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Probing the quality control mechanism of theEscherichia colitwin-arginine translocase with folding variants of ade novo-designed heme protein

Sutherland, George A., Grayson, Katie J., Adams, Nathan B.P., Mermans, Daphne M.J., Jones, Alexander S., Robertson, Angus J., Auman, Dirk B., Brindley, Amanda A., Sterpone, Fabio, Tuffery, Pierre, and others. (2018) Probing the quality control mechanism of theEscherichia colitwin-arginine translocase with folding variants of ade novo-designed heme protein. Journal of Biological Chemistry, 293 . pp. 6672-6681. ISSN 0021-9258. (doi:10.1074/jbc.RA117.000880) (KAR id:66556)

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Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin arginine translocase (Tat). The Tat machinery exports folded and cofactor containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N-terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system’s quality control mechanism.

Item Type: Article
DOI/Identification number: 10.1074/jbc.RA117.000880
Subjects: Q Science > QD Chemistry > QD431 Organic Chemistry- Biochemistry- Proteins, peptides, amino acids
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
Depositing User: Colin Robinson
Date Deposited: 27 Mar 2018 14:32 UTC
Last Modified: 29 May 2019 20:24 UTC
Resource URI: (The current URI for this page, for reference purposes)
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