Tryptophan in the Pore of the Mechanosensitive Channel MscS ASSESSMENT OF PORE CONFORMATIONS BY FLUORESCENCE SPECTROSCOPY
Tryptophan in the Pore of the Mechanosensitive Channel MscS ASSESSMENT OF PORE CONFORMATIONS BY FLUORESCENCE SPECTROSCOPY
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DOI：
10.1074/jbc.m109.071472
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发表时间：
2010-02-19
影响因子：
4.8
通讯作者：
Booth, Ian R.
中科院分区：
文献类型：
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作者：
Rasmussen, Tim;Edwards, Michelle D.;Booth, Ian R.
通道蛋白的结构变化提供了理解支撑功能的门控转换所需的关键见解。色氨酸(Trp，Trp)是唯一对环境敏感的物质，可以作为构象变化的报告分子。在这里，我们使用了在小的机械敏感通道蛋白MSCs的毛孔螺旋内的定点Trp插入来监测构象转变。我们发现Trp可以取代Leu在两个孔密封位置Leu(105)和Leu(109)处插入，从而产生功能通道。使用Trp(105)作为探针，我们证明了A106V突变导致纯化的通道蛋白中的修饰构象与溶液中更开放的状态一致。此外，我们还发现，在磷脂或其溶菌体的存在下，溶解的MSCs转变为更开放的构象。
Structural changes in channel proteins give critical insights required for understanding the gating transitions that underpin function. Tryptophan (Trp) is uniquely sensitive to its environment and can be used as a reporter of conformational changes. Here, we have used site-directed Trp insertion within the pore helices of the small mechanosensitive channel protein, MscS, to monitor conformational transitions. We show that Trp can be inserted in place of Leu at the two pore seal positions, Leu(105) and Leu(109), resulting in functional channels. Using Trp(105) as a probe, we demonstrate that the A106V mutation causes a modified conformation in the purified channel protein consistent with a more open state in solution. Moreover, we show that solubilized MscS changes to a more open conformation in the presence of phospholipids or their lysoforms.