The solid state and solution structure of HAPyU.
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Crystallography, X-Ray↗
Biomedical subjects
T Domke
Publications and source records attributed to T Domke.
NMR determination of the global structure of the 113Cd derivative of desulforedoxin: investigation of the hydrogen bonding pattern at the metal center.
Desulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H-bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341-353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side-chain protons, H-bonded in the X-ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide protons near the metal center confirm the H-bonding pattern seen in the X-ray model (Archer M et al., 1995, J Mol Biol 251:690-702) and also suggest that H-bond lengths may vary between the Fe, Zn, and 113Cd forms. The H-bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH-S H-bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of "H-bond mediated" coupling in Dx indicates that the NH-S H-bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer "through space" coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer "through space" coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a "through space" coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H-bond network varies slightly when Cd is introduced. The similarity between the H-bonding pattern seen at the metal center in Dx, Rd (including H-bonded and through space-mediated coupling), and many zinc-finger proteins suggests that these H-bonds are structurally vital for stabilization of the metal centers in these proteins.
Amino Acid Sequence↗
New saponins from the seeds of Agrostemma githago var. githago.
Two new saponins were isolated from the seeds of Agrostemma githago L. var. githago. On the basis of chemical and spectral evidence their structures were determined to be 3-O-beta-D-xylopyranosyl-(1-->3)-[beta-D-galactopyranosyl-(1-->2)]-beta- D- glucuronopyranosylgypsogenin-28-O-beta-D-xylopyranosyl-(1-->4)- [beta-D-glucopyranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-beta- D-4-O-acetylfucopyranoside and 3-O-beta-D-xylopyranosyl-(1-->3)- [beta-D-galactopyranosyl-(1-->2)]-beta-D-glucuronopyranosyl-gyp sogenin- 28-O-beta-D-xylopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)- [beta-D-glucopyranosyl-(1-->3)]-alpha-L-rhamnopyranosyl-(1-->2)-beta-D- 4-O-acetylfucopyranoside.
Carbohydrate Conformation↗
Purification and characterization of a cytotoxic exolipid of Burkholderia pseudomallei.
Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of B. pseudomallei. After cultivation of B. pseudomallei in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxytetradec anoyl-beta-hydroxytetradecanoate (Rha-Rha-C14-C14), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.
Animals↗
Structure of an acidic exopolysaccharide of Burkholderia pseudomallei.
A recently described water-soluble exopolysaccharide of Burkholderia pseudomallei recognized by the IgG 1 monoclonal antibody 3015 [Steinmetz, I., Rohde, M. & Brenneke, B. (1995) Infect. Immun. 63, 3959-3965] was isolated by repetitive ethanol-precipitation steps and by anion-exchange chromatography. The structure of the polysaccharide was determined by a combination of chemical-derivatization and mass-spectrometric techniques (compositional and methylation analysis, GC/MS, and electrospray-ionization-MS/MS of reduced and permethylated hydrolytic fragments), and two-dimensional 1H-NMR methods (COSY, TOCSY and NOESY) and confirmed by isolation and structural characterization of the depolymerized repeating unit of the polysaccharide. The combined structural data established a linear tetrasaccharide repeating unit consisting of three galactose residues, one bearing a 2-linked O-acetyl group, and a 3-deoxy-D-manno-2-octulosonic acid residue. [-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1- ->5)-beta-Kdo-(2-->]n
Burkholderia pseudomallei↗
Birch pollen profilin: structural organization and interaction with poly-(L-proline) peptides as revealed by NMR.
The secondary structure of birch pollen profilin, a potent human allergen, was elucidated by multidimensional nuclear magnetic resonance (NMR), as a prerequisite to study the interaction of this profilin with ligands for its poly-(L-proline) (PLP)-binding site. The chemical shifts of the 15N-labeled backbone amide groups were used to monitor complex formation with various PLP peptides. Titration with deca-L-proline (P10) yielded a KD of 0.2 mM. P8 was the shortest PLP to provoke a significant reaction. (GP5)3G bound significantly, confirming the interaction between profilins and the protein VASP containing this motif. Birch profilin interacted also with GP6GP5, found in the cyclase-associated protein (CAP), a suspected profilin ligand.
Acanthamoeba↗
Structure of stewartan, the capsular exopolysaccharide from the corn pathogen Erwinia stewartii.
Stewartan, the acidic exopolysaccharide of Erwinia stewartii, was purified from agar grown cells. To facilitate its structural analysis, chemical and enzymatic depolymerizations were carried out using hydrofluoric acid and E. amylovora phage phi-Ea1h, respectively. Structural characterization of the resulting oligosaccharides was performed by a combination of mass spectrometric and one- and two-dimensional (1D/2D) NMR spectroscopic techniques. A branched repeating unit with seven monosaccharide residues, all in their pyranose forms, was found: [Table: see text]
Bacterial Capsules↗
Structure of amylovoran, the capsular exopolysaccharide from the fire blight pathogen Erwinia amylovora.
The acidic exopolysaccharide (EPS) of Erwinia amylovora, amylovoran, was purified from culture supernatants of bacteria in minimal medium and cleaved chemically either by treatment with trifluoracetic acid or hydrofluoric acid, and enzymatically by digestion with depolymerase from E. amylovora phage phi-Ealh. Structural characterization of the resulting oligosaccharides was performed by a combination of mass spectrometric and NMR [one- and two-dimensional (1D and 2D)] spectroscopic techniques. A branched repeating unit with five monosaccharide residues and various substituents was determined: [sequence: see text] The terminal monosaccharide of the side branch, which bears a 4,6-bound pyruvate residue in the R-configuration, was found to be modified with 2-linked (26%), 3-linked (24%), 2-,3-linked (40%) O-acetyl groups, or these were absent (10%). An additional glucose residue is linked to approximately 10% of the core alpha-galactose of the repeating unit.
Bacterial Capsules↗
1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus.
1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)-CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the alpha-protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H alpha chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020-9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C alpha and H alpha frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C alpha chemical shifts and 'TOCSY ladders'. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D 15N NOESY-HMQC data and C alpha secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427-445] shows no major differences.
Amino Acid Sequence↗