The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo
The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo
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DOI：
10.1002/pro.686
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发表时间：
2011-09-01
期刊：
影响因子：
8
通讯作者：
Blount, Paul
中科院分区：
文献类型：
--
作者：
Iscla, Irene;Wray, Robin;Blount, Paul
来自大肠杆菌的大电导机械敏感通道（MscL）作为一个紧急释放阀，允许细胞在急性渗透下休克中存活。它是研究得最好的机械敏感通道之一，并作为蛋白质如何感知和响应膜张力的范例。结核分枝杆菌和金黄色葡萄球菌的两个MscL晶体结构已被解决，分别显示五聚体和四聚体结构。随后进行了几项研究，以了解它们的化学计量差异是物种差异还是结晶过程中蛋白质操作的结果。两项独立研究现在一致认为，全长金黄色葡萄球菌MscL实际上是一个五聚体，而不是四聚体。虽然洗涤剂似乎在改变蛋白质的寡聚状态中起作用，但细胞质螺旋束也有牵连。在这里，我们利用体内二硫捕获技术评估了金黄色葡萄球菌MscL的c端区域在天然膜通道寡聚化中的作用。我们发现，具有不同c端截断的金黄色葡萄球菌MscL的低聚态，包括用于获得四聚体金黄色葡萄球菌MscL晶体结构的金黄色葡萄球菌MscL，在体内都是五聚体。因此，金黄色葡萄球菌蛋白的c端结构域仅在SaMscL蛋白溶于洗涤剂时才在其寡聚状态下发挥关键作用。
The mechanosensitive channel of large conductance (MscL) from E. coli serves as an emergency release valve allowing the cell to survive acute osmotic downshock. It is one of the best studied mechanosensitive channels and serves as a paradigm for how a protein can sense and respond to membrane tension. Two MscL crystal structures of the orthologs M. tuberculosis and S. aureus have been solved showing pentameric and tetrameric structures, respectively. Several studies followed to understand whether the discrepancy in their stoichiometry was a species difference or a consequence of the protein manipulation for crystallization. Two independent studies now agree that the full-length S. aureus MscL is actually a pentamer, not tetramer. While detergents appear to play a role in modifying the oligomeric state of the protein, a cytoplasmic helical bundle has also been implicated. Here, we evaluate the role of the C-terminal region of S. aureus MscL in the oligomerization of the channel in native membranes by using an in vivo disulfide-trapping technique. We find that the oligomeric state of S. aureus MscLs with different C-terminal truncations, including the one used to obtain the tetrameric S. aureus MscL crystal structure, are pentamers in vivo. Thus, the C-terminal domain of the S. aureus protein only plays a critical role in the oligomeric state of the SaMscL protein when it is solubilized in detergent.