Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo–designed heme protein
(2018) In Journal of Biological Chemistry 293(18). p.6672-6681- Abstract
- 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... (More)
- 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-dimensional1H 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. (Less)
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- 2018-05-01
- type
- Contribution to journal
- publication status
- published
- in
- Journal of Biological Chemistry
- volume
- 293
- issue
- 18
- pages
- 6672 - 6681
- publisher
- American Society for Biochemistry and Molecular Biology
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- scopus:85046639493
- ISSN
- 1083-351X
- DOI
- 10.1074/jbc.RA117.000880
- language
- English
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- 9a6721d0-ab8b-48e2-84a0-504e37176c6f
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- 2024-01-25 13:40:56
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@article{9a6721d0-ab8b-48e2-84a0-504e37176c6f,
abstract = {{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-dimensional1H 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.}},
author = {{Sutherland, George A. and Grayson, Katie J. and Adams, Nathan B.P. and Mermans, Daphne M.J. and Jones, Alexander S. and Robertson, Angus J. and Auman, Dirk B. and Brindley, Amanda A. and Sterpone, Fabio and Tuffery, Pierre and Derreumaux, Philippe and Dutton, P. Leslie and Robinson, Colin and Hitchcock, Andrew and Hunter, C. Neil}},
issn = {{1083-351X}},
language = {{eng}},
month = {{05}},
number = {{18}},
pages = {{6672--6681}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{Journal of Biological Chemistry}},
title = {{Probing the quality control mechanism of the <i>Escherichia coli </i>twin-arginine translocase with folding variants of a <i>de novo</i>–designed heme protein}},
url = {{http://dx.doi.org/10.1074/jbc.RA117.000880}},
doi = {{10.1074/jbc.RA117.000880}},
volume = {{293}},
year = {{2018}},
}