RESUMO
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 immunoprecipitation protein purification methods. Subsequent chemical and biochemical analyses identified lipid and protein components, respectively. Two major lipid components, comigrating 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 discontinuous protein boundary. Based on described features, these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet.
Assuntos
Benzoquinonas/química , Lipoproteínas/química , Lipoproteínas/isolamento & purificação , Thermoplasma/química , Benzoquinonas/isolamento & purificação , Microscopia Crioeletrônica , Lipídeos/química , Lipídeos/isolamento & purificação , Lipoproteínas/metabolismo , Proteoma , Vitelogeninas/química , Vitelogeninas/genética , Vitelogeninas/isolamento & purificaçãoRESUMO
A transformation method yielding up to 10(4) 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 Nov(R) gyraseB gene integrated into its chromosomal counterpart by homologous recombination.
Assuntos
Técnicas de Transferência de Genes , Thermoplasma/genética , Transformação Genética , Cromossomos de Archaea/genética , Clonagem Molecular , Meios de Cultura , DNA Arqueal/genética , DNA Circular/genética , Resistência Microbiana a Medicamentos , Vetores Genéticos/genética , Concentração de Íons de Hidrogênio , Novobiocina/farmacologia , Regiões Promotoras Genéticas , Análise de Sequência de DNA , TemperaturaRESUMO
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.
Assuntos
Microscopia Eletrônica/métodos , Espectrometria de MassasRESUMO
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.
Assuntos
Proteínas Arqueais/metabolismo , Citosol/metabolismo , Thermoplasma/metabolismo , Cromatografia em Gel , Cromatografia Líquida , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
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.
