The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Förster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the osmoregulatory type-I ABC importer OpuA from Lactococcus lactis. We present data probing both intradomain distances that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC, and interdomain distances between SBDs or transmembrane domains. Using this methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our work expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

中文翻译：

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ABC进口商OpuA构象状态和动力学的单分子研究

目前通过 ABC 进口商的主动运输模型主要基于结构、生化和遗传数据。我们在这里建立了单分子 Förster 共振能量转移 (smFRET) 分析，以监测来自乳酸乳球菌的渗透调节 I 型 ABC 进口商 OpuA 的构象状态和异质性。我们提供了探测域内距离的数据，这些距离阐明了底物结合域 (SBD) OpuAC 内的构象变化，以及 SBD 或跨膜域之间的域间距离。使用这种方法，我们研究了配体结合机制以及构象变化对 ATP 和甘氨酸甜菜碱的依赖性。我们的工作将 smFRET 调查的范围扩大到一类迄今为止尚未研究过的 ABC 进口商，并为未来全面了解他们的运输周期铺平了道路。