Abstract
Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.
Highlights
Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism
The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes
The heterodisulfide is reduced by the heterodisulfide reductase complex that is supplied with reducing equivalents by the methylviologenreducing hydrogenase [5]
Summary
Cultivation and Preparation of Cell-free Extract of M. thermautotrophicus—M. thermautotrophicus (DSM1053) was cultured in a 12-liter fed-batch fermentor containing 10 liters of medium. Cytosolic and membrane protein preparations were diluted with 2–3 volumes of solubilization buffer (50 mM NaCl, 5 mM 6-aminocaproic acid, 1 mM EDTA, and 50 mM imidazole, pH 7.0) containing either 2% (v/v) laurylmaltoside, 1% (v/v) digitonin, or 1% (v/v) Triton X-100. For the blue native first dimension, protein preparations were separated on gels containing the following bis-/acrylamide gradients: 4 –13, 5–15, 6 –18, 8 –20, and 10 –22%. Protein fragments were extracted by adding 1 l of 0.5% (v/v) trifluoroacetic acid, 5 mM n-octyl glucopyranoside and incubating for 2 h at room temperature and a final 1-min sonication step. MALDI-TOF MS Measurements and MASCOT Search Parameters—For MS analysis, 0.25 l of extracted peptides was pipetted on a MALDI-TOF sample plate and directly mixed with an equal volume of sample buffer containing 20 mg/ml ␣-cyano-4-hydroxycinnamic acid in 0.05% (v/v) TFA, 50% (v/v) acetonitrile. Genes were considered to be part of an operon when the intergenic distance was smaller than 55 bp
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