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A Ejchart

Publications and source records attributed to A Ejchart.

At least 19 recordsLinked to original sources
PubMed2000

Structure of the lipopolysaccharide core region of Hafnia alvei strains 1185 and 1204.

Sugar and methylation analyses using gas chromatography/mass spectrometry and NMR spectroscopy proved that the core oligosaccharides of Hafnia alvei strains 1185 and 1204 have the following formula: carbohydrate sequence [see text] where Kdo = 3-deoxy-oct-2-ulosonic acid and P-PEtN = diphosphorylethanolamine. The structure shown above is a slight modification of the typical core region of H. alvei lipopolysaccharides. The difference refers to one sugar only: terminal galactose is present in the core of strains of 1185 and 1204, while terminal glucose in the typical core.

Carbohydrate Conformation↗
PubMed1999

Alpha-helix nucleation by a calcium-binding peptide loop.

A 12-residue peptide AcDKDGDGYISAAENH2 analogous to the third calcium-binding loop of calmodulin strongly coordinates lanthanide ions (K = 10(5) M-1). When metal saturated, the peptide adopts a very rigid structure, the same as in the native protein, with three last residues AAE fixed in the alpha-helical conformation. Therefore, the peptide provides an ideal helix nucleation site for peptide segments attached to its C terminus. NMR and CD investigations of peptide AcDKDGDGYISAAEAAAQNH2 presented in this paper show that residues A13-Q16 form an alpha-helix of very high stability when the La3+ ion is bound to the D1-E12 loop. In fact, the lowest estimates of the helix content in this segment give values of at least 80% at 1 degreesC and 70% at 25 degreesC. This finding is not compatible with existing helix-coil transition theories and helix propagation parameters, s, reported in the literature. We conclude, therefore, that the initial steps of helix propagation are characterized by much larger s values, whereas helix nucleation is even more unfavorable than is believed. In light of our findings, thermodynamics of the nascent alpha-helices is discussed. The problem of CD spectra of very short alpha-helices is also addressed.

Amino Acid Sequence↗
PubMed1999

NMR measurements of proton exchange between solvent and peptides and proteins.

Scope and limitations of the NMR based methods, equilibration and magnetization transfer, for measuring proton exchange rates of amide protons in peptides and proteins with water protons are discussed. Equilibration is applied to very slow processes detected by hydrogen-deuterium exchange after a solute is dissolved in D2O. Magnetization transfer allows to study moderately rapid processes in H2O. A number of precautions should be undertaken in order to avoid systemic errors inherent in the magnetization transfer method.

Amino Acid Sequence↗
PubMed1998

Solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus.

Cytochrome c6 is a small, soluble electron carrier between the two membrane-bound complexes cytochrome b6f and photosystem I (PSI) in oxygenic photosynthesis. We determined the solution structure of cytochrome c6 from the thermophilic cyanobacterium Synechococcus elongatus by NMR spectroscopy and molecular dynamics calculations based on 1586 interresidual distance and 28 dihedral angle restraints. The overall fold exhibits four alpha-helices and a small antiparallel beta-sheet in the vicinity of Met58, one of the axial heme ligands. The flat hydrophobic area in this cytochrome c6 is conserved in other c6 cytochromes and even in plastocyanin of higher plants. This docking region includes the site of electron transfer to PSI and possibly to the cytochrome b6f complex. The binding of cytochrome c6 to PSI in green algae involves interaction of a negative patch with a positive domain of PSI. This positive domain has not been inserted at the evolutionary level of cyanobacteria, but the negatively charged surface region is already present in S. elongatus cytochrome c6 and may thus have been optimized during evolution to improve the interaction with the positively charged cytochrome f. As the structure of PSI is known in S.elongatus, the reported cytochrome c6 structure can provide a basis for mutagenesis studies to delineate the mechanism of electron transfer between both.

Amino Acid Sequence↗
PubMed1997

Solution structure of porcine delta sleep-inducing peptide immunoreactive peptide A homolog of the shortsighted gene product.

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i' + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed.

Acetylation↗
PubMed1997

The structure of 3'-O-anthraniloyladenosine, an analogue of the 3'-end of aminoacyl-tRNA.

