PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif.

The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans. This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain. Five different mutations of the tyrosine at position 387 (Tyr387-->Phe, Tyr387-->Trp, Tyr387-->His, Tyr387-->Asn and Tyr387-->Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme. Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity. Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation. With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis.

Crystal structure of a bacterial chitinase at 2.3 A resolution.

BACKGROUND: Chitinases cleave the beta-1-4-glycosidic bond between the N-acetyl-D-glucosamine units of which chitin is comprised. Chitinases are present in plants, bacteria and fungi, but whereas structures are available for two prototypic plant enzymes, no structure is available for a bacterial or fungal chitinase. RESULTS: To redress this imbalance, the structure of native chitinase A from Serratia marcescens has been solved by multiple isomorphous replacement and refined at 2.3 A resolution, resulting in a crystallographic R-factor of 16.2%. The enzyme comprises three domains: an all beta-strand amino-terminal domain, a catalytic alpha/beta-barrel domain, and a small alpha+beta-fold domain. There are several residues with unusual geometries in the structure. Structure determination of chitinase A in complex with N,N',N",N"'-tetra-acetylo-chitotetraose, together with biochemical and sequence analysis data, enabled the positions of the active-site and catalytic residues to be proposed. CONCLUSIONS: The reaction mechanism seems to be similar to that of lysozyme and most other glycosylhydrolases, i.e. general acid-base catalysis. The role of the amino-terminal domain could not be identified, but it has similarities to the fibronectin III domain. This domain may possibly facilitate the interaction of chitinase A with chitin.

Crystallization of recombinant chitinase from the cloned chiA gene of Serratia marcescens.

The chiA gene encoding for the chitinase enzyme from Serratia marcescens was efficiently overexpressed under the pL promoter and the enzyme was secreted into the growth medium. The chitinase was purified to homogeneity using affinity chromatography on a Phenyl-Sepharose column and the protein was successfully crystallized. The crystals are presently in the form of small needles in space group C222(1) and have unit cell dimensions a = 204(+/- 0.5) A, b = 134(+/- 0.5) A, c = 60(+/- 0.5) A. The crystals diffract X-rays to about 3 A resolution and are suitable for three-dimensional structural analysis.

Crystallization of recombinant chitobiase from Serratia marcescens.

We are currently investigating the biochemical and structural properties of both chitin degrading enzymes chitinase and chitobiase from Serratia marcescens. Previously we have reported the first crystallization and characterization of chitinase crystals (Vorgias et al., 1992). In this communication we present the first crystallization of chitobiase. The protein was synthesized in Escherichia coli and purified to homogeneity using cation exchange chromatography and fast protein liquid chromatography. The crystals have the shape of small prisms and the space group is P2(1) with beta = 101.0 degrees and unit cell dimensions a = 63.2 A, b = 133.2 A, c = 55.1 A. They diffract X-rays to about 2.5 A resolution and are suitable for three-dimensional structural analysis.

Structures of chitobiase mutants complexed with the substrate Di-N-acetyl-d-glucosamine: the catalytic role of the conserved acidic pair, aspartate 539 and glutamate 540.

The catalytic domain of chitobiase (beta-N-1-4 acetylhexosaminidase) from Serratia marcescens, is an alpha/beta TIM-barrel. This enzyme belongs to family 20 of glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp539-Glu540). It was proposed that catalysis by this enzyme family is carried out by glutamate 540 acting as a proton donor and by the acetamido group of the substrate as a nucleophile. We investigated the role of Asp539 and Glu540 by site-directed mutagenesis, biochemical characterization and by structural analyses of chitobiase -substrate co-crystals. We found that both residues are essential for chitobiase activity. The mutations, however, led to subtle changes in the catalytic site. Our results support the model that Glu540 acts as the proton donor and that Asp539 acts in several different ways. Asp539 restrains the acetamido group of the substrate in a specific orientation by forming a hydrogen bond with N2 of the non-reduced (-1) sugar. In addition, this residue participates in substrate binding. It is also required for the correct positioning of Glu540 and may provide additional negative charge at the active site. Thus, these biochemical and structural studies provide a molecular explanation for the functional importance and conservation of these residues.

Solution structure of the HU protein from Bacillus stearothermophilus.

