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、两个孔形成亚基和一个ATP酶二聚体组成。目前对麦芽糖转运蛋白转运机制的认识主要是基于对大肠杆菌麦芽糖转运蛋白的结构和功能分析。大肠杆菌/沙门氏菌。由于这一制度的一些独特的特点，它是值得怀疑的衍生运输模式是否适用于其他第一类ABC进口商。因此，我们将研究类似的良好表征的组氨酸转运蛋白，HisJ/LAO-QMP 2，S的构象变化。鼠伤寒。我们将专注于调查在运输周期中使用微尺度热泳和表面等离子体共振作为实验工具，其同源结合蛋白的转运蛋白的亲和力的变化。这些研究旨在更好地理解由结合蛋白引发的跨膜结构域的构象变化，进而触发膜相对侧的ATP水解。此外，通过利用我们在经典ABC进口商方面的专业知识，我们将研究BioYMN转运蛋白的构象变化，该转运蛋白是新发现的ECF转运蛋白亚家族的成员。特别是，我们将应用sitedirected化学交联半胱氨酸变体纳入纳米光盘阐明的ATP酶BioM的动力学和BioM和BioN依赖于底物和/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.