Quality control of substrate conformation in the Escherichia coli Twin Arginine protein-targeting pathway

In Escherichia coli, the twin arginine translocase (Tat) is one of the major protein translocation mechanisms. The Tat system has the ability to transport folded proteins across the inner membrane. Therefore, it has the ability to discriminate between folding states. However, it is not well understood how the Tat system senses the folding state of a substrate. In this study we probed the Tat proofreading mechanism and we investigated whether Tat substrates in E. coli are translocated by the Tat system due to their rapid folding kinetics. We demonstrate that the E. coli Tat machinery can process a de-novo designed substrate (BT6 maquette). Moreover the Tat proofreading mechanism can discriminated between different folding states of this substrate. This data and the fact that this simple four helix artificial substrate offers a lot of engineering freedom, suggests that BT6 is an ideal candidate to study the Tat proofreading mechanism (chapter 3). In chapter 4, we focussed on the Tat system’s proofreading ability by substituting substrate surfaces of BT6 maquette. Mutants with substituted surface properties were expressed in order to understand what Tat senses as folded. Expression assays showed whether the mutants were accepted or rejected by Tat. We propose that the proofreading system does not sense a global unfolded state of the substrate but has the ability to sense localised unfolded regions. Finally, we tested whether Tat substrates fold co- or post-translationally to determine the speed of the folding kinetics by using an arrest peptide-mediated force measurements assay (chapter 5). This study was to increase our understanding about the rationale for using the Tat system