Study of the Protein Complex, Pore Diameter and Pore Forming Activity of the Borrelia burgdorferi P13 Porin [Microbiology]

May 13th, 2014 by Barcena–Uribarri, I., Thein, M., Barbot, M., Sans–Serramitȷana, E., Bonde, M., Mentele, R., Lottspeich, F., Bergstrom, S., Benz, R.

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study, the structure and function of P13 was studied in detail. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nS in 1 M KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ± 100 mV. Blue Native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400 Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry and the use of a p13-deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6 nS pore-forming activity in the outer membrane of B. burgdorferi.