Nanofluidic Model Membrane for the Single-Molecule Observation of Membrane Proteins
Nanofluidic Model Membrane for the Single-Molecule Observation of Membrane Proteins
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DOI：
10.1021/acs.langmuir.2c00724
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发表时间：
2022-05-31
期刊：
影响因子：
3.9
通讯作者：
Morigaki，Kenichi
中科院分区：
文献类型：
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作者：
Komatsu，Ryota;Tanimoto，Yasushi;Morigaki，Kenichi
膜蛋白在细胞中发挥着重要作用，是药物最重要的靶点之一。在受控模型膜环境中研究膜蛋白可以提供有关其分子特性和功能的明确的定量信息。然而，在模型系统中重建膜蛋白面临着巨大的技术挑战。在这里，我们开发了一种新型模型膜平台，通过结合微图案脂质膜和纳米流体通道来高灵敏度观察膜蛋白。通过光刻整合聚合脂质双层和天然（流体）脂质双层生成微图案模型膜。通过使用由涂覆有生物相容性聚合物刷的二氧化硅纳米颗粒组成的粘附层将聚合物双层和PDMS板附接，在流体双层和聚二甲基硅氧烷(PDMS)板之间形成具有限定厚度的纳米流体通道。当我们将视紫红质 (Rh)（一种 G 蛋白偶联受体 (GPCR)）从去污剂溶解状态重构到液体双层中时，仅成功地重构了在脂质双层中横向扩散并迁移到纳米间隙连接中的 Rh 分子，在纳米间隙连接中可以以极大改善的信号背景比来观察它们。纳米间隙连接有效地分离了重建和观察的位点，并为在单分子水平上研究膜蛋白的分子特性和功能提供了一个新的平台。
Membrane proteins play essential roles in the cell, and they constitute one of the most important targets of drugs. Studying membrane proteins in a controlled model membrane environment can provide unambiguous, quantitative information on their molecular properties and functions. However, reconstituting membrane proteins in a model system poses formidable technological challenges. Here, we developed a novel model membrane platform for highly sensitive observation of membrane proteins by combining a micropatterned lipid membrane and a nanofluidic channel. A micropatterned model membrane was generated by lithographically integrating a polymerized lipid bilayer and a natural (fluid) lipid bilayer. A nanofluidic channel having a defined thickness was formed between the fluid bilayer and a polydimethylsiloxane (PDMS) slab by attaching the polymeric bilayer and PDMS slab using an adhesion layer composed of silica nanoparticles that are coated with a biocompatible polymer brush. As we reconstituted rhodopsin (Rh), a G-protein-coupled receptor (GPCR), from a detergent-solubilized state into the fluid bilayer, only successfully reconstituted Rh molecules diffused laterally in the lipid bilayer and migrated into the nanogap junction, where they could be observed with a vastly improved signal-to-background ratio. The nanogap junction effectively separates the sites of reconstitution and observation and provides a novel platform for studying the molecular properties and functions of membrane proteins at the single-molecular level.