Abstract：
Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall devoted to efflux effectively cell toxic substances. Recent seminal work revealed the high-resolution structure of such a tripartic composition TolC-AcrA-AcrB suggesting to design inhibitors preventing efflux of antibiotics. To show that electrophysiology can provide supporting information here, we reconstitute single TolC homotrimer into a planar lipid membrane, apply a transmembrane voltage and follow the assembly of AcrA to TolC using the modulation of the ion current through TolC channel during binding. In particular, the presence of AcrA in solution increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we show that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex in presence of putative efflux pump inhibitors. Both characteristics, the average ion current across TolC and the current noise, taken into consideration together, point to a stiffening of the tip of TolC which might reduce the formation of the complex.
摘要：
在单个分子水平上表征蛋白质-蛋白质相互作用是一个挑战，实验以及对数据的解释。例如,革兰氏阴性菌含有跨越外部和内部细胞壁的蛋白质复合物，专门用于有效地流出细胞毒性物质。最近的开创性工作揭示了这种三组分组合物TolC-AcrA-AcrB的高分辨率结构，建议设计防止抗生素流出的抑制剂。为了证明电生理学可以在这里提供支持信息，我们将单个TolC同源三聚体重建为平面脂质膜，应用跨膜体积，并在结合过程中使用通过TolC通道的离子电流的调制将AcrA组装到TolC。特别是,AcrA在溶液中的存在增加了通过TolC的平均离子电流，此外,减少了由TolC闪烁引起的离子电流波动。这里,我们表明，离子电流波动的统计特性（功率谱密度）提供了在假定的外排泵抑制剂存在下TolC-AcrA复合物相互作用的补充量度。这两个特点,跨TolC的平均离子电流和电流噪声，一起考虑，指向TolC尖端的硬化，这可能会减少复合物的形成。