3'-O-Anthraniloyladenosine, an analogue of the 3'- terminal aminoacyladenosine residue in aminoacyl-tRNAs, was prepared by chemical synthesis, and its crystal structure was determined. The sugar pucker of 3'-O-anthraniloyladenosine is 2'-endo resulting in a 3'-axial position of the anthraniloyl residue. The nucleoside is insynconformation, which is stabilized by alternating stacking of adenine and benzoyl residues of the neighboring molecules in the crystal lattice. The conformation of the 5'-hydroxymethylene in 3'-O- anthraniloyladenosine is gauche-gauche. There are two intramolecular and two intermolecular hydrogen bonds and several H-bridges with surrounding water molecules. The predominant structure of 3'-O-anthraniloyladenosine in solution, as determined by NMR spectroscopy, is 2'-endo,gauche-gauche and anti for the sugar ring pucker, the torsion angle around the C4'-C5'bond and the torsion angle around the C1'-N9 bond, respectively. The 2'-endo conformation of the ribose in 2'(3')-O-aminoacyladenosine, which places the adenine and aminoacyl residues in equatorial and axial positions, respectively, could serve as a structural element that is recognized by enzymes that interact with aminoacyl-tRNA or by ribosomes to differentiate between aminoacylated and non-aminoacylated tRNA.

Acylation↗
PubMed1996

Secondary structure and tertiary fold of the birch pollen allergen Bet v 1 in solution.

Bet v 1 is the major birch pollen allergen and therefore the main cause of type I allergies observed in early spring. It is composed of 159 amino acid residues adding up to a molecular mass of 17 kDa. We determined the secondary structure and tertiary fold of full-length Bet v 1 by NMR spectroscopy. Two- and three-dimensional NMR measurements suggest that Bet v 1 is a globular monomer in solution with a high content of well defined secondary structure. Of the total of 159 residues, 135 could be sequentially assigned, using an improved assignment strategy based mainly on heteronuclear experiments. An improved strategy for structure calculation revealed three helices and two beta-sheets as major elements of secondary structure. The globular tertiary structure is mainly stabilized by two antiparallel beta-sheets. The two helices at the C terminus are in accordance with the results from the solution structure of the chemically synthesized peptide Bet v 1-(125-154). This peptide is composed of two helices connected by a hinge. The structural features of Bet v 1 are highly similar to those found in the Ambrosia allergen Amb t V.

Allergens↗
PubMed1995

Structure of human parathyroid hormone 1-37 in solution.

Human parathyroid hormone (hPTH), amino acids Ser1 to Leu37, is biologically active with respect to both receptor binding and activation of adenylate cyclase to influence the serum calcium concentration. It induces DNA synthesis via an unknown signal pathway. We investigated the structure of hPTH(1-37) in H2O/buffer solution under near physiological conditions, that is pH 6.0 and 270 mM salt, by circular dichroism, ultracentrifugation, nuclear magnetic resonance spectroscopy, and molecular dynamics calculations. Complete sequence specific assignments of all 1H resonances were performed by using 1H two-dimensional NMR measurements (double quantum-filtered correlated spectroscopy, nuclear Overhauser effect spectroscopy (NOESY), and total correlation spectroscopy with suppression of NOESY-type cross-peaks spectra). hPTH(1-37) obtained helical structure and showed hydrophobic interactions defining a tertiary structure. The NH2-terminal four amino acids of hPTH(1-37) did not show a stable conformation. Evidence for an alpha-helical region between Ile5 and Asn10 was found. This region was followed by a flexible link (Gly12, Lys13) and a well defined turn region, His14 to Ser17. The latter was stabilized by hydrophobic interactions between Trp23 and Leu15. Ser17 through at least Leu28 formed an alpha-helix. Arg20 and Lys27 were involved in the core built by His14 to Ser17. Unrestrained molecular dynamics simulations indicated that the structure was stable on the 200 ps time scale.

Amino Acid Sequence↗
PubMed1995

1H nuclear-magnetic-resonance investigation of oxidized Fe4S4 ferredoxin from Thermotoga maritima. Hyperfine-shifted resonances, sequence-specific assignments and secondary structure.

