The Tat system translocates folded proteins across energy-transducing prokaryotic membranes. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was tolerated. Length-variation in TatB clearly destabilized TatBC-containing complexes, indicating that the 12-residues-length is crucial for Tat component interactions and translocon assembly. Metal tagging transmission electron microscopy revealed the dimensions of TatA assemblies, which prompted molecular dynamics simulations. These showed that interacting TMHs of larger TatA assemblies can thin the membrane together with laterally aligned tilted APHs that generate a deep V-shaped groove. The conserved hydrophobic mismatch may thus be important for membrane destabilization during Tat transport, and the exact length of 12 hydrophobic residues could be a compromise between functionality and proton leakage minimization.