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W Saenger

Publications and source records attributed to W Saenger.

At least 73 records · Page 4Linked to original sources
PubMed1998

Huntingtin aggregation monitored by dynamic light scattering.

An initial stage of fibrillogenesis in solutions of glutathione S-transferase-huntingtin (GST-HD) fusion proteins has been studied by using dynamic light scattering. Two GST-HD systems with poly-L-glutamine (polyGln) extensions of different lengths (20 and 51 residues) have been examined. For both systems, kinetics of z-average translation diffusion coefficients (Dapp) and their angular dependence have been obtained. Our data reveal that aggregation does occur in both GST-HD51 and GST-HD20 solutions, but that it is much more pronounced in the former. Thus, our approach provides a powerful tool for the quantitative assay of GST-HD fibrillogenesis in vitro.

Glutathione Transferase↗
PubMed1998

Crystal structure of human arylsulfatase A: the aldehyde function and the metal ion at the active site suggest a novel mechanism for sulfate ester hydrolysis.

Human lysosomal arylsulfatase A (ASA) is a prototype member of the sulfatase family. These enzymes require the posttranslational oxidation of the -CH2SH group of a conserved cysteine to an aldehyde, yielding a formylglycine. Without this modification sulfatases are catalytically inactive, as revealed by a lysosomal storage disorder known as multiple sulfatase deficiency. The 2.1 A resolution X-ray crystal structure shows an ASA homooctamer composed of a tetramer of dimers, (alpha 2)4. The alpha/beta fold of the monomer has significant structural analogy to another hydrolytic enzyme, the alkaline phosphatase, and superposition of these two structures shows that the active centers are located in largely identical positions. The functionally essential formylglycine is located in a positively charged pocket and acts as ligand to an octahedrally coordinated metal ion interpreted as Mg2+. The electron density at the formylglycine suggests the presence of a 2-fold disordered aldehyde group with the possible contribution of an aldehyde hydrate, -CH(OH)2, with gem-hydroxyl groups. In the proposed catalytic mechanism, the aldehyde accepts a water molecule to form a hydrate. One of the two hydroxyl groups hydrolyzes the substrate sulfate ester via a transesterification step, resulting in a covalent intermediate. The second hydroxyl serves to eliminate sulfate under inversion of configuration through C-O cleavage and reformation of the aldehyde. This study provides the structural basis for understanding a novel mechanism of ester hydrolysis and explains the functional importance of the unusually modified amino acid.

Aldehydes↗
PubMed1998

Crystallization and preliminary X-ray analysis of the Tet-repressor/operator complex.

Three crystal forms of the repressor protein TetR class D in complex with the palindromic 17 bp operator sequence containing T overhangs on both sides were obtained by hanging-drop vapor-diffusion methods using PEG 4000 and PEG monomethylether 5000 as precipitants. Although the crystallization conditions were very similar, up to three different crystal forms were observed in the same drop. The space groups are monoclinic C2, P21 and hexagonal P6122. The asymmetric units of the latter two crystal forms contain one repressor-operator complex. The crystal structures of these forms were solved by molecular replacement using the Tet-repressor molecule of the complex with tetracycline as a search model.

Base Sequence↗
PubMed1998

Melting points of lysozyme and ribonuclease A crystals correlated with protein unfolding: a Raman spectroscopic study.

The effects of a temperature increase on monoclinic and tetragonal lysozyme single crystals were investigated by polarizing microscopy, X-ray diffraction and laser Raman spectroscopy. To prevent dissolution, the mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was observed around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and higher crystal forces in monoclinic lysozyme. Raman spectral changes in the amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature where birefringence changes, and differences in the S-S band suggest that in monoclinic lysozyme, denaturation is accompanied with disruption of some S-S bonds. Comparison with thermal denaturation and gel formation (beta-aggregation) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme crystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at the unfolding temperature at 333 K, i.e. loss of structure induces breakdown of crystal lattice and macroscopic shape. Although the crystal lattice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of thermal unfolding of the polypeptide chains.

