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Michael J. Danson - One of the best experts on this subject based on the ideXlab platform.
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The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.
FEBS Journal, 2007Co-Authors: Caroline Heath, Mareike G. Posner, Hans C. Aass, Abhishek Upadhyay, David J. Scott, Michael J. DansonAbstract:The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (Mr = 5 × 106) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
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Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum.
FEBS letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
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kinetics and mechanism of the citrate synthase from the thermophilic archaeon Thermoplasma acidophilum
Biochemistry, 2000Co-Authors: Linda C Kurz, Rupert J. M. Russell, George R Drysdale, Marian Riley, Maharaj Alejandro Tomar, Judy Chen, Michael J. DansonAbstract:The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS). All discrete steps in the mechanistic sequence of PCS can be identified in TpCS. The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes. However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible. TpCS is a less efficient catalyst. Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis...
Istvan Nagy - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the Thermoplasma acidophilum protein ta1207
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2017Co-Authors: Ganesh Ramnath Pathare, Dennis Thomas, Agnes Hubert, Istvan Nagy, Andreas BracherAbstract:The crystal structure of the Ta1207 protein from Thermoplasma acidophilum is reported. Ta1207 was identified in a screen for high-molecular-weight protein complexes in T. acidophilum. In solution, Ta1207 forms homopentamers of 188 kDa. The crystal structure of recombinant Ta1207 solved by Se-MAD at 2.4 A resolution revealed a complex with fivefold symmetry. In the crystal lattice, calcium ions induce the formation of a nanocage from two pentamers. The Ta1207 protomers comprise two domains with the same novel α/β topology. A deep pocket with a binding site for a negatively charged group suggests that Ta1207 functions as an intracellular receptor for an unknown ligand. Homologues of Ta1207 occur only in Thermoplasmatales and its function might be related to the extreme lifestyle of these archaea. The thermostable Ta1207 complex might provide a useful fivefold-symmetric scaffold for future nanotechnological applications.
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lipoprotein like particles in a prokaryote quinone droplets of Thermoplasma acidophilum
Fems Microbiology Letters, 2016Co-Authors: Istvan Nagy, Roland Wilhelm Knispel, Christine Kofler, Massimiliano Orsini, Sandor Varga, Elisabeth Weyherstingl, Ruben Fernandezbusnadiego, Jozsef Kukolya, Marius Boicu, Stephan NickellAbstract:Cytosolic, globular droplets with an average diameter of 50 nm were observed in vitrified Thermoplasma acidophilum cells by means of cryo-electron tomography. These droplets were isolated by column chromatography and immuno-precipitation protein purification methods. Subsequent chemical and biochemical analyzes identified lipid and protein components, respectively. Two major lipid components, co-migrating menaquinones at the solvent front and the slower migrating Thermoplasma polar lipid U4, were detected by TLC experiments. The major protein component was identified as the 153 amino acid long Ta0547 vitellogenin-N domain protein. This domain has been found so far exclusively in large lipid transport proteins of vertebrates and non-vertebrates. Blast protein database homology searches with Ta0547 did not return any eukaryal hits, homologous sequences were found only in thermo-acidophilic archaeons. However, a profile-sequence domain search performed with the vitellogenin-N domain (PF01347) hmm-profile against the T. acidophilum proteome returned Ta0547 as hit. Electron microscopy appearance of isolated droplets resembled to lipoprotein particles. However no (tetraether) lipid layer could be detected on the droplets surface, rather hydrophobic compounds of the electron dense lumen were surrounded by a denser dis-continuous protein boundary. Based on described features these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet.
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Protein complex purification from Thermoplasma acidophilum using a phage display library
Journal of Microbiological Methods, 2013Co-Authors: Agnes Hubert, Yasuo Mitani, Tomohiro Tamura, Marius Boicu, Istvan NagyAbstract:Abstract We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli . E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available.
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the first transformation method for the thermo acidophilic archaeon Thermoplasma acidophilum
Journal of Microbiological Methods, 2013Co-Authors: Erzsebet Baka, Roland Wilhelm Knispel, Sandor Varga, Cedric F V Hobel, Csaba Fekete, Milan Ivanics, Balazs Kriszt, Istvan Nagy, Jozsef KukolyaAbstract:A transformation method yielding up to 104 transformants per μg circular DNA was developed for Thermoplasma acidophilum. The method is based on a natural DNA uptake process in which T. acidophilum cells keep their integrity and turn competent at pH 3.5 and 58 °C. Shuttle vector maintenance could not be detected, since the used NovR gyraseB gene integrated into its chromosomal counterpart by homologous recombination.
