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

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-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.

Engineering of the Bacillus subtilis PhoD signal peptide to direct high-level, Tat-specific export of a single-chain antibody fragment to the periplasm in Eschericha coli

“…It should be noted that we have not tested the 'quality'of the exported proteins, in terms of folding delity or activity. However, several heterologous proteins (including an scFv)have been exported by Tat and shown to be active in previous studies [7,11,12,13] and there is no obvious reason why export mediated by the PhoDn signal should be different. Moreover, a range of studies have shown that the Tat system is predisposed to export correctly folded proteins [5][6][7][8][9][10][11][12][13][14][15][16][17][18]10].…”

The Twin-Arginine Pathway for Protein Secretion

“…Generally, Tat substrates only exit the cytosol once they are fully folded and contain their cofactor (138,139,140,141,142). The respective proofreading is extremely tight in B. subtilis (166,167), but 10 less stringent in the thylakoidal Tat system as unfolded proteins are also transported (35).…”

Designed for life: biocompatible de novo designed proteins and components

“…The functionalities of natural systems may be harnessed, for example, by transporting man-designed elements to the desired location within the cell. In recent research the E. coli twin-arginine translocation (TAT) apparatus, whose quality control mechanism will only allow the export of fully folded proteins across the cytoplasmic membrane, could ‘read’ the folding state of a completely artificial heme-binding protein and translocate it to the periplasm [ 65 ]. While the bacterial Sec system has been proved capable of transporting de novo proteins in an unfolded state [ 24 ], the TAT system may be able to transport other de novo proteins that must fold in the cytoplasm prior to translocation.…”