Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF
Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF
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DOI：
10.1093/protein/gzh078
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发表时间：
2004-09-01
影响因子：
2.4
通讯作者：
Delcour, AH
中科院分区：
文献类型：
--
作者：
Baslé, A;Qutub, R;Delcour, AH
大肠杆菌porin活性的电压和pH依赖性的分子基础尚不清楚。以前显示L3环不参与电压依赖性。在这里，我们使用了七个OMPF突变体，其中单个细胞外回路（除L3以外）一次被删除。蛋白质以与野生型相当的水平表达，并纯化为三聚体。将野生型和突变蛋白插入平面脂质双层中，以测量其活性。当前的电压关系显示出大于临界电压的典型的孔林通道闭合。七个缺失突变体的临界电压的测量结果与野生型相对于野生型没有显着差异，因此消除了单个环在电压敏感性中的作用。然而，环路L1，L7或L8的缺失影响了在酸性pH下关闭的通道的趋势。野生型通道在酸性pH时更容易关闭，相对于pH 7.0，在pH 4.0时，其开放概率降低了60％。对于缺乏循环L1，L7或L8的突变体，发现通道的打开概率在pH 4.0时与pH 7.0时没有显着差异。其他缺失突变体保留了与野生型通道相似的pH敏感性。根据这些结果讨论了大肠杆菌OMPF孔蛋白电压和pH依赖性的可能机械场景。
The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by similar to60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.