Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes.
Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes.
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平面脂质双层膜中细菌外膜 TonB 依赖性转运蛋白的重建。
DOI：
10.1073/pnas.0910023106
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发表时间：
2009
影响因子：
11.1
通讯作者：
Finkelstein,Alan
中科院分区：
文献类型：
--
作者：
Udho,Eshwar;Jakes,KarenS;Buchanan,SusanK;James,KarronJ;Jiang,Xiaoxu;Klebba,PhillipE;Finkelstein,Alan
铁载体结合的铁和维生素 B12 等微量营养素通过一组 22 链 β 桶蛋白穿过革兰氏阴性细菌的外膜。它们具有一个不寻常的特征，即它们的 N 末端从周质侧插入 β 桶并堵塞管腔。运输是由 TonB 蛋白的能量驱动运动引起的，它要么将塞子从桶中拔出，要么使其在桶内重新排列。迄今为止，在脂质双层膜中以离子传导状态重建天然堵塞通道的尝试尚未成功。然而，我们发现，如果溶液含有 4 M 尿素，那么，当通道的周质侧面向该溶液时，可以观察到宏观电导和单通道事件。这些结果是通过 FhuA、Cir 和 BtuB 获得的；对于前两者，通过去除 4 M 尿素来关闭通道。 4 M 尿素暴露产生的通道不是一般蛋白质变性的结果，因为它们的配体结合特性得以保留。因此，使用FhuA，将铁铬（其铁载体）添加到面向细胞外的反侧可逆地抑制4M尿素诱导的通道开放并阻断通道。对于 Cir，将大肠菌素 Ia（针对 Cir 的微生物毒素）添加到面向细胞外的反面可防止 4 M 尿素诱导的通道开放。我们假设 4 M 尿素可逆地展开 FhuA 和 Cir 栓塞，从而打开通过这些通道的离子传导通路，并且这在某种程度上模拟了 TonB 对这些栓塞的体内作用。
Micronutrients such as siderophore-bound iron and vitamin B12cross the outer membrane of Gram-negative bacteria through a group of 22-stranded β-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the β-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if thecissolution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to thetrans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to thetrans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.