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

TitleProbing the quality control mechanism of the twin-arginine translocase with folding variants of a -designed heme protein.
Publication TypeJournal Article
Year of Publication2018
AuthorsSutherland GA, Grayson KJ, Adams NBP, Mermans DMJ, Jones AS, Robertson AJ, Auman DB, Brindley AA, Sterpone F, Tuffery P, Derreumaux P, P Dutton L, Robinson C, Hitchcock A, C Hunter N
JournalJ Biol Chem
Volume293
Issue18
Pagination6672-6681
Date Published2018 05 04
ISSN1083-351X
KeywordsAmino Acid Sequence, Bacterial Proteins, Circular Dichroism, Escherichia coli, Escherichia coli Proteins, Heme-Binding Proteins, Hemeproteins, Membrane Transport Proteins, Methylamines, Models, Molecular, Oxidoreductases, N-Demethylating, Periplasm, Protein Folding, Protein Sorting Signals, Protein Stability, Protein Transport, Proton Magnetic Resonance Spectroscopy, Substrate Specificity, Temperature
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 Tat system to recognize and translocate -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 cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the trimethylamine--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 -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.

DOI10.1074/jbc.RA117.000880
Alternate JournalJ. Biol. Chem.
Citation Key2018|2129
PubMed ID29559557
PubMed Central IDPMC5936819
Grant ListBB/M000265/1 / / Biotechnology and Biological Sciences Research Council / United Kingdom
T32 GM008275 / GM / NIGMS NIH HHS / United States