Biomedical subjects

H Schindelin

Publications and source records attributed to H Schindelin.

26 records · Page 2Linked to original sources

PubMed1995

Crystal structure of an RNA dodecamer containing the Escherichia coli Shine-Dalgarno sequence.

The synthetic dodecameric RNA fragment rUAAGGAGGUGAU resembles a region upstream of the initiation site in prokaryotic mRNAs whereas the pyrimidine-rich complementary strand is identical to the last 12 nucleotides of Escherichia coli 16 S rRNA. The complex thus serves as a model for the Shine-Dalgarno interaction which is required for proper initiation of translation. The crystal structure of rUAAGGAGGUGUA.rAUCACCUCCUUA has been determined at 2.6 A resolution and refined against 2957 1 sigma(F) structure amplitudes to an R-value of 0.195. The unit cell of the triclinic crystals contains two double-stranded RNA molecules. The conformation of the two duplexes is similar, with a root-mean-square deviation of 0.683 A between equivalent atoms, and resembles calf thymus A-DNA as determined by X-ray fiber diffraction methods. Both molecules from continuous helices that penetrate the entire crystal, but the dinucleotide step in between dodecameric duplexes has an unusual geometry with a negative twist angle. The long helices cross over each other in a characteristic manner by inserting the backbone of one molecule into the minor groove of another. These contacts are stabilized by several direct intermolecular hydrogen bonds most of which are mediated by 2'-hydroxyl groups of the ribose sugars suggesting a general mode for the interaction between RNA molecules which is different from DNA-DNA interactions.

Base Sequence↗

PubMed1994

Crystal structure of CspA, the major cold shock protein of Escherichia coli.

The major cold shock protein of Escherichia coli, CspA, produced upon a rapid downshift in growth temperature, is involved in the transcriptional regulation of at least two genes. The protein shares high homology with the nucleic acid-binding domain of the Y-box factors, a family of eukaryotic proteins involved in transcriptional and translational regulation. The crystal structure of CspA has been determined at 2-A resolution and refined to R = 0.187. CspA is composed of five antiparallel beta-strands forming a closed five-stranded beta-barrel. The three-dimensional structure of CspA is similar to that of the major cold shock protein of Bacillus subtilis, CspB, which has recently been determined at 2.45-A resolution. However, in contrast to CspB, no dimer is formed in the crystal. The surface of CspA is characteristic for a protein interacting with single-stranded nucleic acids. Due to the high homology of the bacterial cold shock proteins with the Y-box factors, E. coli CspA and B. subtilis CspB define a structural framework for the common cold shock domain.

Amino Acid Sequence↗

PubMed1994

Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds.

Single crystals of three different isoenzymes of (R)-(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2(1) with unit cell parameters a = 69.9, b = 95.1, c = 95.6 A, and beta = 118.5 degrees. A complete set of diffraction data has been collected to 2.6 A resolution on native crystals of isoenzyme III.

Aldehyde-Lyases↗

PubMed1993

The complex between ribonuclease T1 and 3'GMP suggests geometry of enzymic reaction path. An X-ray study.

The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 N epsilon H+ activates O2'-H, (d) Glu58 carboxylate acts as base and His92 N epsilon H+ as acid in a general acid-base catalysis. The crystals have the monoclinic space group P2(1), a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, beta = 90.62 degrees with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11,338 data > or = 1 sigma (Fo) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle epsilon in trans. The O2'-H group is activated by accepting and donating hydrogen bonds from His40 N epsilon H+ and to Glu58 O epsilon 1; the phosphate is hydrogen bonded to Glu58 O epsilon 2H, Arg77 N epsilon H+ and N eta 2H+, Tyr38 O eta H, His92 N eta H+. The conformation of ribose phosphate is such that O2' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 N epsilon H+; we infer from a model building study that this bond is equivalent to the scissile P-O5' in a substrate GpN.

Binding Sites↗

PubMed1993

Conformations and conformational changes of four Phe-->Trp variants of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis studied by circular dichroism and fluorescence spectroscopy.