The histone-like protein HU from Bacillus stearothermophilus is a dimer with a molecular mass of 19.5 kDa that is capable of bending DNA. An X-ray structure has been determined, but no structure could be established for a large part of the supposed DNA-binding beta-arms. Using distance and dihedral constraints derived from triple-resonance NMR data of a 13C/15N doubly-labelled HU protein 49 distance geometry structures were calculated, which were refined by means of restrained Molecular Dynamics. From this set a total of 25 refined structures were selected having low constraint energy and few constraint violations. The ensemble of 25 structures display a root-mea-square co-ordinate deviation of 0.36 A with respect to the average structure, calculated over the backbone heavy atoms of residues 2 to 54 and 75 to 90 (and residues 2' to 54' and 75' to 90' of the second monomer). The structure of the core is very similar to that observed in the X-ray structure, with a pairwise r.m.s.d. of 1.06 A. The structure of the beta-hairpin arm contains a double flip-over at the prolines in the two strands of the beta-arm. Strong 15N-NH heteronuclear nuclear Overhauser effects indicate that the beta-arm and especially the tip is flexible. This explains the disorder observed in the solution and X-ray structures of the beta-arm, in respect of the core of the protein. Overlayed onto itself the beta-arm is better defined, with an r.m.s.d. of 1.0 A calculated over the backbone heavy atoms of residues 54 to 59 and 69 to 74. The tip of the arm adopts a well-defined 4:6 beta-hairpin conformation similar to the iron co-ordinating beta-arms of rubredoxin.

Modular structure, local flexibility and cold-activity of a novel chitobiase from a psychrophilic Antarctic bacterium.

The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature chitobiase ArChb possesses four functionally independent domains: a catalytic domain stabilized by Ca(2+), a galactose-binding domain and an immunoglobulin-like domain followed by a cell-wall anchorage signal, typical of cell-surface proteins from Gram-positive bacteria. Binding of saccharides was analyzed by differential scanning calorimetry, allowing to distinguish unequivocally the catalytic domain from the galactose-binding domain and to study binding specificities. The results suggest that ArChb could play a role in bacterium attachment to natural hosts. Kinetic parameters of ArChb demonstrate perfect adaptation to catalysis at low temperatures, as shown by a low activation energy associated with unusually low K(m) and high k(cat) values. Thermodependence of these parameters indicates that discrete amino acid substitutions in the catalytic center have optimized the thermodynamic properties of weak interactions involved in substrate binding at low temperatures. Microcalorimetry also reveals that heat-lability, a general trait of psychrophilic enzymes, only affects the active site domain of ArChb.

N-Acetylglucosaminidase (chitobiase) from Serratia marcescens: gene sequence, and protein production and purification in Escherichia coli.

The chitobiase (Chb) encoding gene (chb) from Serratia marcescens (Sm) has been cloned, sequenced and expressed in Escherichia coli (Ec). Sequencing has revealed an open reading frame encodinga protein of 885 amino acids (aa). Ec cells harbouring plasmids containing chb can produce enzymatically active Sm Chb protein which is secreted into the periplasm. An efficient purification scheme using cation-exchange chromatographyis presented. This yields about 3 mg of > 95% pure Sm Chb per litre of Ec culture. The deduced aa sequence is 27-aa longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the cell membrane. Comparison with the other members of the family 20 of glycosyl hydrolases revealed that Chb has a conserved central region which aligns with almost all members of this family. According to the crystal structure of Sm Chb, this region comprises the catalytic domain of Chb which has an alpha/beta barrel fold.

The DNA-binding protein HU from mesophilic and thermophilic bacilli: gene cloning, overproduction and purification.

The major histone-like bacterial protein (HU)-encoding genes (hup) from five different Bacilli have been cloned, sequenced and overexpressed in Escherichia coli. The five Bacilli selected are closely related, but have different optimum growth temperatures: greater than 70 degrees C for Bacillus caldolyticus and B. caldotenax; 60-65 degrees C for B. stearothermophilus (Bst); 37 degrees C for B. subtilis and 30 degrees C for B. globigii. The deduced amino acid (aa) sequences from the three thermophiles are identical. Those from the two mesophiles are also identical and differ from those of the thermophiles at eleven aa positions. The mesophilic proteins have an extra two aa at the C terminus. Cells harbouring plasmids containing the hup genes can produce HU. An efficient purification scheme using cation-exchange chromatography and fast protein liquid chromatography is presented. This gives approx. 30-40 mg of more than 95% pure Bst HU per litre of E. coli culture.

Cloning, overexpression, purification and crystallisation of ribosomal protein L9 from Bacillus stearothermophilus.

The cloning, sequencing and overexpression of the gene coding for Bacillus stearothermophilus ribosomal protein L9 is described. The sequence corresponds directly to that presented for the protein itself by classical methods, differing at only a few amino acid positions. The purification and crystallisation of the corresponding L9 protein is presented. The crystals are isomorphous to those described for L9 obtained by conventional methods.

Efficient degradation in vitro of all intermediate filament subunit proteins by the Ca2+-activated neutral thiol proteinase from Ehrlich ascites tumor cells and porcine kidney.

