Henrich Erik, Sörmann Janina, Eberhardt Peter, Peetz Oliver, Mezhyrova Julija, Morgner Nina, Fendler Klaus, Dötsch Volker, Wachtveitl Josef, Bernhard Frank, Bamann Christian
Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Frankfurt am Main, Germany.
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
Biophys J. 2017 Sep 19;113(6):1331-1341. doi: 10.1016/j.bpj.2017.03.026. Epub 2017 Apr 24.
Nanodiscs that hold a lipid bilayer surrounded by a boundary of scaffold proteins have emerged as a powerful tool for membrane protein solubilization and analysis. By combining nanodiscs and cell-free expression technologies, even completely detergent-free membrane protein characterization protocols can be designed. Nanodiscs are compatible with various techniques, and due to their bilayer environment and increased stability, they are often superior to detergent micelles or liposomes for membrane protein solubilization. However, transport assays in nanodiscs have not been conducted so far, due to limitations of the two-dimensional nature of nanodisc membranes that offers no compartmentalization. Here, we study Krokinobacter eikastus rhodopsin-2 (KR2), a microbial light-driven sodium or proton pump, with noncovalent mass-spectrometric, electrophysiological, and flash photolysis measurements after its cotranslational insertion into nanodiscs. We demonstrate the feasibility of adsorbing nanodiscs containing KR2 to an artificial bilayer. This allows us to record light-induced capacitive currents that reflect KR2's ion transport activity. The solid-supported membrane assay with nanodisc samples provides reliable control over the ionic condition and information of the relative ion activity of this promiscuous pump. Our strategy is complemented with flash photolysis data, where the lifetimes of different photointermediates were determined at different ionic conditions. The advantage of using identical samples to three complementary approaches allows for a comprehensive comparability. The cell-free synthesis in combination with nanodiscs provides a defined hydrophobic lipid environment minimizing the detergent dependence often seen in assays with membrane proteins. KR2 is a promising tool for optogenetics, thus directed engineering to modify ion selectivity can be highly beneficial. Our approach, using the fast generation of functional ion pumps incorporated into nanodiscs and their subsequent analysis by several biophysical techniques, can serve as a versatile screening and engineering platform. This may open new avenues for the study of ion pumps and similar electrogenic targets.
由支架蛋白边界包围着脂质双层的纳米圆盘已成为一种用于膜蛋白溶解和分析的强大工具。通过将纳米圆盘与无细胞表达技术相结合，甚至可以设计出完全无去污剂的膜蛋白表征方案。纳米圆盘与各种技术兼容，并且由于其双层环境和更高的稳定性，在膜蛋白溶解方面它们通常优于去污剂胶束或脂质体。然而，由于纳米圆盘膜的二维性质限制了其分区功能，到目前为止尚未在纳米圆盘中进行转运测定。在这里，我们研究了嗜盐栖热袍菌视紫红质-2（KR2），一种微生物光驱动的钠或质子泵，在其共翻译插入纳米圆盘后，通过非共价质谱、电生理和闪光光解测量进行研究。我们证明了将含有KR2的纳米圆盘吸附到人工双层膜上的可行性。这使我们能够记录反映KR2离子转运活性的光诱导电容电流。使用纳米圆盘样品的固体支持膜测定法可对离子条件进行可靠控制，并提供这种多功能泵的相对离子活性信息。我们的策略辅以闪光光解数据，其中在不同离子条件下测定了不同光中间体的寿命。对相同样品采用三种互补方法的优势在于可实现全面的可比性。无细胞合成与纳米圆盘相结合提供了明确的疏水脂质环境，最大限度地减少了膜蛋白测定中常见的对去污剂的依赖。KR2是光遗传学中有前景的工具，因此进行定向工程改造以改变离子选择性可能非常有益。我们的方法利用快速生成整合到纳米圆盘中的功能性离子泵，并随后通过几种生物物理技术对其进行分析，可作为一个通用的筛选和工程平台。这可能为离子泵和类似的电生靶标的研究开辟新途径。
