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Wolfgang Baumeister - One of the best experts on this subject based on the ideXlab platform.
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Blotting protein complexes from native gels to electron microscopy grids
Nature methods, 2012Co-Authors: Roland Wilhelm Knispel, Christine Kofler, Marius Boicu, Wolfgang Baumeister, Stephan NickellAbstract:We report a simple and generic method for the direct transfer of protein complexes separated by native gel electrophoresis to electron microscopy grids. After transfer, sufficient material remains in the gel for identification and characterization by mass spectrometry. The method should facilitate higher-throughput single-particle analysis by substantially reducing the time needed for protein purification, as demonstrated for three complexes from Thermoplasma acidophilum.
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vat the Thermoplasma homolog of mammalian p97 vcp is an n domain regulated protein unfoldase
Journal of Biological Chemistry, 2005Co-Authors: Alexandra Gerega, Tomohiro Tamura, Wolfgang Baumeister, Beate Rockel, Jurgen Peters, Peter ZwicklAbstract:Abstract The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg2+ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VATΔN complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.
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Thermoplasma acidophilum TAA43 is an archaeal member of the eukaryotic meiotic branch of AAA ATPases.
Biological chemistry, 2004Co-Authors: Leticia Santos, Wolfgang Baumeister, Jurgen Peters, Andrei N Lupas, Tancred Frickey, Peter ZwicklAbstract:Sequencing of the Thermoplasma acidophilum genome revealed a new gene, taa43 , which codes for a 43-kDa protein containing one AAA domain; we therefore termed it Thermoplasma AAA ATPase of 43 kDa (TAA43). Close homologs of TAA43 are found only in related Thermoplasmales, e.g. T. volcanium and Ferroplasma acidarmanus , but not in other Archaea. A detailed phylogenetic analysis showed that TAA43 and its homologs belong to the 'meiotic' branch of the AAA family. Although AAA proteins usually assemble into high-molecular-weight complexes, native TAA43 is predominantly dimeric except for a minor fraction eluting in the void volume of a sizing column. Wild-type and mutant TAA43 proteins were overexpressed in Escherichia coli , purified as dimers and characterized functionally. Since the canonical proteasome activating nucleotidase is not present in Thermoplasmales, TAA43 was tested for stimulation of proteasome activity, which was, however, not detected. Interestingly, immunoprecipitation analysis with TAA43 specific antibodies found a fraction of native TAA43 associated with Thermoplasma ribosomal proteins.
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The Chaperones of the archaeon Thermoplasma acidophilum.
Journal of structural biology, 2001Co-Authors: Andreas Ruepp, Wolfgang Baumeister, Beate Rockel, Irina Gutsche, Andrei N LupasAbstract:Abstract Chaperonesare an essential component of a cell's ability to respond to environmental challenges. Chaperones have been studied primarily in bacteria, but in recent years it has become apparent that some classes of chaperones either are very divergent in bacteria relative to archaea and eukaryotes or are missing entirely. In contrast, a high degree of similarity was found between the chaperonins of archaea and those of the eukaryotic cytosol, which has led to the establishment of archaeal model systems. The archaeon most extensively used for such studies is Thermoplasma acidophilum, which thrives at 59°C and pH 2. Here we review information on its chaperone complement in light of the recently determined genome sequence.
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the genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum
Nature, 2000Co-Authors: Andreas Ruepp, Werner Graml, Marthaleticia Santosmartinez, Kristin K Koretke, Craig Volker, Werner H Mewes, Dmitrij Frishman, Susanne Stocker, Andrei N Lupas, Wolfgang BaumeisterAbstract:Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at 59 degrees C and pH 2, which was isolated from self-heating coal refuse piles and solfatara fields. Species of the genus Thermoplasma do not possess a rigid cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function of their more complex eukaryotic homologues. Our interest in protein folding and degradation led us to seek a more complete representation of the proteins involved in these pathways by determining the genome sequence of the organism. Here we have sequenced the 1,564,905-base-pair genome in just 7,855 sequencing reactions by using a new strategy. The 1,509 open reading frames identify Thermoplasma as a typical euryarchaeon with a substantial complement of bacteria-related genes; however, evidence indicates that there has been much lateral gene transfer between Thermoplasma and Sulfolobus solfataricus, a phylogenetically distant crenarchaeon inhabiting the same environment. At least 252 open reading frames, including a complete protein degradation pathway and various transport proteins, resemble Sulfolobus proteins most closely.
Michael J. Danson - One of the best experts on this subject based on the ideXlab platform.
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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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the crystal structure of glucose dehydrogenase from Thermoplasma acidophilum
Structure, 1994Co-Authors: Jamie John, Michael J. Danson, David W Hough, Susan J Crennell, Garry L TaylorAbstract:Abstract Background The archaea are a group of organisms distinct from bacteria and eukaryotes. Structures of proteins from archaea are of interest because they function in extreme environments and because structural studies may reveal evolutionary relationships between proteins. The enzyme glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum is of additional interest because it is involved in an unusual pathway of sugar metabolism. Results We have determined the crystal structure of this glucose dehydrogenase to 2.9 a resolution. The monomer comprises a central nucleotide-binding domain, common to other nucleotide-binding dehydrogenases, flanked by the catalytic domain. Unexpectedly, we observed significant structural homology between the catalytic domain of horse liver alcohol dehydrogenase and T. acidophilum glucose dehydrogenase. Conclusions The structural homology between glucose dehydrogenase and alcohol dehydrogenase suggests an evolutionary relationship between these enzymes. The quaternary structure of glucose dehydrogenase may provide a model for other tetrameric alcohol/polyol dehydrogenases. The predicted mode of nucleotide binding provides a plausible explanation for the observed dual- cofactor specificity, the molecular basis of which can be tested by site-directed mutagenesis.
