Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin.
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.
From the Rudolf-Virchow-Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany, School of Engineering and Science, Jacobs University Bremen, Campusring 1, D-28759 Bremen, Germany, i.barcenauribarri@jacobs-university.de.
Iván
I
Bárcena-Uribarri
From the Rudolf-Virchow-Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
Marcus
M
Thein
From the Rudolf-Virchow-Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
Mariam
M
Barbot
From the Rudolf-Virchow-Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
Eulalia
E
Sans-Serramitjana
Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden, and.
Mari
M
Bonde
Max-Planck Institute of Biochemistry, Protein Analysis Department, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Reinhard
R
Mentele
Max-Planck Institute of Biochemistry, Protein Analysis Department, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Friedrich
F
Lottspeich
Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden, and.
Sven
S
Bergström
From the Rudolf-Virchow-Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany, School of Engineering and Science, Jacobs University Bremen, Campusring 1, D-28759 Bremen, Germany.
Roland
R
Benz
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D025065:Borrelia burgdorferi; D004305:Dose-Response Relationship, Drug; D015640:Ion Channel Gating / Q000187:drug effects; D008051:Lipid Bilayers / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000378:metabolism*; D016062:Porosity; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D018272:Porins; D012492:Salts
Antibiotics; Antigen; Bacterial Pathogenesis; Black Lipid Bilayer; Blue Native PAGE; Membrane Protein; Membrane Transport