Blount P, Sukharev S I, Schroeder M J, Nagle S K, Kung C
Laboratory of Molecular Biology, University of Wisconsin, Madison 53706, USA.
Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11652-7. doi: 10.1073/pnas.93.21.11652.
MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.
MscL是一种通道蛋白，在受到机械应力时会在大肠杆菌细胞质膜上打开一个大孔。此前，我们通过使用膜片钳作为功能检测方法高度富集了MscL蛋白，并克隆了相应的基因。预测的蛋白质包含一个跨越分子三分之二的大部分为疏水的核心区域和一个更亲水的羧基末端尾巴。由于MscL与已表征的蛋白质没有同源性，通过简单检查无法预测该蛋白质的功能区域。在此，我们通过诱变寻找了该分子的功能重要区域。我们发现，从氨基末端缺失3个氨基酸的短缺失，以及从该蛋白质羧基末端缺失27个氨基酸的较大缺失，对通道特性几乎没有影响。因此，我们将核心机械敏感机制的研究范围缩小到了这个136个氨基酸的蛋白质中的106个残基。相比之下，在假定的第一个跨膜结构域中的一个赖氨酸或周质环中的一个谷氨酰胺的单残基替换导致机械敏感性曲线发生明显偏移和/或通道门控动力学发生巨大变化，这表明这些区域的构象结构对于正常的机械敏感通道门控至关重要。
