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首页> 外文期刊>Biochemistry >TonB-dependent transporter fhua in planar lipid bilayers: Partial exit of its plug from the barrel
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TonB-dependent transporter fhua in planar lipid bilayers: Partial exit of its plug from the barrel

机译：平面脂质双层中依赖于TonB的转运蛋白fhua：塞子从枪管中部分退出

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摘要

TonB-dependent transporters (TBDTs), which transport iron-chelating siderophores and vitamin B12 across the outer membrane of Gram-negative bacteria, share a conserved architecture of a 22-stranded β-barrel with an amino-terminal plug domain occluding the barrel. We previously reported that we could induce TBDTs to reversibly open in planar lipid bilayers via the use of urea and that these channels were responsive to physiological concentrations of ligands. Here we report that in the presence of urea, trypsin can cleave the amino-terminal 67 residues of the plug of the TonB-dependent transporter FhuA, as assessed by gel shift and mass spectrometry assays. On the bilayer, trypsin treatment in the presence of urea resulted in the induced conductance no longer being reversed upon removal of urea, suggesting that urea opens intact FhuA channels by pulling the plug at least partly out of the barrel and that removal of the urea then allows reinsertion of the plug into the barrel. When expressed separately, the FhuA plug domain was found to be a mostly unfolded structure that was able to occlude isolated FhuA β-barrels inserted into the membrane. Thus, although folded in the barrel, the plug need not be folded upon exiting the barrel. The rate of insertion of the β-barrels into the membrane was tremendously increased in the presence of an osmotic gradient provided by either urea or glycerol. Negative staining electron microscopy showed that FhuA in a detergent solution formed vesicles, thus explaining why an osmotic gradient promoted the insertion of FhuA into membranes.
机译：依赖TonB的转运蛋白（TBDTs）将螯合铁的铁载体和维生素B12转运至革兰氏阴性细菌的外膜，它们具有22链β桶的保守结构，其氨基末端的塞结构域封闭了桶。我们以前曾报道过，我们可以通过使用尿素诱导TBDT在平面脂质双层中可逆地打开，并且这些通道对配体的生理浓度有反应。在这里我们报告说，在尿素的存在下，胰蛋白酶可以裂解TonB依赖性转运蛋白FhuA的栓塞的氨基末端67个残基，如通过凝胶位移和质谱分析所评估的。在双层膜上，在存在尿素的情况下进行胰蛋白酶处理导致去除尿素后诱导的电导不再反转，这表明尿素通过将塞子至少部分从机筒中拉出而打开了完整的FhuA通道，然后去除了尿素允许将塞子重新插入枪管。当单独表达时，发现FhuA插入结构域是大部分未折叠的结构，能够封闭插入膜中的分离的FhuAβ-桶。因此，尽管在筒中折叠，但是插头在离开筒时无需折叠。在由尿素或甘油提供的渗透梯度的存在下，β-桶插入膜的速率大大增加。负染色电子显微镜显示，去污剂溶液中的FhuA形成囊泡，从而解释了为什么渗透梯度会促进FhuA插入膜中。

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