Characterization and engineering of the twin-arginine translocation pathway of Escherichia coli

The twin-arginine translocation (Tat) pathway of Escherichia coli provides a novel method for the export of proteins from the cytoplasm to the periplasm. Remarkably, it allows for large, folded proteins to cross the inner membrane, with no apparent effect on cell viability. Protein export from the cytoplasm is employed in a variety of biotechnological applications including manufacturing and protein engineering. However, until the discovery of the Tat pathway, such applications relied on the Sec pathway, in which proteins transverse the lipid bilayer membrane in an extended form and protein folding takes place after export. Many proteins of biotechnological interest are not compatible with export via the “classical” Sec pathway. Thus, the export of such Sec-incompatible proteins via the Tat pathway could open the way for new biotechnology applications. This work explores several mechanistic, physiological and technology-related aspects of Tat export In all organisms, proteins are secreted by virtue of a peptide extension, or signal peptide, comprising 15-45 amino acids. The signal peptide serves as a “zip code” and is cleaved after export. E. coli contains 29 putative Tat-specific signal peptides but their ability to mediate export via the Tat pathway has not been confirmed experimentally. The export pathway (Tat or Sec) utilized by this set of 29 signal peptides was characterized using fusions to protein reporters. The reporter proteins chosen for this study are functional only when translocated across the membrane either via the Sec or Tat pathways. Surprisingly, it was found that while 11/29 signal peptides are Tat-specific and 2/29 are Sec-specific, a set of 16/29 signal peptides were able to direct export via both the Tat and Sec pathways. Interestingly, increasing the charge of the region surrounding the cleavage site – particularly the N-terminus of the mature protein – resulted in Tat specificity. In separate studies we showed that in addition to an appropriate signal sequence, proteins destined for export via the Tat pathway must complete their folding in the cytoplasm. Partially folded proteins are not competent for export via this pathway. The requirement for folding in the cytoplasm prior to export was demonstrated by using an elegant system whereby the conformation of the polypeptide chain in the cytoplasm could be controlled using conditions that supported or prevented the formation of disulfide bonds. These results led us to propose that the Tat pathway contains an intrinsic folding quality control mechanism, a concept that has since been widely adopted in the literature. Finally, a new methodology was developed for the engineering of proteins that require the cytoplasmic machinery to fold but must then be exported into the bacterial periplasmic space. Specifically, we created a Tat-based system to enable the display of proteins on filamentous phage, a prerequisite for the high-throughput screening of protein libraries. This system relies on the forced dimerization of the phage coat protein p3, which is exported into the periplasm via the Sec pathway, and the protein that is to be displayed, which is exported via the Tat pathway. Forced dimerization of p3 and the desired protein in the periplasm was mediated by coiled-coil interactions. We further demonstrated that this Tat-dependent display platform shows promise for use in engineering ligand-binding loops into green fluorescent protein (GFP) for sensor applications.