Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.
J Am Chem Soc. 2015 Apr 22;137(15):4884-7. doi: 10.1021/jacs.5b00040. Epub 2015 Apr 9.
The natural mechanisms that direct proteins to membranes are typically complex, requiring multiple steps and accessory components. It would be advantageous to develop simplified methods to direct proteins of interest to phospholipid membranes in a single step. Here we report a modular method for membrane localization of proteins by using chemically modified phospholipid anchors capable of covalent attachment to O(6)-methylguanine DNA methyltransferase (SNAP-tag) fusion proteins. To our knowledge, this is the first use of SNAP-tag reactions to modify benzylguanine-functionalized lipid membranes. We demonstrate that photocaged lipid precursors enable light-triggered spatial and temporal control over protein localization. The anchoring system is compatible with cell-free expression, allowing for genetic targeting of proteins to lipid membranes of giant unilamellar vesicles. This technique can be used to control membrane curvature effects, similar to what has been previously observed with certain membrane-bound proteins. This work addresses a current need in synthetic biology for simplified and robust methods to control membrane localization of expressed proteins and shows promise as a general tool for protein targeting to lipid vesicles and cellular membranes.
引导蛋白质到膜上的自然机制通常很复杂，需要多个步骤和辅助组件。开发将感兴趣的蛋白质一步直接导向磷脂膜的简化方法将是有利的。在这里，我们报告了一种通过使用能够与 O(6)-甲基鸟嘌呤 DNA 甲基转移酶 (SNAP 标签) 融合蛋白发生共价连接的化学修饰的磷脂锚定来实现蛋白质膜定位的模块化方法。据我们所知，这是首次将 SNAP 标签反应用于修饰苄基鸟嘌呤功能化的脂质膜。我们证明了光笼脂质前体能够实现对蛋白质定位的光触发时空控制。该锚定系统与无细胞表达兼容，允许对质膜进行基因靶向，以实现巨单层囊泡的脂质膜。该技术可用于控制膜曲率效应，类似于先前观察到的某些膜结合蛋白的情况。这项工作满足了合成生物学中对简化和稳健的方法来控制表达蛋白的膜定位的需求，并有望成为将蛋白靶向脂质囊泡和细胞膜的通用工具。