Circular dichroic spectra in the region 180-260 nm of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis and of four mutants with a Phe residue replaced by Trp, i.e. [F29W]HBsu, [F47W]HBsu, [F50W]HBsu and [F79W]HBsu, show minor differences only and demonstrate the general similarity of the conformations of these proteins. Fluorescence maxima at 315-320 nm and 330-335 nm indicate a more hydrophobic environment or a more effective stacking of Trp residues in mutants [F29W]HBsu and [F50W]HBsu in comparison to [F47W]HBsu and [F79W]HBsu, respectively. Unfolding of the mutants in high-ionic-strength buffers by increasing concentrations of urea results in a red shift of the fluorescence emission maxima to about 350 nm; the fluorescence intensities decrease strongly for [F29W]HBsu and [F50W]HBsu but show a small increase for [F47W]HBsu and [F79W]HBsu. The data suggest complex unfolding patterns with subtle differences between the single mutants. The circular dichroic spectra in the region 250-320 nm are dominated by the effects of the Trp residues and signal position-dependent differences in the environment of the Trp residues. The conformations of the mutant proteins depend on the ionic strength of the buffer and become more stable against unfolding by denaturants or increasing temperatures at higher ionic strength. At low ionic strength a pronounced protein-concentration dependence of the conformation of the mutants is seen.

Bacillus subtilis↗

PubMed1993

Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein.

The cold-shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature. It leads to the increased production of the major cold-shock proteins, CS7.4 and CspB, respectively. CS7.4 is a transcriptional activator of two genes. CS7.4 and CspB share 43 per cent sequence identity with the nucleic acid-binding domain of the eukaryotic gene-regulatory Y-box factors. This cold-shock domain is conserved from bacteria to man and contains the RNA-binding RNP1 sequence motif. As a prototype of the cold-shock domain, the structure of CspB has been determined here from two crystal forms. In both, CspB is present as an antiparallel five-stranded beta-barrel. Three consecutive beta-strands, the central one containing the RNP1 motif, create a surface rich in aromatic and basic residues that are presumably involved in nucleic acid binding. Preferential binding of CspB to single-stranded DNA is observed in gel retardation experiments.

Amino Acid Sequence↗

PubMed1992

Determination of DNA-binding parameters for the Bacillus subtilis histone-like HBsu protein through introduction of fluorophores by site-directed mutagenesis of a synthetic gene.

A synthetic gene encoding the histone-like DNA-binding protein HBsu from Bacillus subtilis has been expressed in Escherichia coli. Yields of the purified protein are at least 20 mg/l culture medium. The recombinant HBsu protein is chromatographically, immunologically and functionally identical with the authentic wild-type protein. N-terminal sequencing of the purified protein confirms the fidelity of expression of the synthetic gene in E. coli. Site-directed mutagenesis of the synthetic gene was employed to replace several amino acid residues of HBsu protein with tryptophan to facilitate the determination of DNA-binding parameters by fluorescence spectroscopy. According to gel-retardation experiments, the mutant protein [Phe47----Trp]HBsu shows identical DNA binding to wild-type HBsu protein. Analysis of fluorescence binding data reveals that [Phe47----Trp]HBsu binds double-stranded DNA with a dissociation constant in the micromolar range. Computer-assisted fit of binding models to the experimental data renders positive cooperativity of binding unlikely. A dimer of [Phe47----Trp]HBsu appears to contact three or four base pairs of DNA. These results are in partial disagreement with earlier measurements on closely homologous proteins which tended to show cooperative binding and a longer DNA contact region.

Amino Acid Sequence↗

PubMed1992

Overproduction, crystallization, and preliminary X-ray diffraction studies of the major cold shock protein from Bacillus subtilis, CspB.

The major cold shock protein from Bacillus subtilis (CspB) was overexpressed using the bacteriophage T7 RNA polymerase/promoter system and purified to apparent homogeneity from recombinant Escherichia coli cells. CspB was crystallized in two different forms using vapor diffusion methods. The first crystal form obtained with ammonium sulfate as precipitant belongs to the trigonal crystal system, space group P3(1)21 (P3(2)21) with unit cell dimensions a = b = 59.1 A and c = 46.4 A. The second crystal form is tetragonal, space group P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 56.9 A and c = 53.0 A. These crystals grow with polyethylene glycol 4000 as precipitant.

Bacillus subtilis↗