Calorimetry↗
PubMed1998

Interaction of Tet repressor with operator DNA and with tetracycline studied by infrared and Raman spectroscopy.

Tet repressor (TetR) is involved in the most abundant mechanism of tetracycline (Tc) resistance of gram-negative bacteria. Raman spectra were measured for the class D TetR protein, for an oligodeoxyribonucleotide with sequence corresponding to operator site O1, and for the TetR:oligonucleotide complex. TetR forms a complex with [Ni-Tc]+, which does not bind to operator DNA. Raman and infrared measurements indicate nearly identical conformations of TetR with and without [Ni-Tc]+. Differences between the experimental spectrum of the TetR:operator DNA complex and the computed sum of the component spectra provide direct spectroscopic evidence for changes in DNA backbone torsions and base stacking, rearrangement of protein backbone, and specific contacts between TetR residues and DNA bases. Complex formation is connected with intensity decrease at 1376 cm(-1) (participation of thymine methyl groups), intensity increase at 1467 cm(-1) (hydrogen bond formation at guanine N7), decreased intensity ratio I854/I823 (increased hydrophobicity of tyrosine environment), increased intensity at 1363 cm(-1) (increased hydrophobicity of tryptophan ring environment), differences in the range 670-833 cm(-1) (changes in B-DNA backbone torsions and base stacking), and decreased intensity of the amide I band (structural rearrangement of TetR backbone consistent with a reduction of the distance between the two binding helices).

Base Sequence↗
PubMed1998

Crystal structure of alpha-cyclodextrin-acetonitrile-hexahydrate.

Crystals of the ternary alpha-cyclodextrin-acetonitrile hexahydrate inclusion complex belong to the orthorhombic space group P212121 with unit cell dimensions a = 9.479(2), b = 14.323(4), c = 37.397(9) A. Refinement against 4202 X-ray diffraction data converged at R=0.059. The alpha-cyclodextrin macrocycle forms a regular torus which is stabilized by intramolecular O-3-O-2 hydrogen bonds between neighboring glucose units. Within the cavity, one acetonitrile and one water molecule are located with occupancy factors of 0.8 and 0.7. respectively. They are hydrogen bonded to disordered O65B, and acetonitrile is stabilized in position by C-5-H...N interactions. The remaining five water molecules link symmetry related cyclodextrin molecules.

Acrylonitrile↗
PubMed1998

Aggregation of A beta Alzheimer's disease-related peptide studied by dynamic light scattering.

The aggregation behavior of the major component of Alzheimer's disease-related, amyloid peptides, Abeta-(1-40) and Abeta-(1-42), was studied in solution using dynamic light scattering. With most solvents employed, we found fibrils coexisting with oligomeric Abeta species. Pronounced differences were observed in aggregation of Abeta-(1-40) and (1-42) sequences in acetonitrile-water mixtures. Cofactors such as Zn2+ were found to induce deaggregation of Abeta instead of aggregation. The results indicated that the initial state of the peptide immediately after synthesis is rather poorly defined. Using freezing instead of lyophilization after the final peptide synthesis step, may partially relieve these problems.

Acetonitriles↗
PubMed1998

Raman spectroscopic analysis of Tet repressor-operator DNA interaction in deuterium oxide.

Tet repressor (TetR) plays a central role in the regulation of its own gene and in that of TetA, a resistance protein against the antibiotic tetracycline (Tc). In the absence of Tc, the TetR dimer binds with two alpha-helix-turn-alpha-helix motifs to two successive major grooves of operator DNA. In order to elucidate structural features of the TetR:operator complex, we measured the Raman spectra of the TetR protein, a 18-mer oligonucleotide with sequence corresponding to TetR operator DNA, and the TetR:operator complex in D2O. The spectra confirm and extend previously obtained results in H2O: i) B-DNA conformation is conserved with only small perturbations of the backbone geometry; ii) TetR and operator DNA interact at major groove sites, as evident from intensity changes of thymine and guanine bands; iii) Minor changes of TetR secondary structure are indicated upon operator binding, and iv) Local environments of aromatic amino acids are altered in the complex. These spectroscopic findings are consistent with a molecular model proposed of the basis of genetic and biochemical studies.

