Nanopore-based sensors for nucleic acid sequencing and single-molecule detection typically employ pore-forming membrane proteins with hydrophobic external surfaces, suitable for insertion into a lipid bilayer. In contrast, hydrophilic pore-containing molecules, such as DNA origami, have been shown to require chemical modification to favor insertion into a lipid environment. In this work, we describe a strategy for inserting polar proteins with an inner pore into lipid membranes, focusing here on a circular 12-subunit assembly of the thermophage G20c portal protein. X-ray crystallography, electron microscopy, molecular dynamics, and thermal/chaotrope denaturation experiments all find the G20c portal protein to have a highly stable structure, favorable for nanopore sensing applications. Porphyrin conjugation to a cysteine mutant in the protein facilitates the protein’s insertion into lipid bilayers, allowing us to probe ion transport through the pore. Finally, we probed the portal interior size and shape using a series of cyclodextrins of varying sizes, revealing asymmetric transport that possibly originates from the portal’s DNA-ratchet function.

中文翻译：

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卟啉辅助将嗜热噬菌体门户蛋白对接到脂质双层中：纳米孔工程和表征。

用于核酸测序和单分子检测的基于纳米孔的传感器通常使用具有疏水性外表面的成孔膜蛋白，适合插入脂质双层。相反，已显示亲水的含孔分子，例如DNA折纸，需要进行化学修饰以利于插入脂质环境。在这项工作中，我们描述了一种将带有内孔的极性蛋白插入脂质膜的策略，此处重点关注嗜热噬菌体G20c门控蛋白的圆形12亚基组装。X射线晶体学，电子显微镜，分子动力学和热/离液剂变性实验都发现G20c门禁蛋白具有高度稳定的结构，有利于纳米孔传感应用。卟啉与蛋白质中的半胱氨酸突变体结合有助于蛋白质插入脂质双层中，从而使我们能够探测离子通过孔的转运。最后，我们使用一系列大小不一的环糊精探测了门的内部大小和形状，揭示了可能源自门的DNA棘轮功能的不对称转运。