Bárcena-Uribarri Iván, Thein Marcus, Barbot Mariam, Sans-Serramitjana Eulalia, Bonde Mari, Mentele Reinhard, Lottspeich Friedrich, Bergström Sven, Benz Roland
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,
From the Rudolf-Virchow-Center, Deutsche Forschungsgemeinschaft Research Center for Experimental Biomedicine, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
J Biol Chem. 2014 Jul 4;289(27):18614-24. doi: 10.1074/jbc.M113.539528. Epub 2014 May 13.
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.
P13是伯氏疏螺旋体的主要外膜蛋白之一。先前的研究将P13描述为一种孔蛋白。在本研究中，对P13的一些结构和功能方面进行了研究。根据脂质双层研究，P13在1 m KCl中显示出0.6纳西门子的通道形成活性。单通道和选择性测量表明，P13对阳离子或阴离子均无偏好，并且在±100 mV范围内均未显示电压门控。蓝色天然聚丙烯酰胺凝胶电泳用于分离和表征天然状态下的P13蛋白复合物。该复合物具有约300 kDa的高分子量，并且仅由P13单体组成。使用非电解质研究通道大小，显示表观直径约为1.4 nm，截留分子量为400 Da。用不同底物进行的多通道滴定强化了P13形成一般扩散通道的观点。通过二维SDS-PAGE、蛋白质印迹、质谱分析以及使用p13缺失突变株，证实了复合物中P13的身份。结果表明，P13是负责伯氏疏螺旋体外膜中0.6纳西门子孔形成活性的蛋白质。