Bacterial Proteins↗
PubMed1997

Lysozyme aggregation studied by light scattering. I. Influence of concentration and nature of electrolytes.

Static and dynamic light scattering have been employed to investigate the behaviour of lysozyme solutions when varying the concentration of (NH(4))(2)SO(4) and NaCl for screening the repulsive forces between the monomers. At the initial aggregation stages clusters, which can be classified as mass-fractals undergoing diffusion limited-like aggregation, coexist with monomers or small lysozyme oligomers. The kinetics of fractal growth deliver observables that exhibit distinct tendencies when examined as a function of the concentration and nature of the electrolyte. The behaviour of the observables changes drastically above 0.84 M (NH(4))(2)SO(4) and 0.60 M NaCl. Static light scattering revealed a progressive restructuring of the fractals to compact structures at the latter stages of the reaction. Based on the correlations between the various observables an attempt is made to predict the long-term fate of the nucleating solutions.

Journal Article↗
PubMed1997

Lysozyme aggregation studied by light scattering. II. Variations of protein concentration.

Static and dynamic light scattering have been employed to investigate the behaviour of nucleating lysozyme solutions in the range between 0.34 and 3.08 mM. Preselected concentrations of NaC1 and (NH(4))(2)SO(4) have been used to screen the repulsive Coulombic interactions and trigger aggregation. Initially, mass-fractals undergoing diffusion limited-like aggregation coexist with monomers or small lysozyme oligomers. The growth kinetics of the fractals deliver observables that exhibit distinct tendencies when examined as a function of lysozyme concentration. The behaviour of the observables changes drastically around 2.0 mM lysozyme. Static light scattering experiments revealed progressive restructuring or growth of compact structures at later stages of the aggregation. Based on the correlations between the observables an attempt is made to predict whether the examined solutions will crystallize or not. A tentative scheme, involving the most prominent structures observed in nucleating lysozyme solutions, is discussed.

Journal Article↗
PubMed1997

Photosystem I of Synechococcus elongatus at 4 A resolution: comprehensive structure analysis.

An improved structural model of the photosystem I complex from the thermophilic cyanobacterium Synechococcus elongatus is described at 4 A resolution. This represents the most complete model of a photosystem presently available, uniting both a photosynthetic reaction centre domain and a core antenna system. Most constituent elements of the electron transfer system have been located and their relative centre-to-centre distances determined at an accuracy of approximately 1 A. These include three pseudosymmetric pairs of Chla and three iron-sulphur centres, FX, FA and FB. The first pair, a Chla dimer, has been assigned to the primary electron donor P700. One or both Chla of the second pair, eC2 and eC'2, presumably functionally link P700 to the corresponding Chla of the third pair, eC3 and eC'3, which is assumed to constitute the spectroscopically-identified primary electron acceptor(s), A0, of PSI. A likely location of the subsequent phylloquinone electron acceptor, QK, in relation to the properties of the spectroscopically identified electron acceptor A1 is discussed. The positions of a total of 89 Chla, 83 of which constitute the core antenna system, are presented. The maximal centre-to-centre distance between antenna Chla is < or = 16 A; 81 Chla are grouped into four clusters comprising 21, 23, 17 and 20 Chla, respectively. Two "connecting" Chla are positioned to structurally (and possibly functionally) link the Chla of the core antenna to those of the electron transfer system. Thus the second and third Chla pairs of the electron transfer system may have a dual function both in energy transfer and electron transport. A total of 34 transmembrane and nine surface alpha-helices have been identified and assigned to the 11 subunits of the PSI complex. The connectivity of the nine C-terminal (seven transmembrane, two "surface") alpha-helices of each of the large core subunits PsaA and PsaB is described. The assignment of the amino acid sequence to the transmembrane alpha-helices is proposed and likely residues involved in co-ordinating the Chla of the electron transfer system discussed.

Bacterial Proteins↗
PubMed1997

Crystallization and preliminary X-ray crystallographic and electron microscopic study of a bacterial DNA helicase (RSF1010 RepA).

Helicases are ATP-driven enzymes essential for DNA unwinding. The broad host range plasmid RSFI010 harbours a gene (repA) encoding for one of the smallest known oligomeric helicases, RepA, a homo-hexamer with 30 kDa subunits. Electron micrographs indicate that the overall shape of RepA resembles a hexagon with globular monomers at the corners, diameter 140 A, and a central channel. Below pH 6, the molecules aggregate into tubular structures. The enzyme has been purified and crystallized using the hanging-drop vapour-diffusion method with polyethyleneglycol monomethylether as precipitating agent. The crystals exhibit the monoclinic space group P2(1) with unit-cell parameters a = 105.8, b = 180.3, c = 115.4 A, beta = 95.2 degrees, and diffract to 3.5 A resolution using rotating-anode Cu Kalpha radiation. Assuming two 180 kDa molecules per asymmetric unit, the volume per unit weight is V(m) = 3.06 A Da(-1), equivalent to a solvent content of 60%. A self-rotation search indicates that the sixfold axis of the hexamer is parallel to the ac plane and inclined at about 2 degrees to the c axis. The two hexamers are oriented head-to-head with point-group symmetry D(6).

Journal Article↗
PubMed1997

Substrate specificity of hybrid modules from peptide synthetases.

Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions. Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine-activating module of surfactin synthetase were constructed by combining the two reading frames at various highly conserved consensus sequences. The resulting DNA fragments were expressed in Escherichia coli as C-terminal fusions to the gene encoding for the maltose-binding protein. The fusion proteins were purified, and the amino acid specificities, the acceptance of different nucleotide analogues, and the substrate binding affinities were analyzed. We found evidence for a large N-terminal domain and a short C-terminal domain of about 19 kDa within the two modules, which are separated by the sequence motif GELCIGG. The two domains could be reciprocally transferred between the two modules, and the constructed hybrid proteins showed amino acid adenylating activity. Hybrid proteins fused at various consensus motifs within the two domains were inactive, indicating that the domains may fold independently and represent complex functional units. The N-terminal domain was found to be responsible for the amino acid specificity of the modules, and it is also involved in the recognition of the ribosyl and the phosphate moieties of the nucleotide substrate. For tyrocidine synthetase I, we could confine the sites for amino acid specificity to a region of 330 residues. The C-terminal domain is essential for the enzymatic activity and has a strong impact on the specific activity of the modules.

Escherichia coli↗
PubMed1997

Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 A resolution.

Trimeric calf spleen purine nucleoside phosphorylase has been complexed with hypoxanthine via phosphorolysis of inosine in the presence of phosphate. The resulting, "Michaelis" complex (three hypoxanthine molecules per trimer), presumed to be formed under these conditions, crystallized in the cubic space group P2(1)3, with unit cell dimension a = 94.11 A and one monomer in the asymmetric crystal unit; the biologically active trimer is located on the crystallographic 3-fold axis. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL outstation, Hamburg, c/o DESY). The crystal structure has been determined by molecular replacement and refined at 2.15 A resolution to an R-value of 0.18. In the hypoxanthine binding site, a cis-peptide bond between Asn243 and Lys244 is observed. Side-chains of GIu201 and Asn243, as well as one integral water molecule located in the base binding site, form hydrogen bonds with the hypoxanthine N-1 H, N-7 H and O-6. A second water molecule links the base positions N-3 and N-9 with an adjacent pocket, which presumably is the phosphate-binding site. This pocket is filled completely by a cluster of six water molecules. Hence all possible donor/acceptor-positions of hypoxanthine are saturated by hydrogen-bonding to protein side-chains or integral water molecules. Purine nucleoside phosphorylase isolated form human tissues is a primary target for chemotherapeutic intervention, and the more stable calf enzyme has similar physico-chemical and kinetic properties, as well as response to inhibitors. Hence the high-resolution structure presented here may serve for design of inhibitors with potential pharmacological applications.

