The twin-arginine translocation (Tat) pathway utilizes the proton-motive force to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, we examined a complete set of 19-amino-acid substitutions. Unexpectedly, although the polar amino acid is preferred overall, our experiments suggest that it is not necessary for a functional TatA. Hydrophilicity and helix-stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophilicity, which was demonstrated to impact the Tat transport activity. Furthermore, we identified a four-residue motif at the N terminus of the TatA TMH by sequence alignment. Using a biochemical approach, we found that the N-terminal motif was functionally significant, with evidence indicating a potential role in the preference for utilizing different proton-motive force components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoids, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits: TatA, TatB, and TatC. TatA and TatB possess short transmembrane alpha helices (TMHs), both of which are only 15 residues long in Escherichia coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer, although the functional significance of this mismatch is unclear. Here, we sought to address the functional importance of the hydrophobic mismatch in the Tat transport mechanism in E. coli. We conducted three different assays to evaluate the effect of TMH length mutants on Tat activity and observed that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off following any modification of this length. Surprisingly, TatA and TatB with as few as 11 residues in their TMHs can still insert into the membrane bilayer, albeit with a decline in membrane integrity. These findings support a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, we conclude that the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

The twin arginine translocation (Tat) pathway transports folded protein across the cytoplasmic membrane in bacteria, archaea, and across the thylakoid membrane in plants as well as the inner membrane in some mitochondria. In plant chloroplasts, the Tat pathway utilizes the protonmotive force (PMF) to drive protein translocation. However, in bacteria, it has been shown that Tat transport depends only on the transmembrane electrical potential (Δψ) component of PMF in vitro. To investigate the comprehensive PMF requirement in Escherichia coli, we have developed the first real-time assay to monitor Tat transport utilizing the NanoLuc Binary Technology in E. coli spheroplasts. This luminescence assay allows for continuous monitoring of Tat transport with high-resolution, making it possible to observe subtle changes in transport in response to different treatments. By applying the NanoLuc assay, we report that, under acidic conditions (pH = 6.3), ΔpH, in addition to Δψ, contributes energetically to Tat transport in vivo in E. coli spheroplasts. These results provide novel insight into the mechanism of energy utilization by the Tat pathway.

Although tremendous advances have been made in preparing porous crystals from molecular precursors^1,2, there are no general ways of designing and making topologically diversified porous colloidal crystals over the 10-1,000 nm length scale. Control over porosity in this size range would enable the tailoring of molecular absorption and storage, separation, chemical sensing, catalytic and optical properties of such materials. Here, a universal approach for synthesizing metallic open-channel superlattices with pores of 10 to 1,000 nm from DNA-modified hollow colloidal nanoparticles (NPs) is reported. By tuning hollow NP geometry and DNA design, one can adjust crystal pore geometry (pore size and shape) and channel topology (the way in which pores are interconnected). The assembly of hollow NPs is driven by edge-to-edge rather than face-to-face DNA-DNA interactions. Two new design rules describing this assembly regime emerge from these studies and are then used to synthesize 12 open-channel superlattices with control over crystal symmetry, channel geometry and topology. The open channels can be selectively occupied by guests of the appropriate size and that are modified with complementary DNA (for example, Au NPs).