Study of the Protein Complex, Pore Diameter, and Pore-forming Activity of the Borrelia burgdorferi P13 Porin

Iván Bárcena-Uribarri,Marcus Thein,Mariam Barbot,Eulalia Sans-Serramitjana,Mari Bonde,Reinhard Mentele,Friedrich Lottspeich,Sven Bergström,Roland Benz

doi:10.1074/jbc.m113.539528

Abstract

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens 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-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

Highlights

B. burgdorferi P13 has unique characteristics compared with other porins
Faint traces of the P13 protein were observed in Western blots of this first BN-PAGE especially in the low molecular mass range
To minimize this effect and increase the homogeneity of the sample, the P13 complex was extracted from the first blue native PAGE (BNPAGE) and reloaded again on a BN-PAGE of identical characteristics

Summary

Background

B. burgdorferi P13 has unique characteristics compared with other porins. Results: P13 is a homo-oligomer with a 0.6 nS conductance in 1 M KCl and a substrate cut-off of 400 Da. Most of the porins found in the outer membranes of Gram-negative bacteria are composed of antiparallel ␤-sheets forming ␤-barrel cylinders They are arranged as oligomers, usually trimers, which provide a high stability to the protein complex [11, 12]. P13, the subject of this study, represents an atypical type of porin It forms channels in the outer membrane of Borrelia despite its small 13-kDa molecular mass and its ␣-helical secondary structure [24]. The reason for the high copy number of genes coding for P13-like proteins is still not understood, but it reinforces the idea that P13 could be an indispensable outer membrane protein Another protein called Oms, which is exported to the periplasm, was initially described as a porin with a 0.6-nanosiemen (nS) single channel conductance [29]. The 0.6-nS single channel conductance of P13 described in this study was completely different to the 3.5-nS conductance previously reported [24]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION