2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.     

1H and (13)C NMR of multilamellar dispersions of polyunsaturated (22:6) phospholipids

The polyunsaturated fatty acid docosahexaenoic acid (DHA) makes up approximately 50% of the lipid chains in the retinal rod outer segment disk membranes and a large fraction of the lipid chains in the membranes of neuronal tissues. There is an extensive literature concerned with the dietary requirements for essential fatty acids and the importance of DHA to human health, but relatively little research has been done on the physical properties of this important molecule. Using (1)H and (13)C MAS NMR measurements of dispersions of 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine in excess phosphate buffer, we have unambiguously assigned most of the resonances in both the (1)H and (13)C NMR spectra. We were able to use cross-polarization spectroscopy to follow the transfer of polarization from specific (1)H nuclei not only to their directly bonded (13)C but also to those (13)C that are in close proximity, even though they are not directly bonded. Cross-peaks in two-dimensional cross-polarization spectra revealed a close association between the choline headgroup and at least part of the DHA chain but not with the palmitate chain. Finally, we examined the dynamics of the different parts of this lipid molecule, using rotating frame spin-lattice relaxation measurements, and found that methylene groups of both chains experience important motions with correlation times in the 10-micros range, with those for the palmitate chain being approximately 50% longer than those of the DHA chain. The choline headgroup and the chain terminal groups have significantly shorter correlation times, and that part of the dipolar interaction that is fluctuating at these correlation times is significantly smaller for these groups than it is for the palmitate and DHA chain methylenes.     

1H-, 13C- and 31P-NMR studies of dioctanoylphosphatidylcholine and dioctanoylthiophosphatidylcholine

Coupling constants and chemical shifts were measured for dioctanoylphosphatidylcholine and its thio analogue in a CDCl3/CD3OD solvent mixture. Replacing the bridging oxygen atom of the CH-CH2-O-P portion of the phosphatidylcholine molecule with a sulfur atom affects chemical shifts and coupling constants in the glycerol backbone portion of the molecule as well as in the choline head group region. Preferred conformations about selected bonds in the phospholipids were determined from the vicinal 1H-1H, 31P-1H and 31P-13C coupling constants. A reduction of the 31P T2* (effective spin-spin relaxation time) for the thio analogue, as well as changes in the relative chemical shifts of 13C nuclei in the acyl chains, suggest a somewhat greater degree of aggregation for the thio analogue. The quadrupolar coupling constant 1J(14N-13C) for the choline methyls of either analogue, however, indicates that aggregation of these phospholipids in the CDCl3/CD3OD solvent mixture is not significant. Differences in conformation between dioctanoylphosphatidylcholine and its thio analogue may be responsible for their differences in chemical and physical properties.     

Carbon-13 NMR studies of chloroplast membranes: carbon-13 enrichment without 13C-13C couplings

A novel approach to carbon-13 (13C) enrichment of chloroplast membranes (and plant materials in general) is presented for 13C-nuclear magnetic resonance (13C-NMR) studies. The method minimizes the occurrence of spectral complications arising from 13C-13C couplings resulting from a statistical distribution of 13C within the molecule with low probability of encountering two 13C atoms adjacent to each other. This is achieved by growing the plants in light surrounded by an atmosphere containing 1/3rd 12CO2 and 2/3rd 13CO2, liberated by weak acid-treatment of a mixture of corresponding barium carbonate salts.     

Quantitative deuterium isotopic profiling at natural abundance indicates mechanistic differences for delta 12-epoxidase and delta 12-desaturase in Vernonia galamensis

Quantitative (2)H NMR spectroscopy can determine the natural abundance ((2)H/(1)H) ratio at each site of a molecule. In natural products, variation in these values is related to the reaction mechanisms in the pertinent biosynthetic pathway. For the first time, this novel approach has been exploited to probe for mechanistic differences in the introduction of different functionalities into a long-chain fatty acid. Vernolic acid, a major component of the seed oil of Vernonia galamensis, contains both an epoxide and a desaturation. The site-specific isotopic distribution ((2)H/(1)H)(i) has been determined for both vernolic acid and linoleic acid isolated from the same V. galamensis oil. It is found that the ((2)H/(1)H) ratio of vernolic acid shows a pattern along the entire length of the chain, consistent with linoleic acid being its immediate precursor. Notably, the C13 relates to the C13 of linoleic acid but not to the C13 of oleic acid. Furthermore, the C12 and C13 positions in vernolic acid are less depleted, consistent with a change in hybridization state from sp(2) to sp(3). However, the C11 position shows a marked relative enrichment in the vernolic acid, implying that it plays a role in the epoxidase but not the desaturase mechanism. Thus, although it can be concluded that the catalytic mechanisms for the epoxidase and desaturase activities are similar, marked differences in the residual ((2)H/(1)H) patterns indicate that the reaction mechanisms are not identical.     

Carbon-13 NMR study of switch variant anti-dansyl antibodies: antigen binding and domain-domain interactions

A 13C NMR study is reported of switch variant anti-dansyl antibodies, which possess the identical VH, VL, and CL domains in conjunction with highly homologous but not identical heavy-chain constant regions. Each of these antibodies has been selectively labeled with 13C at the carbonyl carbon of Trp, Tyr, His, or Cys residue by growing hybridoma cells in serum-free medium. Spectral assignments have been made by following the procedure described previously for the switch variant antibodies labeled with [1-13C]Met [Kato, K., Matsunaga, C., Igarashi, T., Kim, H., Odaka, A., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 270-278]. On the basis of the spectral data collected for the antibodies and their proteolytic fragments, we discuss how 13C NMR spectroscopy can be used for the structural analyses of antigen binding and also of domain-domain interactions in the antibody molecule.     

Spectroscopic (FTIR,FT-Raman,NMR and UV) and molecular structure investigations of 1,5-diphenylpenta-2,4-dien-1-one: a combined experimental and theoretical approach

The title molecule 1,5-diphenylpenta-2,4-dien-1-one (cinnamylideneacetophenone, CA) has been synthesised and characterised by FTIR, FT-Raman, NMR and UV–vis spectral analyses. The possible stable conformers of the CA molecule were searched by potential energy surface scan at B3LYP level of theory. The molecular geometry from X-ray determination of the CA molecule in the ground state has been compared using the density functional theory (DFT) with 6-31G(d,p) basis set. The harmonic vibrational modes, the corresponding wavenumbers and IR and Raman intensities of most stable conformer were calculated by the DFT method. The assignments of the fundamentals were proposed on the basis of total energy distribution calculations. The calculated ¹³C and ¹H NMR chemical shifts using gauge including atomic orbitals approach are in good agreement with the observed chemical shifts. The molecular stability and bond strength have been investigated by applying natural bond orbital analysis. Using the time-dependent DFT method, the electronic absorption spectrum of the title compound has been predicted and the electronic transitions within the molecule have been interpreted. The molecular electrostatic potential map was used for predicting possible hydrogen and oxygen bonding sites in the CA molecule.     

Biosynthesis of violacein: intact incorporation of the tryptophan molecule on the oxindole side, with intramolecular rearrangement of the indole ring on the 5-hydroxyindole side

Feeding experiments with a mixture of [2-13C]- and [indole-3-13C]tryptophans, of [3-13C]- and [indole-3-13 C]tryptophans (1:1 molar ratio) and of others have proved that the 1,2-shift of the indole ring occurred via an intramolecular process for formation of the left part (5-hydroxyindole side) of the violacein skeleton and demonstrated that the C-C bond from C2 of the indole ring to C2 of the side chain was completely retained for formation of the right part (oxindole side) during the entire biosynthetic process. Due to the involvement of transaminase, it has remained unresolved whether indolylpyruvic acid is the biosynthetic intermediate and/or from where the nitrogen atom of the pyrrolidone ring originates. An incorporation experiment with a mixture of [2-13C]- and [alpha-15N]tryptophans (1:1 molar ratio) verified that the nitrogen atom in the central ring was exclusively derived from the right-side tryptophan. Thus, all the carbon and nitrogen atoms in the right part of the violacein skeleton were constructed by intact incorporation of the tryptophan molecule, with decarboxylation probably occurring at a later biosynthetic stage.     

