The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel
The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel
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DOI：
10.1093/protein/11.9.797
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发表时间：
1998-09-01
期刊：
影响因子：
--
通讯作者：
Delcour, AH
中科院分区：
文献类型：
--
作者：
Liu, NZ;Delcour, AH
孔蛋白是大肠杆菌外膜的三聚体通道形成蛋白，每个亚基含有16条P链，形成一个跨膜的β-桶，其孔被第三个胞外环(L3)收缩。我们研究了OMPC孔蛋白两个关键区域的定点突变的影响：(I)D315A突变针对连接L3环与相邻桶壁的氢键网络的关键成分；(Ii)D118Q、R174Q和R92Q突变针对L3环根部的可能盐桥。我们纯化了每个突变体的外膜部分，并将它们重组到适合电生理的脂质体中。膜片钳实验表明，D315A、D118Q和R92Q突变体的开放和关闭状态之间的自发转换频率在D315A、D118Q和R92Q突变体中增加，而在R174Q突变体中没有变化。这些转变不受跨膜电压变化的驱动，代表了不同功能构象之间的热振荡。然而，通道的不对称电压依赖失活不受突变的影响，这表明自发门控过程和电压依赖门控过程的分子机制不同。我们认为，L3环在孔道上的位置或灵活性，如假定的氢键网络和盐桥所控制的，在决定自发通道门控的频率中发挥作用。
Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli, Each subunit contains 16 P-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology, Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.