Comparative analysis of the dynamics of the binding protein-dependent histidine ABC import system and the ECF-type biotin transporter

在原核生物中，介导溶质摄取的典型ABC转运系统由一个胞质外溶质结合蛋白、两个成孔亚基和一个atp酶二聚体组成。我们目前对麦芽糖转运机制的了解主要是基于对大肠杆菌/沙门氏菌麦芽糖转运蛋白的结构和功能分析。由于该系统的一些独特特征，派生的运输模型是否适用于其他I型ABC进口商是值得怀疑的。因此，我们将研究鼠伤寒沙门氏菌中具有相似特征的组氨酸转运体HisJ/LAO-QMP2的构象变化。我们将利用微尺度热泳术和表面等离子体共振作为实验工具，重点研究转运蛋白对其同源结合蛋白的亲和力在转运周期中的变化。这些研究旨在更好地了解由结合蛋白启动的跨膜结构域的构象变化，并反过来在膜的另一侧触发ATP水解。此外，通过利用我们在典型ABC导入体方面的专业知识，我们将研究BioYMN转运体的构象变化，BioYMN转运体是新发现的ecf转运体亚家族的一员。特别地，我们将应用位点定向化学交联对纳入纳米圆盘的半胱氨酸变体，以阐明atp酶BioM的动力学以及BioM和BioN依赖底物和/或核苷酸的相互作用。

Canonical ABC transport systems mediating the uptake of solutes in prokaryotes are composed of an extracytoplasmic solute binding protein, two pore-forming subunits and an ATPase dimer. Our current knowledge on the transport mechanism is largely based on structural and functional analysis of the maltose transporter of E. coli/Salmonella. Due to some unique features of this system it is questionable whether the derived transport model holds for other type I ABC importers. Thus, we will study conformational changes of the similarly well-characterized histidine transporter, HisJ/LAO-QMP2, of S. typhimurium. We will focus on investigating changes in affinity of the transporter for its cognate binding protein during the transport cycle using microscale thermophoresis and surface plasmon resonance as experimental tools. These studies are aimed to better understand conformational changes of the transmembrane domains that are initiated by the binding protein and in turn trigger ATP hydrolysis at the opposite side of the membrane. Furthermore, by taking advantage of our expertise on canonical ABC importers, we will study conformational changes of the BioYMN transporter, a member of the newly discovered subfamily of ECF-transporters. In particular, we will apply sitedirected chemical cross-linking on cysteine variants incorporated into nano-discs to elucidate the dynamics of the ATPase BioM and the interaction of BioM and BioN dependent on substrate and/or nucleotides.