Vimentin, desmin, glial fibrillary acidic protein, neurofilament triplet proteins, and a mixture of cytokeratins were digested with Ca2+-activated neutral thiol proteinase isolated from Ehrlich ascites tumor (EAT) cells and porcine kidney. All intermediate filament proteins were degraded by the proteinase, although with different rates and Ca2+ optima. These results are in part at variance with our previous statement that the Ca2+-activated proteinase from EAT cells is specific for vimentin and desmin.

Colchicine, colcemide and cytochalasin B do not affect translocation of the glucocorticoid hormone-receptor in rat thymocytes or Ehrlich ascites cells.

The involvement of intracellular cytoskeletal elements in the translocation of the dexamethasone-receptor complex from the cytoplasm to the nucleus was studied using the cytoskeleton-disrupting agents colcemide, colchicine and cytochalasin B. These compounds did not affect the translocation of the hormone-receptor complex. We conclude that microfilaments and microtubules do not play a role in the translocation of the glucocorticoid hormone-receptor complex from the cytoplasm to the nucleus.

Nucleic acid-binding activities of the intermediate filament subunit proteins desmin and glial fibrillary acidic protein.

In analogy to experimental results previously obtained with vimentin and neurofilament triplet proteins, the intermediate filament (IF) proteins desmin and glial fibrillary acidic protein (GFAP) were also found to have high capacities to associate with nucleic acids. Employing a collection of native and heat-denatured pro- and eukaryotic DNAs, a series of naturally occurring single-stranded (ss)RNAs and a variety of synthetic polynucleotides of the RNA and DNA type, both proteins could be shown to bind preferentially to single-stranded polynucleotides. In the case of ssDNA and synthetic polyribonucleotides, a clear dependency of the binding activity on the G-content of the nucleic acids was demonstrated. The interaction of desmin with ssDNA and tRNA was characterized by strong cooperativity. When a mixture of desmin and vimentin was offered to excess ssDNA, the cooperativity effect brought about segregation of both protein species into two distinct populations of deoxyribonucleoprotein particles with substantially different sedimentation rates; this segregation is in sharp contrast to the ability of desmin and vimentin to form heteropolymers in filament assembly. In general, desmin and GFAP were found to be similar to vimentin and neurofilament proteins in their nucleic acid-binding properties. However, there were also striking differences between individual non-epithelial IF proteins at this level.

Isolation of glial fibrillary acidic protein from bovine brain white matter and its purification by affinity chromatography on single-stranded DNA-cellulose.

Myelinated axons, which had been prepared from bovine brain white matter employing the flotation method, were extracted with Triton X-100 in a low ionic strength buffer containing 4 mM Mg2+. After delipidation of the detergent-resistant, residual material with chloroform-methanol, glial fibrillary acidic protein (GFAP) was solubilized with 6 M urea. It was enriched by ion exchange chromatography in the presence of 6 M urea on CM-Sepharose CL-6B at pH 5 and on DE52-cellulose at pH 7.6, respectively. The final purification was achieved by affinity chromatography on single-stranded DNA-cellulose in 6 M urea. Employing SDS-polyacrylamide gel electrophoresis, the molecular weight of the purified GFAP was determined to be 51,000. 2D-polyacrylamide gel electrophoresis revealed a major protein constituent of pI 4.7 to 4.8, accompanied by 3 acidic isoelectric variants. Upon incubation at 37 degrees C in the presence of 150 mM KCl, GFAP assembled into 10 nm-filaments.

Isolation, purification and characterization of the intermediate filament protein desmin from porcine smooth muscle.

Desmin was isolated from porcine stomach smooth muscle, which had been treated with 0.6 M KCl in the presence of Triton X-100, by extraction with 6 M urea and chromatographed on CM-Sepharose CL-6B at pH 5. After delipidation with chloroform-methanol, the protein was purified by affinity chromatography on arginine methylester Sepharose 4B and single-stranded DNA-cellulose, respectively. Chromatography on single-stranded DNA-cellulose removed a considerable amount of vimentin which had been extracted and enriched together with desmin. The molecular weight of the purified desmin was 55,000 as determined by polyacrylamide gradient slab gel electrophoresis in the presence of Na-dodecylsulfate. Two-dimensional polyacrylamide gel electrophoresis revealed one major polypeptide of pI 5.3 to 5.4 accompanied by two to three acidic, isoelectric variants. During incubation in the presence of 150 mM KCl, desmin assembled into 10 nm filaments. This method allows the isolation of large amounts of pure desmin in a relatively short time with only minimal denaturation of the protein.

Involvement of the N-terminal polypeptide of vimentin in the formation of intermediate filaments.

