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.
Müller
Sonja D
SD
Forschungszentrum Karlsruhe, POB 3640, D-76021 Karlsruhe, Germany.
De Angelis
Anna A
AA
Walther
Torsten H
TH
Grage
Stephan L
SL
Lange
Christian
C
Opella
Stanley J
SJ
Ulrich
Anne S
AS
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins