Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo -designed heme protein.

Autor:	Sutherland GA; From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom., Grayson KJ; From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom., Adams NBP; From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom., Mermans DMJ; the School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom., Jones AS; the School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom., Robertson AJ; From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom., Auman DB; the Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104., Brindley AA; From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom., Sterpone F; the Laboratoire de Biochimie Théorique, UPR 9080 CNRS, Université Paris Diderot, Sorbonne Paris Cité, 75005 Paris, France, and., Tuffery P; INSERM U973, Université Paris Diderot, Sorbonne Paris Cité, 75013 Paris, France., Derreumaux P; the Laboratoire de Biochimie Théorique, UPR 9080 CNRS, Université Paris Diderot, Sorbonne Paris Cité, 75005 Paris, France, and., Dutton PL; the Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104., Robinson C; the School of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom., Hitchcock A; From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom., Hunter CN; From the Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom, c.n.hunter@sheffield.ac.uk.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2018 May 04; Vol. 293 (18), pp. 6672-6681. Date of Electronic Publication: 2018 Mar 20.
DOI:	10.1074/jbc.RA117.000880
Abstrakt:	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 1 H 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. Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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