Monoclonal antibodies to human protein C: effects on the biological activity of activated protein C and the thrombin-catalyzed activation of protein C1

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.     

Crystal structure and solution conformation of S,S'-bis(Boc-Cys-Ala-OMe): intramolecular antiparallel beta-sheet conformation of an acyclic cystine peptide

The conformation of the acyclic biscystine peptide S,S'-bis(Boc-Cys-Ala-OMe) has been studied in the solid state by x-ray diffraction, and in solution by 1H- and 13C-nmr, ir, and CD methods. The peptide molecule has a twofold rotation symmetry and adopts an intramolecular antiparallel beta-sheet structure in the solid state. The two antiparallel extended strands are stabilized by two hydrogen bonds between the Boc CO and Ala NH groups [N...O 2.964 (3) A, O...HN 2.11 (3) A, and NH...O angle 162 (3) degrees]. The disulfide bridge has a right-handed conformation with the torsion angle C beta SSC beta = 95.8 (2) degrees. In solution the presence of a twofold rotation symmetry in the molecule is evident from the 1H- and 13C-nmr spectra. 1H-nmr studies, using solvent and temperature dependencies of NH chemical shifts, paramagnetic radical induced line broadening, and rate of deuterium-hydrogen exchange effects on NH resonances, suggest that Ala NH is solvent shielded and intramolecularly hydrogen bonded in CDCl3 and in (CD3)2SO. Nuclear Overhauser effects observed between Cys C alpha H and Ala NH protons and ir studies provide evidence of the occurrence of antiparallel beta-sheet structure in these solvents. The CD spectra of the peptide in organic solvents are characteristic of those observed for cystine peptides that have been shown to adopt antiparallel beta-sheet structures.     

Mutational spectrum and recombinogenic effects induced by aminofluorene adducts in bacteriophage M13

Double-stranded replicative form (RFI) DNA of bacteriophage M13 strain M13mp10 which carries partial lacZ gene has been modified in vitro to various extents with N-hydroxy-2-amino-fluorene (N-OH-AF) and then transfected into E. coli cells. High-performance liquid chromatography (HPLC) analysis results demonstrate that the sole adduct (95%) formed in modified DNA is N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF). Approximately 20 adducts per RFI molecule constitute 1 lethal event when plaque-forming ability is assayed on E. coli cells which have received no prior SOS induction. The mutagenicity of dG-C8-AF adducts was assayed by measuring loss of beta-galactosidase activity as a function of adducts per molecule. A dose-dependent increase in Lac- mutants was observed, with a 4-fold increase in mutants per survivor at 30 adducts/molecule. The mutations produced, characterized by DNA sequencing, occur predominantly at either G or C positions different from those observed in the spontaneous mutant spectrum. Restriction-mapping results show that in our assay system, dG-C8-AF adducts induce a previously unreported recombinogenic activity.     

Conformational flexibility of luteinizing hormone-releasing hormone in aqueous solution. A carbon-13 spin-lattice relaxation time study.

The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the T1 data the rates of overall molecular motion and intramolecular motion of side chains have been estimated. LH-RH is a flexible molecule in solution, having segmental motion along the backbone as well as in the nonaromatic side chains.     

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.     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997     

Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In (1)H (15)N HSQC ((1)H (15)N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.     

