The relative intensities of the pair of {sup 1}H{sup {epsilon}1} peaks from the same residue. A soluble-mutant enzyme (nuclease G79S + H124L), which exhibited a drastically altered (N{sub a1}) to (N{sub a2}) ratio, provided additional evidence that the pair of {sup 1}H{sup {delta}2} peaks and the pair of {sup 1}H{sup {epsilon}1} peaks of His{sup 121} report on the same conformational equilibrium (N{sub a1} {r equilibrium} N{sub a2}). The unusual chemical shift of the {sup 1}H{sup {delta}2} of His{sup 121} is attributed to diamagnetic shielding by the aromatic ring of Tyr{sup 91} as verified by ring-current calculations based on two X-ray structures for wild-type staphylococcal nuclease.

31P NMR relaxation measurements of the phosphate backbone of a double stranded hexadeoxynucleotide in solution: determination of the chemical shift anisotropy

The five phosphates of the deoxynucleotide d(CpGpTpApCpG)2 have been assigned by two-dimensional heteronuclear NMR spectroscopy. The chemical shift anisotropy and correlation time of each phosphate group has been determined from measurements of the spin-lattice, spin-spin relaxation rate constants and the 31P-{1H} nuclear Overhauser enhancement (NOE) at three magnetic field strengths (4.7 T, 9.4 T, and 11.75 T) and two temperatures (288 K and 298 K). As expected, the relaxation data require two mechanisms to account for the observed rate constants, i.e. dipole-dipole and chemical shift anisotropy. At 9.4 T and 11.75 T, the latter mechanism dominates the relaxation, leading to insignificant NOE intensities. The correlation time, chemical shift anisotropy and effective P-H distance were obtained from least-squares fitting to the data. Comparison of the fitted value for the correlation time with that obtained from 1H measurements shows that the molecule behaves essentially as rigid rotor on the nanosecond timescale. Large amplitude motions observed in long segments of DNA are due to bending motions that do not contribute significantly to relaxation in short oligonucleotides.

ChemInform Abstract: A Synthetic Entry into the Quino(2,1‐b) (1,3)benzoxazine Ring System.

The double ring closure of N-(2-hydroxybenzyl)anthranilic acids 1 with acetic anhydride or its acyclic analogous (RCH2CO)2O 2b and 2e lead to the formation of novel 12H-quino[2,1-b][1,3]benzoxazin-5-ones. The structure of the obtained compounds was proved with the use of their ir, mass, 1H and 13C nmr spectra. Homo- and heteronuclear two-dimensional nmr experiments were performed to unambiguously assign all proton and carbon chemical shifts.