Vizualisation of “pore-like” structures of twin arginine translocation (Tat) system in supported lipid bilayers by AFM.

Olga Nolandt, Hartmut Gliemann, Anne Ulrich

Karlsruhe Institute of Technology (KIT), Germany

The twin arginine translocation (Tat) system can transport fully folded proteinsacross bacterial or thylakoid membranes. The Tat system of Bacillus subtilis, which serves to export the phosphodiesterase (PhoD) consists of only two membrane proteins, TatA_d and TatC_d. The larger component TatC_d has a molecular weight of 28 kDa and several membrane-spanning segments. The TatA_d, with molecular weight of 7.3 kDa, consists of an N-terminal transmembrane segment, followed by an amphipathic helix and a highly charged, possibly unstructured C-terminus. Multiple copies of TatA_d are supposed to form the transmembrane channel.

The aim of our study was to visualize in the supported lipid bilayers the protein structures responsible for the Tat membrane transport.

The recombinant TatA_d, TatA_d without unstructured C-terminus, and TatC_d proteins were expressed in E.coli, purified and reconstituted into the lipid vesicles. The samples for the AFM investigations were established by the fusion of the vesicles on the surface of mica according to the standard procedure.

Figure 1. AFM (Tapping Mode) Amplitude Image (800 nm × 800 nm) DMPC lipid bilayer with reconstituted TatA_d (1:1000 mol/mol protein to lipid ratio) on mica.

Some samples demonstrate the “pore-like” structures with diameter of 20-60 nm. (See for instance Fig.1). The dynamic of the “pore” formation is under investigation.

We expect that this approach will provide more detailed information about the structure and functionality of the Tat translocase.