Blount P, Sukharev S I, Moe P C, Schroeder M J, Guy H R, Kung C
Laboratory of Molecular Biology, University of Wisconsin-Madison 53706, USA.
EMBO J. 1996 Sep 16;15(18):4798-805.
We have studied the membrane topology and multimeric structure of a mechanosensitive channel, MscL, which we previously isolated and cloned from Escherichia coli. We have localized this 15-kDa protein to the inner membrane and, by PhoA fusion, have shown that it contains two transmembrane domains with both the amino and carboxyl termini on the cytoplasmic side. Mutation of the glutamate at position 56 to histidine led to changes in channel kinetics which were dependent upon the pH on the periplasmic, but not cytoplasmic side of the membrane, providing additional evidence for the periplasmic positioning of this part of the molecule. Tandems of two MscL subunits expressed as a single polypeptide formed functional channels, suggesting an even number of transmembrane domains per subunit (amino and carboxyl termini on the same side of the membrane), and an even number of subunits per functional complex. Finally, cross-linking studies suggest that the functional MscL complex is a homohexamer. In summary, these data are all consistent with a protein domain assignment and topological model which we propose and discuss.
我们研究了一种机械敏感通道MscL的膜拓扑结构和多聚体结构，该通道是我们之前从大肠杆菌中分离并克隆出来的。我们已将这种15 kDa的蛋白质定位到内膜上，并且通过PhoA融合实验表明它含有两个跨膜结构域，其氨基和羧基末端均位于细胞质一侧。将第56位的谷氨酸突变为组氨酸会导致通道动力学发生变化，这种变化取决于膜周质侧而非细胞质侧的pH值，这为该分子这一部分位于膜周质提供了额外证据。以单一多肽形式表达的两个MscL亚基串联形成了功能性通道，这表明每个亚基的跨膜结构域数量为偶数（氨基和羧基末端在膜的同一侧），并且每个功能复合物的亚基数量也为偶数。最后，交联研究表明功能性MscL复合物是一个同型六聚体。总之，这些数据都与我们提出并讨论的蛋白质结构域分配和拓扑模型一致。
