大电导的细菌机械敏感通道(MscL)用作生物紧急释放阀,防止急性渗透应激引起的细胞裂解的发生。其易于处理的性质使其成为蛋白质如何直接感知膜张力的范例。尽管很多人都知道在通道门控中特定残基的疏水性的重要性,目前尚不清楚膜上的静电是否起任何作用。我们研究了源自功能不同的直系同源物的MscL嵌合体:大肠杆菌和金黄色葡萄球菌。一组解剖导致观察到改变单个残留物的电荷,大肠杆菌(Ec)-MscL的K101,影响通道表型:当突变为负残基时,该通道的机械敏感性较低,开放停留时间较长。假设静电相互作用,我们通过在蛋白质的其他地方进行定点诱变并将通道重构为确定的脂质,来确定它们是否是由于蛋白质-蛋白质或蛋白质-脂质的相互作用,有和没有负头组。我们发现,尽管这两种相互作用似乎都起着一定的作用,通道表型的主要决定因素似乎是蛋白质-脂质静电。数据表明了静电相互作用在MscL门控动力学中的作用模型。
The bacterial mechanosensitive channel of large conductance (MscL) serves as a biological emergency release valve, preventing the occurrence of cell lysis caused by acute osmotic stress. Its tractable nature allows it to serve as a paradigm for how a protein can directly sense membrane tension. Although much is known of the importance of the hydrophobicity of specific residues in channel gating, it has remained unclear whether electrostatics at the membrane plays any role. We studied MscL chimeras derived from functionally distinct orthologues: Escherichia coli and Staphylococcus aureus. Dissection of one set led to an observation that changing the charge of a single residue, K101, of E. coli (Ec)-MscL, effects a channel phenotype: when mutated to a negative residue, the channel is less mechanosensitive and has longer open dwell times. Assuming electrostatic interactions, we determined whether they are due to protein-protein or protein-lipid interactions by performing site-directed mutagenesis elsewhere in the protein and reconstituting channels into defined lipids, with and without negative head groups. We found that although both interactions appear to play some role, the primary determinant of the channel phenotype seems to be protein-lipid electrostatics. The data suggest a model for the role of electrostatic interactions in the dynamics of MscL gating.