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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes
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Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

机译：平面脂质双层膜中细菌外膜TonB依赖转运蛋白的重构。

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摘要

Micronutrients such as siderophore-bound iron and vitamin B_(12) cross 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 the cis solution 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 the trans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin la (the microbial toxin that targets Cir) to the trans, 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.
机译：微量营养元素（如铁载体结合的铁和维生素B_（12））通过一组22链β-桶形蛋白质穿过革兰氏阴性细菌的外膜。它们共有一个不寻常的特征，即它们的N末端从周质侧插入β桶并堵塞内腔。转运是由能量驱动的TonB蛋白运动引起的，该运动要么将塞子拉出枪管，要么使其在枪管内重新排列。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均未成功。但是，我们发现，如果顺式溶液含有4 M尿素，则通道的周质面朝向该溶液，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。对于前两个，通过除去4 M尿素关闭通道。暴露于4 M尿素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，在FhuA中，向反式，面向细胞外的一面添加铁铬酸（其铁载体）可逆地抑制4 M尿素诱导的通道开放并阻塞通道。使用Cir时，将大肠菌素la（靶向Cir的微生物毒素）添加到反式，面向细胞外的一侧可防止4 M尿素诱导的通道打开。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途径，并且在某种程度上模拟TonB在这些栓塞上的体内作用。

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    Eshwar Udho; Karen S. Jakes; Susan K. Buchanan; Karron J. James; Xiaoxu Jiang; Phillip E. Klebba; Alan Finkelstein;

  • 作者单位

    Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461;

    Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461;

    Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;

    Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada H3A 2B4;

    Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019;

    Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019;

    Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
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  • 关键词

    BtuB; Cir; FhuA; urea-denaturation;

    机译：BtuB;西尔FhuA;尿素变性;

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