Synthesis and utilization of reversible and irreversible light-activated nanovalves derived from the channel protein MscL
Synthesis and utilization of reversible and irreversible light-activated nanovalves derived from the channel protein MscL
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DOI：
10.1038/nprot.2007.196
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Feringa, Ben L.
中科院分区：
文献类型：
--
作者：
Kocer, Armagan;Walko, Martin;Feringa, Ben L.
该方案详细描述了将大电导（MscL）通道蛋白的机械敏感通道化学修饰成光激活纳米阀及其在合成脂质体囊泡中用于触发递送的用途。它是基于电荷诱导的激活，否则机械敏感通道共价连接到蛋白质的合理设计的合成功能。在黑暗中，这些功能将不带电，通道将保持关闭，但紫外线照射将导致它们的电离和触发通道活动。在可逆激活的情况下，随后用可见光照射将中和电荷，导致通道关闭。该方案包括光响应化合物的合成，蛋白质分离及其化学标记，蛋白质重组成人工膜，其在单分子水平上的分析及其在脂质体递送中的应用。整个过程需要4天。与诱变不同，该方法允许引入定制设计的官能团。
This protocol details the chemical modification of the mechanosensitive channel of large-conductance (MscL) channel protein into a light-activated nanovalve and its utilization for triggered delivery in synthetic liposomal vesicles. It is based on charge-induced activation of this otherwise mechanosensitive channel by covalent attachment to the protein of rationally designed synthetic functionalities. In the dark, these functionalities will be uncharged and the channel will stay closed, but UV illumination will cause their ionization and trigger channel activity. In the case of reversible activation, subsequent illumination with visible light will neutralize the charge, causing the channel to close. The protocol includes synthesis of light-responsive compounds, protein isolation and its chemical labeling, reconstitution of the protein into artificial membranes, its analysis at the single-molecule level and its application in liposomal delivery. The whole protocol takes 4 days. Unlike mutagenesis, this method allows the introduction of custom-designed functional groups.