The crystal and molecular structure of the alpha-helical nonapeptide antibiotic leucinostatin A

The crystal and molecular structure of the nonapeptide antibiotic leucinostatin A, containing some uncommon amino acids and three Aib residues, has been determined by x-ray diffraction analysis. The molecule crystallizes in the orthorhombic space group P2(1)2(1)2(1), a = 10.924, b = 17.810, c = 40.50 A, C62H111N11O13, HCl.H2O, Z = 4. The peptide backbone folds in a regular right-handed alpha-helix conformation, with six intramolecular i----(i + 4) hydrogen bonds, forming C13 rings. The nonapeptide chain includes at the C end an unusual beta-Ala residue, which also adopts the helical structure of the other eight residues. In the crystal the helices are linked head to tail by electrostatic and hydrogen-bond interactions, forming continuous helical rods. The crystal packing is formed by adjacent parallel and antiparallel helical rods. Between adjacent parallel helical columns there are only van der Waals contacts, while between adjacent antiparallel helical columns hydrogen-bond interactions are formed.     

Physical studies of the interaction between detergent-solubilized cholesterol and PC vesicles

The ¹³C NMR spectrum of [4-¹³C] cholesterol solubilized in aqueous solutions of sodium dodecyl sulfate indicates that the molecule has considerably more rotational freedom than when it is intercalated into phosphatidylcholine (PC) vesicles. The interaction between detergent-solubilized cholesterol and PC vesicles has been studied by both NMR and gel permeation chromatography. Our results indicate that greater that 90% of the cholesterol is slowly transferred into the PC bilayer when the detergent-solubilized cholesterol is mixed with PC vesicles.     

Low-temperature 1H-NMR evidence of the folding of isolated ribonuclease S-peptide

The temperature (-7 degrees C to 45 degrees C, pH 5.4) and pH (0 degrees C) dependence of 1H chemical shifts of ribonuclease S-peptide (5 mM, 1 M NaCl) has been measured at 360 MHz. The observed variations evidence the formation of a partial helical structure, involving the fragment Thr-3-Met-13. Two salt-bridges stabilize the helix: those formed by Glu-9- ...His-12+ and Glu-2- ...Arg-10+. The structural features deduced from the 1H-NMR at low temperature for the isolated S-peptide are compatible with the structure shown by the same molecule in the ribonuclease S crystal.     

Didemnin B. Conformation and dynamics of an antitumour and antiviral depsipeptide studied in solution by 1H and 13C. n.m.r. spectroscopy

The solution conformation of didemnin B, the most potent member of a family of depsipeptides that shows antitumour, antiviral, and immunosuppressive activity, has been studied in chloroform solution using n.m.r. spectroscopy. 1H and 13C spectra have been assigned from analysis of a number of two-dimensional homonuclear and heteronuclear chemical shift correlation experiments which confirm the recently corrected primary structure of the molecule. The conformation of the peptide has been deduced from measurements of the temperature dependence of the NH chemical shifts, analysis of coupling constant data and primarily through NOE effects observed in the rotating frame. Interproton distance bounds determined from a quantitative analysis of the ROE data provide 41 constraints from which a family of closely related structures were calculated using distance geometry algorithms. A type II beta-turn involving residues Thr6, Leu7, and Pro8 is well represented in the computed conformers as is a hydrogen bonding interaction between the NH of Leu3 and the carbonyl oxygen of Thr6. This latter interaction causes the linear portion of the structure to fold back over the depsipeptide ring, imparting to it a degree of structural stability as well as giving the molecule a somewhat globular character. Only one transannular hydrogen bond, between Ist1 NH and Leu3 carbonyl, stabilizes the conformation of the depsipeptide, which has an irregular non-planar configuration. The small temperature coefficients (less than 2.0 x 10(-3) ppm/degrees C) for the NHs of Ist1 and Leu3 are consistent with their involvement in these hydrogen bonding interactions. We find that many of the structural features observed in the crystalline form of didemnin B are conserved in solution. Analysis of the 13C spin-lattice relaxation rates of the protonated carbons reveals small variations in effective correlation times at specific sites in the molecule. The data suggests that the peptide segment encompassing residues Leu3 through to Thr6 is in a more motionally restricted part of the structure.