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crystallization and preliminary crystallographic study of citrate synthase from the thermophilic archaeon Thermoplasma acidophilum
Journal of Molecular Biology, 1993Co-Authors: Rupert J. M. Russell, Michael J. Danson, David Byrom, G.l. TaylorAbstract:Abstract Single crystals of citrate synthase from the Archaeon Thermoplasma acidophilum were obtained in two forms rasing the hanging drop vapour diffusion method and polyethylene glycol 3350 as precipitant. Type 1 crystals belong to the orthorhombic space group P2221, with unit cell dimensions a = 80·9 A, b = 103·8 A, c = 98·3 A and one dimer in the asymmetric unit. Type 2 crystals belong to the monoclinic space group P21, with unit cell dimensions a = 53·8 A, b = 173·8 A, c = 86·7 A and β = 97·1° and two dimers in the asymmetric unit.
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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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.
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Complete Polar Lipid Composition of Thermoplasma acidophilum HO-62 Determined by High-Performance Liquid Chromatography with Evaporative Light-Scattering Detection
Journal of bacteriology, 2002Co-Authors: Haruo Shimada, Naoki Nemoto, Yasuo Shida, Tairo Oshima, Akihiko YamagishiAbstract:Polar ether lipids of Thermoplasma acidophilum HO-62 were purified by high-performance liquid chromatography with an evaporative light-scattering detector. Structures of purified lipids were investigated by capillary gas chromatography, mass spectrometry, and nuclear magnetic resonance. Three types of ether lipids were found: phospholipids, glycolipids, and phosphoglycolipids. The two phospholipids had glycerophosphate as the phosphoester moiety. The seven glycolipids had different combinations of gulose, mannose, and glucose, which formed mono- or oligosaccharides. The eight phosphoglycolipids with two polar head groups contained glycerophosphate as the phosphoester moiety and gulose alone or gulose and mannose, which formed mono- or oligosaccharides, as the sugar moiety. Although gulose is an unusual sugar in nature, several glyco- and phosphoglycolipids contained gulose as one of the sugar moieties in Thermoplasma acidophilum. All the ether lipids had isopranoid chains of C40 or C20 with zero to three cyclopentane rings. The structures of these lipids including four new glycolipids and three new phosphoglycolipids were determined, and a glycosylation process for biosynthesis of these glycolipids was suggested.
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quinone profiles of Thermoplasma acidophilum ho 62
Journal of Bacteriology, 2001Co-Authors: Haruo Shimada, Naoki Nemoto, Yasuo Shida, Tairo Oshima, Akihiko YamagishiAbstract:Quinones of Thermoplasma acidophilum HO-62 were analyzed by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. Menaquinone, methionaquinone, and 2-trans and 2-cis forms of Thermoplasmaquinone were identified. The relative amount of Thermoplasmaquinone increased under anaerobic conditions, and those of menaquinone and methionaquinone increased under aerobic conditions.
Caroline Heath - One of the best experts on this subject based on the ideXlab platform.
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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.
Istvan Nagy - One of the best experts on this subject based on the ideXlab platform.
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proteomics analysis of Thermoplasma quinone droplets
Proteomics, 2019Co-Authors: Istvan Nagy, Marius Boicu, Sandor Varga, Na Sun, Luca Zinzula, Jozsef KukolyaAbstract:A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin-N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D-SDS-PAGE/LC-MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods-column chromatography, immunoprecipitation, and LD ultracentrifugation-are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.
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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, Istvan Nagy, Agnes Hubert, Dennis Thomas, 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, Marius Boicu, Massimiliano Orsini, Sandor Varga, Elisabeth Weyherstingl, Ruben Fernandezbusnadiego, Jozsef Kukolya, 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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the first transformation method for the thermo acidophilic archaeon Thermoplasma acidophilum
Journal of Microbiological Methods, 2013Co-Authors: Erzsebet Baka, Roland Wilhelm Knispel, Istvan Nagy, Sandor Varga, Cedric F V Hobel, Csaba Fekete, Milan Ivanics, Balazs Kriszt, 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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size distribution of native cytosolic proteins of Thermoplasma acidophilum
Proteomics, 2009Co-Authors: Na Sun, Noriko Tamura, Tomohiro Tamura, Roland Wilhelm Knispel, Christine Kofler, Stephan Nickell, Thomas Hrabe, Istvan NagyAbstract:We used molecular sieve chromatography in combination with LC-MS/MS to identify protein complexes that can serve as templates in the template matching procedures of visual proteomics approaches. By this method the sample complexity was lowered sufficiently to identify 464 proteins and - on the basis of size distribution and bioinformatics analysis - 189 of them could be assigned as subunits of macromolecular complexes over the size of 300 kDa. From these we purified six stable complexes of Thermoplasma acidophilum whose size and subunit composition - analyzed by electron microscopy and MALDI-TOF-MS, respectively - verified the accuracy of our method.