Animals↗
PubMed1997

Differential binding of S-adenosylmethionine S-adenosylhomocysteine and Sinefungin to the adenine-specific DNA methyltransferase M.TaqI.

The crystal structures of the binary complexes of the DNA methyltransferase M.TaqI with the inhibitor Sinefungin and the reaction product S-adenosyl-L-homocysteine were determined, both at 2.6 A resolution. Structural comparison of these binary complexes with the complex formed by M.TaqI and the cofactor S-adenosyl-L-methionine suggests that the key element for molecular recognition of these ligands is the binding of their adenosine part in a pocket, and discrimination between cofactor, reaction product and inhibitor is mediated by different conformations of these molecules; the methionine part of S-adenosyl-L-methionine is located in the binding cleft, whereas the amino acid moieties of Sinefungin and S-adenosyl-L-homocysteine are in a different orientation and interact with the active site amino acid residues 105NPPY108. Dissociation constants for the complexes of M.TaqI with the three ligands were determined spectrofluorometrically. Sinefungin binds more strongly than S-adenosyl-L-homocysteine or S-adenosyl-L-methionine, with KD=0.34 microM, 2.4 microM and 2.0 microM, respectively.

Adenosine↗
PubMed1997

Ribonuclease T1 is active when both catalytic histidines are replaced by aspartate.

The enzymatic activity of many ribonucleases (RNases) depends on two histidines, as in RNase A, or one histidine and/or glutamate, as in microbial RNases belonging to the T1 family. In RNase T1, substitution of either one or both of the two histidines at positions 40 and 92 by a variety of other amino acids reduces the activity more than 100-fold. However, the double variant His40-->Asp/His92-->Asp retains a significant residual enzymatic activity towards RNA and guanylyl-3',5'-cytidine, indicating that a pair of substituted side chains in these positions, both with acid functionality, can confer enzymatic activity. It was shown that the substitution of histidine with glutamate in the variant His40-->Glu yields an enzyme with drastically reduced activity and leads to inactivation in the His92-->Glu, His40-->Glu/His92-->Glu variants. For the variants where histidine is substituted with aspartate we found measurable activity from 1% (His40-->Asp) to 6% (His40-->Glu/His92-->Asp) towards RNA.

Aspartic Acid↗
PubMed1996

Crystal structure of anhydrous hexakis-(2,3,6-tri-O-methyl)-cyclomaltohexaose (permethyl-alpha-cyclodextrin) grown from hot water and from cold NaCl solutions.

An X-ray diffraction study was carried out on anhydrous permethyl-alpha-cyclodextrin crystallized from aq solution at 40 degrees C, space group P2(1)2(1)2(1) with a = 15.424(9), b = 18.167(7), c = 23.128(8) A, vol = 6481(5) A3. The molecule is boat-shaped with the narrow end of the cyclodextrin cavity closed by two diametrically opposing glucose residues. These are strongly tilted towards the molecular axis, so that their primary methoxy groups form van der Waals contacts across the molecular opening. The remaining cavity volume is occupied by the methoxy group of a neighboring molecule (self-inclusion). From solutions containing 1 M NaCl, anhydrous permethyl-alpha-cyclodextrin crystallizes isomorphously at room temperature.

Carbohydrate Conformation↗