Investigation of the transport mechanism of ECF transporters by static and dynamic structure determination

在这个项目中，能量偶联因子(ECF)转运体最近被归类为ATP结合盒(ABC)转运体家族的成员，本项目将以来自不同乳杆菌的叶酸ECF转运体(ECF-Folt)为例来研究ECF转运体的功能的结构基础。根据最近的晶体结构推测，ECF转运蛋白使用一种史无前例的转运机制，其中一个完整的亚基在脂质双层(Topples Over)中旋转。为了验证这一有趣的假说，我将用X射线结晶学研究ECF-Folt在新构象状态下的结构，并用单分子荧光光谱研究转运蛋白在运输过程中的动态构象变化。对于ECF-Folt的定点荧光标记，将使用体内非天然氨基酸的掺入结合叠氮-炔点击化学和定义的半胱氨酸残基的经典马来酰亚胺交联。此外，ECF-Folt重组为蛋白脂质体不仅可以在膜环境中动态确定结构，还可以对转运蛋白进行生化表征，并建立体外系统来研究配体与蛋白质的相互作用。在分子水平上了解转运机制可能有助于开发针对人类病原体的新型抗生素，由于缺乏生物合成途径，这些病原体的维生素需求依赖于ECF转运体。

In this project, the structural basis for the function of energy-coupling factor (ECF) transporters, which were recently classified as members of the ATP-binding cassette (ABC) transporter family, will be investigated using the example of the folate ECF transporter (ECF-FolT) from different Lactobacillus species. Based on recent crystal structures it was hypothesized that ECF transporters use an unprecedented transport mechanism in which a complete subunit rotates in the lipid bilayer (Topples over). To test this intriguing hypothesis I will study structures of ECF-FolT in novel conformational states by x-ray crystallography and investigate the dynamic conformational changes of the transporter during the transport cycle using single molecule fluorescence spectroscopy. For site-directed fluorescence labelling of ECF-FolT the in vivo incorporation of non-natural amino acids in combination with azide-alkyne click chemistry and classical maleimide crosslinking at defined cysteine residues will be applied. In addition, reconstitution of ECF-FolT into proteoliposomes allows not only the dynamic structure determination in the membrane environment, but also the biochemical characterization of the transporter and the establishment of an in vitro system to investigate ligand-protein interactions. The understanding of the transport mechanism at molecular level may enable the development of novel antibiotics against human pathogens, which are dependent on ECF transporters for their vitamin requirements due to lack of biosynthetic pathways.