Abstract
The twin arginine translocase (Tat) exports folded proteins across bacterial membranes. The putative pore-forming or membrane-weakening component (TatA(d) in B. subtilis) is anchored to the lipid bilayer via an unusually short transmembrane α-helix (TMH), with less than 16 residues. Its tilt angle in different membranes was analyzed under hydrophobic mismatch conditions, using synchrotron radiation circular dichroism and solid-state NMR. Positive mismatch (introduced either by reconstitution in short-chain lipids or by extending the hydrophobic TMH length) increased the helix tilt of the TMH as expected. Negative mismatch (introduced either by reconstitution in long-chain lipids or by shortening the TMH), on the other hand, led to protein aggregation. These data suggest that the TMH of TatA is just about long enough for stable membrane insertion. At the same time, its short length is a crucial factor for successful translocation, as demonstrated here in native membrane vesicles using an in vitro translocation assay. Furthermore, when reconstituted in model membranes with negative spontaneous curvature, the TMH was found to be aligned parallel to the membrane surface. This intrinsic ability of TatA to flip out of the membrane core thus seems to play a key role in its membrane-destabilizing effect during Tat-dependent translocation.