Structural characterization of the pore forming protein TatAd of the twin-arginine translocase in membranes by solid-state 15N-NMR.

The transmembrane protein TatA is the pore forming unit of the twin-arginine translocase (Tat), which has the unique ability of transporting folded proteins across the cell membrane. This ATP-independent protein export pathway is a recently discovered alternative to the general secretory (Sec) system of bacteria. To obtain insight in the translocation mechanism, the structure and alignment in the membrane of the well-folded segments 2-45 of TatAd from Bacillus subtilis was studied here. Using solid-state NMR in bicelles containing anionic lipids, the topology and orientation of TatAd was determined in an environment mimicking the bacterial membrane. A wheel-like pattern, characteristic for a tilted transmembrane helix, was observed in 15N chemical shift /15N-1H dipolar coupling correlation NMR spectra. Analysis of this PISA wheel revealed a 14-16 residue long N-terminal membrane-spanning helix which is tilted by 17 degrees with respect to the membrane normal. In addition, comparison of uniformly and selectively 15N-labeled TatA2-45 samples allowed determination of the helix polarity angle.

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