Integrated Approach to Study Structure, Function and Dynamics of TRAP Transporters

三重ATP非依赖性周质转运蛋白（TRAP）是细菌和古细菌中一类重要且广泛存在的转运蛋白。典型地，TRAP由两个跨膜结构域和可溶性底物结合蛋白（SBP）组成。单个成员转运多种底物，包括C4-二羧酸、氨基酸或唾液酸。后一种分子在致病菌如霍乱弧菌或流感嗜血杆菌的感染过程中起重要作用。再加上TRAP转运蛋白在真核生物中不存在的事实，这使得它们成为药物发现的有趣靶点。然而，与其他转运蛋白类别（例如ABC转运蛋白或次级活性转运蛋白）相比，TRAPs的研究并不充分，特别是从结构的角度来看。因此，本研究拟采用PELDOR光谱、X射线晶体学和单分子荧光显微镜相结合的方法，对TRAP转运蛋白的结构、功能和动力学进行研究，并利用PELDOR光谱研究TRAP转运蛋白的转运机制。目前的工作假设的运输周期包括多种构象变化所引发的底物易位或转运蛋白和SBP之间的相互作用。这些过程将通过PELDOR对自旋标记蛋白质的测量进行分析。与此同时，纯化的转运蛋白将被结晶化，X射线晶体学将被用来收集高分辨率的信息。最后，自旋标记突变体将被重新用于单粒子荧光显微镜实验。为此，将荧光标记物连接到转运蛋白和SBP上，并将转运蛋白重组为巨大的单层囊泡（GUV）。然后通过单粒子追踪和共定位实验分析不同条件下转运蛋白和SBP之间的相互作用。所有三种方法的综合结果将提供TRAP转运蛋白的非常详细的图片。

The tripartite ATP-independent periplasmic transporters (TRAP) are an important and widespread class of transporters in bacteria and archaea. Typically TRAPs consist of two transmembrane domains and a soluble substrate binding protein (SBP). The individual members translocate a diverse array of substrates, including C4-dicarboxylates, amino acids or sialic acid. The latter molecule plays an important role in infection processes of pathogenic bacteria, such as Vibrio cholerae or Haemophilus influenzae. Together with the fact that TRAP transporters are absent in eukaryotes, this makes them an interesting target for drug discovery. However, in contrast to other transporter classes (e.g. ABC transporters or secondary active transporters), TRAPs are not as well studied, especially from a structural point of view. Therefore, this proposal aims to study the structure, function and dynamics of TRAP transporters by an integrated approach, consisting of PELDOR spectroscopy, X-ray crystallography and single molecule fluorescence microscopy.The transport mechanism of TRAPs will be investigated by PELDOR spectroscopy. The current working hypothesis for the transport cycle includes multiple conformational changes which are triggered by substrate translocation or interactions between transporter and the SBP. These processes will be analysed by PELDOR measurements on the spin labelled proteins. At the same time, the purified transporter will be crystallised and X-ray crystallography will be employed to gather high-resolution information. Finally, the spin labelling mutants will be reused for single particle fluorescence microscopy experiments. For this purpose, fluorescence labels will be attached to both transporter and SBP and the transporter will be reconstituted into giant unilamellar vesicles (GUVs). The interaction between transporter and SBP will then be analysed under different conditions by single particle tracking and co-localization experiments. The combined results from all three methods will deliver a very detailed picture of TRAP transporters.