Purification of the small mechanosensitive channel of Escherichia coli (MscS):: the subunit structure, conduction, and gating characteristics in liposomes
Purification of the small mechanosensitive channel of Escherichia coli (MscS):: the subunit structure, conduction, and gating characteristics in liposomes
复制标题
DOI：
10.1016/s0006-3495(02)75169-2
复制
发表时间：
2002-07-01
影响因子：
3.4
通讯作者：
Sukharev, S
中科院分区：
文献类型：
--
作者：
Sukharev, S
小的机械敏感通道，MSCs，是膨胀驱动的溶质外流系统的一部分，该系统保护细菌在渗透压降档时不被溶解。它在大肠杆菌中已被鉴定为孤儿yggB基因的产物，现在被称为MSCs(Levina等人，1999，EMBO J.18：1730)。在这里，我证明了分离的31 kDa的MSCs蛋白足以形成一个由脂质双层中的张力直接门控的功能性机械敏感通道。在辛基糖苷和脂类存在下纯化的MSCS-6His复合体在高分辨率凝胶过滤柱中以类似200 kDa的颗粒迁移。与此一致的是，蛋白质的交叉连接模式预测了一个六角体。在20-60 mm Hg的压力下，大豆抗菌素脂质体中重组的通道被激活，表现出与原位相同的不对称的I-V曲线和轻微的阴离子选择性。同时，在较宽的盐浓度范围内，单通道电导与缓冲电导成正比。随着压力梯度的增加，通道激活的速度慢于压力梯度减小的关闭速度。因此，打开概率曲线是随着压力的下降而记录的。通过视频成像确定斑块的曲率，可以测量作为膜张力(伽马)函数的通道活动。Po(Gamma)曲线的中点为5.5+/-0.1dyne/cm，当用二态Boltzmann模型拟合时，估计的开环能量为DeltaG=11.4+/-0.5kT，与跃迁相关的面积变化DeltaA=8.4+/-0.4 nm(2)。讨论了自然系统和重构系统中的信道特性之间的对应关系。
The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31 -kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of similar to200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma). Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 0.4 nm(2) when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.