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quantitative proteome and transcriptome analysis of the archaeon Thermoplasma acidophilum cultured under aerobic and anaerobic conditions
Journal of Proteome Research, 2010Co-Authors: Na Sun, Wolfgang Baumeister, Stephan Nickell, Cuiping Pan, Matthias Mann, Istvan NagyAbstract:A comparative proteome and transcriptome analysis of Thermoplasma acidophilum cultured under aerobic and anaerobic conditions has been performed. One-thousand twenty-five proteins were identified covering 88% of the cytosolic proteome. Using a label-free quantitation method, we found that approximately one-quarter of the identified proteome (263 proteins) were significantly induced (>2 fold) under anaerobic conditions. Thirty-nine macromolecular complexes were identified, of which 28 were quantified and 15 were regulated under anaerobiosis. In parallel, a whole genome cDNA microarray analysis was performed showing that the expression levels of 445 genes were influenced by the absence of oxygen. Interestingly, more than 40% of the membrane protein-encoding genes (145 out of 335 ORFs) were up- or down-regulated at the mRNA level. Many of these proteins are functionally associated with extracellular protein or peptide degradation or ion and amino acid transport. Comparison of the transcriptome and proteome s...
Akihiko Yamagishi - One of the best experts on this subject based on the ideXlab platform.
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distinct trna modifications in the thermo acidophilic archaeon Thermoplasma acidophilum
FEBS Letters, 2013Co-Authors: Chie Tomikawa, Takuya Kawamura, Akihiko Yamagishi, Takayuki Ohira, Yasushi Inoue, Tsutomu Suzuki, Hiroyuki HoriAbstract:Thermoplasma acidophilum is a thermo-acidophilic archaeon. We purified tRNALeu (UAG) from T. acidophilum using a solid-phase DNA probe method and determined the RNA sequence after determining via nucleoside analysis and m7G-specific aniline cleavage because it has been reported that T. acidophilum tRNA contains m7G, which is generally not found in archaeal tRNAs. RNA sequencing and liquid chromatography–mass spectrometry revealed that the m7G modification exists at a novel position 49. Furthermore, we found several distinct modifications, which have not previously been found in archaeal tRNA, such as 4-thiouridine9, archaeosine13 and 5-carbamoylmethyuridine34. The related tRNA modification enzymes and their genes are discussed.
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effects of ph and temperature on the composition of polar lipids in Thermoplasma acidophilum ho 62
Journal of Bacteriology, 2008Co-Authors: Haruo Shimada, Naoki Nemoto, Yasuo Shida, Tairo Oshima, Akihiko YamagishiAbstract:Thermoplasma acidophilum HO-62 was grown at different pHs and temperatures, and its polar lipid compositions were determined. Although the number of cyclopentane rings in the caldarchaeol moiety increased when T. acidophilum was cultured at high temperature, the number decreased at low pHs. Glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. The amount of caldarchaeol with more than two sugar units on one side increased under low-pH and high-temperature conditions. The amounts of glycolipids increased and those of phosphoglycolipids decreased under these conditions. The proton permeability of the liposomes obtained from the phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes obtained from the phosphoglycolipids that contained one sugar unit. From these results, we propose the hypothesis that T. acidophilum adapts to low pHs and high temperatures by extending sugar chains on their cell surfaces, as well as by varying the number of cyclopentane rings.
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an actin homolog of the archaeon Thermoplasma acidophilum that retains the ancient characteristics of eukaryotic actin
Journal of Bacteriology, 2007Co-Authors: Futoshi Hara, Naoki Nemoto, Kan Yamashiro, Shin-ichi Yokobori, Yoshinori Ohta, Takuo Yasunaga, Shinichi Hisanaga, Akihiko YamagishiAbstract:Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon Thermoplasma acidophilum, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol. 358:145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg2+, critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.
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Structural analysis of the plasmid pTA1 isolated from the thermoacidophilic archaeon Thermoplasma acidophilum.
Extremophiles : life under extreme conditions, 2006Co-Authors: Kan Yamashiro, Tairo Oshima, Shin-ichi Yokobori, Akihiko YamagishiAbstract:Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH1.8 and 56°C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.
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Characterization of the DNA Gyrase from the Thermoacidophilic Archaeon Thermoplasma acidophilum
Journal of bacteriology, 2005Co-Authors: Kan Yamashiro, Akihiko YamagishiAbstract:Thermoplasma acidophilum is sensitive to the antibiotic drug novobiocin, which inhibits DNA gyrase. We characterized DNA gyrases from T. acidophilum strains in vitro. The DNA gyrase from a novobiocin-resistant strain and an engineered mutant were less sensitive to novobiocin. The novobiocin-resistant gyrase genes might serve as T. acidophilum genetic markers.
