Tetrabrachion: A Filamentous Archaebacterial Surface Protein Assembly of Unusual Structure and Extreme Stability

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The surface (S-) layer of the hyperthermophilic archaebacterium Staphylothermus marinuswas isolated, dissected into separate domains cy chemical and proteolytic methods, and analyzed by spectroscopic, electron microscopic and biochemical techniques. The S-layer is formed by a poorly ordered meshwork of branched, filiform morphological subunits resembling dandelion seed-heads. A morphological subunit (christened by us tetrabrachion) consists of a 70 nm long, almost perfectly straight stalk ending in four straight arms of 24 nm length that provide lateral connectivity by end-to-end contacts. At 32 nm from the branching point, tetrabrachion carries two globular particles of 10 nm diameter that have both tryptic and chymotryptic protease activity. Tetrabrachion is built by a tetramer of M r92,000 polypeptides that form a parallel, four-stranded α-helical rod and separate at one end into four strands. These strands interact in a 1:1 stoichiometry with polypeptides of M r85,000 to form the arms. The arms are composed entirely of β-sheets. All S-layer components contain bound carbohydrates (glucose, mannose, and glucosamine) at a ratio of 38 g/100 g protein for the complete tetrabrachion-protease complex. The unique structure of tetrabrachion is reflected in an extreme thermal stability in the presence of strong denaturants (1% (w/v) SDS of 6M guanidine): the arms, which are stabilized by intramolecular disulphide bridge, melt around 115°C under non-reducing conditions, whereas the stalk sustains heating up to about 130°C. Complete denaturation of the stalk domain requires treatment with 70% (v/v) sulfuric acid or with fumin trifluoromethanesulfonic acid. The globular protease can be heated to 90°C in 6M guanidine and to 120°C in 1% SDS and represents one of the most stable proteases characterized to date.