The oxidized Fe4S4 ferredoxin from the hyperthermophilic bacterium Thermotoga maritima has been investigated by one- and two-dimensional NMR in order to characterize its hyperfine-shifted resonances originating from the cysteinyl cluster ligands and to assign its resonances in the diamagnetic shift range. The chemical shift and relaxation time pattern of the hyperfine-shifted signals is very similar to other oxidized Fe4S4 ferredoxins. A tentative sequence-specific assignment of these resonances according to a general pattern of chemical shift of cysteine protons versus sequence position of cluster ligand is presented. Furthermore, sequence-specific assignments for 85% of the amino acid residues that were obtained without any guidance by known X-ray structures of ferredoxins are given. They reveal the formation of at least two elements of secondary structure by the polypeptide chain of T. maritima ferredoxin: an alpha-helix comprising residues C43-D49 and a double-stranded antiparallel beta-sheet consisting of the N- and C-terminal parts of the protein. This folding pattern is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibrio gigas [Kissinger, C.R., Sieker, L.C., Adman E.T. & Jensen, L.H. (1991) J. Mol. Biol. 219, 693-715] and therefore suggesting different mechanisms of stabilization for T. maritima ferredoxin and the ferredoxin from the hyperthermophilic archaeon Pyrococcus furiosus that was recently investigated by NMR [Teng, Q., Zhou, Z.H., Smith, E.T., Busse, S. C., Howard, J.B., Adams M.W.W. & La Mar, G.N. (1994) Biochemistry 33, 6316-6326].

Amino Acid Sequence↗
PubMed1995

Structural studies of HIV-1 Tat protein.

Tat (trans-activator) proteins are early RNA binding proteins regulating lentiviral transcription. These proteins are necessary components in the life cycle of all known lentiviruses, such as the human immunodeficiency viruses (HIV) or the equine infectious anemia virus (EIAV). Tat proteins are thus ideal targets for drugs intervening with lentiviral growth. The consensus RNA binding motif (TAR, trans-activation responsive element) of HIV-1 is well characterized. Structural features of the 86 amino acid HIV-1, Zaire 2 isolate (HV1Z2) Tat protein in solution were determined by two dimensional (2D) nuclear magnetic resonance (NMR) methods and molecular dynamics (MD) calculations. In general, sequence regions corresponded to structural domains of the protein. It exhibited a hydrophobic core of 16 amino acids and a glutamine-rich domain of 17 amino acids. Part of the NH2 terminus, Val4 to Pro14, was sandwiched between these domains. Two highly flexible domains corresponded to a cysteine-rich and a basic sequence region. The 16 amino acid sequence of the core region is strictly conserved among the known Tat proteins, and the three-dimensional fold of these amino acids of HV1Z2 Tat protein was highly similar to the structure of the corresponding EIAV Tat domain. HV1Z2 Tat protein contained a well defined COOH-terminal Arg-Gly-Asp (RGD) loop similar to the recently determined decorsin RGD loop.

Amino Acid Sequence↗
PubMed1995

Studies on the mechanism of mutagenicity and genotoxicity induced by dihydralazine.

Dihydralazine was found to be mutagenic towards S. typhimurium TA1537, TA97, TA1538 and TA98 and genotoxic towards E. coli PQ37. Using the nitro blue tetrazolium reduction method we have found that dihydralazine can generate active oxygen species. The possible role of active oxygen species in mutagenicity (Ames test) and genotoxicity (SOS Chromotest) of dihydralazine was studied by testing the influence of the different active oxygen species scavengers on these two processes. Of the active oxygen scavengers tested, only superoxide dismutase suppressed partially the mutagenic and genotoxic activity of dihydralazine. This result seems to indicate that superoxide anion play a role in these two biological events.

Catalase↗
PubMed1994

Trifluoroethanol stabilizes a helix-turn-helix motif in equine infectious-anemia-virus trans-activator protein.

The solution structure of the 75-amino-acid trans-activator (Tat) protein of the equine infectious-anemia virus in trifluoroethanol-containing solution was determined by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear-Over-hauser-enhancement distance restraints, and restrained molecular-dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol-free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA-binding region of the protein showed well-defined alpha-helical secondary structure in trifluoroethanol-containing solution. The helical regions comprise those parts of the molecule whose helix-forming tendencies were noted earlier in trifluoroethanol-free solution. Two helices (Gln38-Arg43 and Asp48-Ala64) are connected by a tight type-II turn centered at the strictly conserved Gly46 leading to a helix-turn-helix motif in the core and basic region of the protein. A third helix (Thr9-Asn13) is located in the less well defined N-terminal part of the protein. These observations may support the notion that the protein adopts a helical structure in the RNA-binding region on complex formation. Although the secondary-structure elements become better defined in trifluoroethanol-containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol-containing solution in general.