Caroline Heath - One of the best experts on this subject based on the ideXlab platform.
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The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization.
FEBS Journal, 2007Co-Authors: Caroline Heath, Mareike G. Posner, Hans C. Aass, Abhishek Upadhyay, David J. Scott, Michael J. DansonAbstract:The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (Mr = 5 × 106) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
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discovery of the catalytic function of a putative 2 oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum
FEBS Letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an α2β2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
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Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum.
FEBS letters, 2004Co-Authors: Caroline Heath, Alex C Jeffries, Michael J. DansonAbstract:Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.
Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.
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quantitative proteome and transcriptome analysis of the archaeon Thermoplasma acidophilum cultured under aerobic and anaerobic conditions
Journal of Proteome Research, 2010Co-Authors: Na Sun, Wolfgang Baumeister, Stephan Nickell, Cuiping Pan, Matthias Mann, Istvan NagyAbstract:A comparative proteome and transcriptome analysis of Thermoplasma acidophilum cultured under aerobic and anaerobic conditions has been performed. One-thousand twenty-five proteins were identified covering 88% of the cytosolic proteome. Using a label-free quantitation method, we found that approximately one-quarter of the identified proteome (263 proteins) were significantly induced (>2 fold) under anaerobic conditions. Thirty-nine macromolecular complexes were identified, of which 28 were quantified and 15 were regulated under anaerobiosis. In parallel, a whole genome cDNA microarray analysis was performed showing that the expression levels of 445 genes were influenced by the absence of oxygen. Interestingly, more than 40% of the membrane protein-encoding genes (145 out of 335 ORFs) were up- or down-regulated at the mRNA level. Many of these proteins are functionally associated with extracellular protein or peptide degradation or ion and amino acid transport. Comparison of the transcriptome and proteome s...
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proteomics analysis of Thermoplasma acidophilum with a focus on protein complexes
Molecular & Cellular Proteomics, 2007Co-Authors: Na Sun, Wolfgang Baumeister, Frank Siedler, Roland Wilhelm Knispel, Stephan Nickell, Florian Beck, Beatrix Scheffer, Istvan NagyAbstract:Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.
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electron cryo microscopy of vat the archaeal p97 cdc48 homologue from Thermoplasma acidophilum
Journal of Molecular Biology, 2002Co-Authors: Beate Rockel, Joanita Jakana, Wah Chiu, Wolfgang BaumeisterAbstract:VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.
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purification crystallization and preliminary x ray diffraction analysis of the tricorn protease hexamer from Thermoplasma acidophilum
Journal of Structural Biology, 2001Co-Authors: Jurgen Bosch, Wolfgang Baumeister, Tomohiro Tamura, Gleb Bourenkov, Larsoliver EssenAbstract:Abstract Tricorn protease from Thermoplasma acidophilum is a hexameric enzyme; in vivo the hexamers assemble further to form large icosahedral capsids of 14.6 MDa. Recombinant Tricorn protease was purified as an enzymatically active hexamer of 0.72 MDa that formed crystals of octahedral morphology under low-ionic-strength conditions. These crystals belong to space group C 2 with unit cell dimensions a = 307.5 A, b = 163.2 A, c = 220.9 A, β = 105.5° and diffract to 2.2-A resolution using high-brilliance synchrotron radiation. Based on analysis of the self-rotation function and the presence of a pseudo-origin peak in the native Patterson map, a packing model was derived for the complex, comprising 1.5 hexamers per asymmetric unit with a solvent content of 43%. Due to the ninefold noncrystallographic symmetry the Tricorn crystals represent an interesting case for phasing X-ray crystallographic data by electron microscopic phase information.
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the thermosome alternating alpha and beta subunits within the chaperonin of the archaeon Thermoplasma acidophilum
Journal of Molecular Biology, 1997Co-Authors: Michael Nitsch, Andrei N Lupas, Martin Klumpp, Wolfgang BaumeisterAbstract:Abstract The thermosome of the archaeon Thermoplasma acidophilum is composed of two subunits, α and β, which are arranged in two stacked, eight-membered rings. Electron cryo-microscopy in conjunction with image analysis revealed a 4-fold symmetry in the heterooligomeric α+β thermosome isolated from Thermoplasma, but not in the homooligomeric α-only thermosome expressed in Escherichia coli. This indicates that α and β-subunits alternate within the rings of the Thermoplasma thermosome rather than forming two different homooligomeric rings. In addition, a small subpopulation of 9-fold symmetric complexes was found among the recombinant α-only thermosomes, and a central mass most likely representing bound substrate molecules was observed in about half of the native and recombinant thermosome particles.
