Danelon Christophe, Brando Thérèse, Winterhalter Mathias
Institut de Pharmacologie et de Biologie Structurale, Université Paul Sabatier, 31077 Toulouse, France.
J Biol Chem. 2003 Sep 12;278(37):35542-51. doi: 10.1074/jbc.M305434200. Epub 2003 Jun 30.
Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.
最近我们发现，重构于黑色脂质膜中的麦芽糖孔蛋白通道在离子传导和糖结合方面均具有明显的不对称特性。这种不对称性还表明麦芽糖孔蛋白的插入具有方向性。然而，其在脂质双层中的取向仍是一个悬而未决的问题。为了阐明取向，我们在通道的每一侧进行了点突变，并分析了由不对称添加麦芽六糖引起的离子电流波动。在第二个系列实验中，我们使用了一种化学修饰的麦芽六糖分子，其从周质侧进入的可能性受到抑制。与细菌的天然外细胞壁不同，我们发现麦芽糖孔蛋白在人工脂质双层中的插入方式是，长的细胞外环暴露于与添加蛋白质时相同的膜侧。基于这种取向，糖结合的方向性特性与生理条件相关。我们发现，自然界通过降低孔两端的激活屏障来优化麦芽糖孔蛋白通道，以便从外部介质捕获糖分子并将它们最有效地排到周质侧。
