‡Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland.
†Institute of Applied Physics and Center for Functional Nanostructures (CFN) and Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany.
Nano Lett. 2015 May 13;15(5):3624-33. doi: 10.1021/acs.nanolett.5b01223. Epub 2015 Apr 21.
Single-molecule force spectroscopy (SMFS) provides detailed insight into the mechanical (un)folding pathways and structural stability of membrane proteins. So far, SMFS could only be applied to membrane proteins embedded in native or synthetic membranes adsorbed to solid supports. This adsorption causes experimental limitations and raises the question to what extent the support influences the results obtained by SMFS. Therefore, we introduce here SMFS from native purple membrane freely spanning across nanopores. We show that correct analysis of the SMFS data requires extending the worm-like chain model, which describes the mechanical stretching of a polypeptide, by the cubic extension model, which describes the bending of a purple membrane exposed to mechanical stress. This new experimental and theoretical approach allows to characterize the stepwise (un)folding of the membrane protein bacteriorhodopsin and to assign the stability of single and grouped secondary structures. The (un)folding and stability of bacteriorhodopsin shows no significant difference between freely spanning and directly supported purple membranes. Importantly, the novel experimental SMFS setup opens an avenue to characterize any protein from freely spanning cellular or synthetic membranes.
单分子力谱（SMFS）提供了对膜蛋白机械（解）折叠途径和结构稳定性的详细了解。到目前为止，SMFS 只能应用于嵌入天然或合成膜并吸附在固体支撑物上的膜蛋白。这种吸附会导致实验限制，并提出了一个问题，即支撑物在多大程度上影响了 SMFS 获得的结果。因此，我们在这里介绍了从天然紫色膜中自由跨越纳米孔的 SMFS。我们表明，正确分析 SMFS 数据需要扩展描述多肽机械拉伸的蠕虫状链模型，增加描述暴露于机械应力的紫色膜弯曲的立方延伸模型。这种新的实验和理论方法允许对膜蛋白细菌视紫红质的逐步（解）折叠进行表征，并分配单个和分组二级结构的稳定性。细菌视紫红质的（解）折叠和稳定性在自由跨越和直接支撑的紫色膜之间没有显著差异。重要的是，新的实验 SMFS 装置为从自由跨越的细胞或合成膜中表征任何蛋白质开辟了一条途径。