The potential to form intermediate filaments of a 54 X 10(3) molecular weight (Mr) polypeptide derived from vimentin by cleavage by the intermediate filament-specific, Ca2+-activated proteinase was investigated. Under physiological conditions of assembly, the breakdown product did not form intermediate filaments. Electron microscopy revealed short, rod-like structures similar to those described by Geisler et al. for a 38 X 10(3) Mr alpha-helical core particle derived from desmin. Since the specific, Ca2+-activated proteinase degrades vimentin preferentially from its N terminus, this result suggests the involvement of the basic, N-terminal polypeptide of vimentin in the assembly of intermediate filaments. This was supported by the observation that arginine inhibits the formation of intermediate filaments from intact vimentin. Whereas lysine had very little effect on the assembly process, guanidinium hydrochloride was effective at the same concentration as arginine. On the basis of these findings, an affinity chromatography method for the identification and isolation of intermediate filament subunit proteins was developed. Beside vimentin, desmin, the 68 X 10(3) Mr neurofilament triplet protein, the glial fibrillary acidic protein and cytokeratins also bound to arginine methylester Sepharose 4B in a salt-stable manner and could be eluted with arginine. The 145 X 10(3) Mr neurofilament triplet protein exhibited reduced binding activity, whereas the 210 X 10(3) Mr subunit did not bind to the affinity matrix. Among the degradation products of vimentin produced by the specific, Ca2+-activated proteinase, only those with molecular weights higher than 40 X 10(3) bound to arginine methylester Sepharose 4B. The same applied to the high molecular weight degradation products of desmin with a protein-resistant 37 X 10(3) Mr polypeptide as the major component. The results suggest that arginine residues of the non-alpha-helical, N-terminal polypeptides of intermediate filament subunit proteins play an important role in filament assembly.

Differential effect of arginine modification with 1,2-cyclohexanedione on the capacity of vimentin and desmin to assemble into intermediate filaments and to bind to nucleic acids.

When the intermediate filament proteins vimentin and desmin were reacted for a short period of time with the arginine-specific reagent 1,2-cyclohexanedione, the modification had a severe, inhibitory effect on the assembly of intermediate filaments and on the susceptibility of the basic, amino-terminal polypeptide of both proteins to degradation by the intermediate filament-specific, Ca2+-activated proteinase. However, it had only a slightly inhibitory effect on the binding of vimentin and desmin to ribosomal RNA from Ehrlich ascites tumour cells. Since the Ca2+-activated proteinase is very likely to be a trypsin-like enzyme, with a preference for arginyl and lysyl peptide bonds, the results indicate that the arginine residues of the amino-terminal polypeptide of vimentin and desmin are highly essential for filament assembly but largely dispensable for the binding of both proteins to nucleic acids. This was supported by the observation that two breakdown products of vimentin lacking a 5 X 10(3) Mr and an 8 X 10(3) Mr polypeptide from the amino terminus, respectively, did not assemble into intermediate filaments but were still capable of binding to rRNA. Both polypeptides also bound to single-stranded DNA-cellulose under non-denaturing conditions, but passed the affinity column in the presence of 6 M-urea. Thus, the binding of vimentin to nucleic acids appears to be based on two components: a non-specific electrostatic interaction mediated by the positively charged arginine residues of the amino-terminal polypeptide that is insensitive to denaturation by urea, and a specific interaction that is sensitive to denaturation by urea.

A rapid purification procedure of recombinant integration host factor from Escherichia coli.

A rapid procedure for the large-scale isolation of recombinant integration host factor (IHF) protein from Escherichia coli is presented. The protein was overproduced in the E. coli K5746 strain, whose construction has already been described. The procedure consists of a mild extraction of protein and fractionation by ammonium sulfate. A single-step affinity chromatography on heparin-Sepharose provided very pure IHF protein. A Mono-S FPLC column was used to highly concentrate the pure IHF for crystallization trials. Attempts to crystallize IHF produced small stable crystals that have a large number of molecules in the asymmetric unit and to date diffract poorly. Further attempts to crystallize IHF under other conditions as well as in a complex with the putative DNA binding site are underway.

Cloning, overproduction, purification and crystallization of the DNA binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima.

The humar gene encoding for the histone-like DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima was efficiently overexpressed in Escherichia coli under the T7 promoter. The HU protein was purified using SP-Sepharose ion-exchange and heparin-affinity chromatography and was successfully crystallized in ammonium sulfate. The crystals were grown in the tetragonal form in space group P43 or P41 and have unit-cell dimensions a = b = 46.12, c = 77.56 A, alpha = beta = gamma = 90 degrees. The crystals diffract X-rays to 1.6 A resolution using synchrotron radiation and are suitable for determination of the HU structure at high resolution.