Amino Acid Sequence↗
PubMed1993

Equine infectious anemia virus Tat is a predominantly helical protein.

Nuclear magnetic resonance (NMR) spectroscopy revealed features of the secondary structure of the equine infectious anemia virus (EIAV) Tat protein in solution. We could show that this protein, which is required in the replication cycle of lentiviruses, forms a predominantly helical structure in trifluoroethanol/water (40% by vol.) solution. In particular, the basic RNA-binding region and the adjacent core domain, which are highly conserved among lentiviral Tat proteins, show helix-type secondary structure under these conditions. Our observations, in concert with recent biochemical data from other laboratories, suggest that the core sequence region and the basic sequence region form interdependent structural domains, both possibly necessary for correct RNA binding.

Amino Acid Sequence↗
PubMed1993

1H- and 13C-NMR investigations on cis-trans isomerization of proline peptide bonds and conformation of aromatic side chains in H-Trp-(Pro)n-Tyr-OH peptides.

1H and 13C high-resolution nmr spectra of cationic, zwitterionic, and anionic forms of the peptides: H-Trp-(Pro)n-Tyr-OH, n = 0-5, and H-Trp-Pro-OCH3 were obtained in D2O solution. Analysis of H alpha (Pro1), H alpha (Trp), C gamma (Pro), H epsilon (Tyr), and H delta (Trp) resonances provided evidence for the presence of two predominant backbone isomers: the all-trans one and another with the Trp-Pro peptide bond in cis conformation; the latter constituted about 0.8 molar fraction of the total peptide (n > 1) concentration. Relative content of these isomers varied in a characteristic way with the number of Pro residues and the ionization state of the peptides. The highest content of the cis (Trp-Pro) isomer, 0.74, was found in the anionic form of H-Trp-Pro-Tyr-OH; it decreased in the order of: anion >> zwitterion approximately cation, and with the number of Pro residues to reach the value of 0.42 in the cationic form of H-Trp-(Pro)5-Tyr-OH. Isomerization equilibria about Pro-Pro bond(s) were found to be shifted far (> or = 0.9) in favor of the trans conformation. Interpretation of the measured vicinal coupling constants J alpha-beta' and J alpha-beta" for C alpha H-C beta H2 proton systems of Trp and Tyr side chains in terms of relative populations of g+, g-, and t staggered rotamers around the chi 1 dihedral angle indicated that in all the peptides studied (a) rotation of Trp indole ring in cis (Trp-Pro) isomers is strongly restricted, and (b) rotation of Tyr phenol ring is relatively free. The most preferred chi 1 rotamer of Trp (0.8-0.9 molar fraction) was assigned as the t one on the basis of a large value of the vicinal coupling constant between the high-field H beta and carbonyl carbon atoms of Trp, estimated for the cis (Pro1) form of H-Trp-Pro-Tyr-OH from a 1H, 13C correlated spectroscopy 1H-detected multiple quantum experiment. This indicates that cis<-->trans equilibrium in the Trp-Pro fragment is governed by nonbonding interactions between the pyrrolidine (Pro) and indole (Trp) rings. A molecular model of the terminal cis Trp-Pro dipeptide fragment is proposed, based on the presented nmr data and the results of our molecular mechanics modeling of low-energy conformers of the peptides, reported elsewhere.

Amino Acid Sequence↗
PubMed1993

Evaluation of the SOS chromotest for detection of genotoxic drugs.

In the present investigation, the SOS Chromotest with E. coli PQ37 was evaluated. The potential of the test to identify genotoxic properties of different cytostatics was examined. Only intercalating agents showed good activity. The SOS Chromotest appeared to be less effective than the Salmonella mutagenicity test in the detection of alkylating agents.

Alkylating Agents↗
PubMed1992

Evaluation of mutagenic, genotoxic and transforming properties of Ukrain.

Evaluation of mutagenic and genotoxic properties of Ukrain was on the basis of the Ames and micronucleus tests. Ukrain was investigated for its ability to induce morphological transformation of embryonic cells of the Syrian hamster. Under the experimental condition used in this study, Ukrain was found to be non-mutagenic and non-genotoxic, and furthermore did not induce morphological cell transformation.

Alkaloids↗