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

通过静态和动态结构测定研究 ECF 转运蛋白的转运机制

• 批准号: 282894753
• 负责人: Dr. Marianne Kluth
• 金额: --
• 依托单位: Department of Biochemistry
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/282894753?language=en
• 关键词: Investigation; transport; mechanism; ECF; transporters

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.

在这个项目中，能量偶联因子（ECF）转运蛋白，这是最近被归类为ATP结合盒（ABC）转运蛋白家族的成员的功能的结构基础，将被调查使用的例子叶酸ECF转运蛋白（ECF-FolT）从不同的乳酸杆菌物种。基于最近的晶体结构，假设ECF转运蛋白使用一种前所未有的转运机制，其中一个完整的亚基在脂质双层中旋转（翻转）。为了验证这一有趣的假设，我将研究结构的ECF-FolT在新的构象状态的X-射线晶体学和研究的动态构象变化的转运蛋白在运输周期中使用单分子荧光光谱。对于ECF-FolT的定点荧光标记，将应用非天然氨基酸的体内掺入与叠氮化物-炔点击化学和在限定的半胱氨酸残基处的经典马来酰亚胺交联的组合。此外，重组ECF-FolT进入蛋白脂质体不仅允许在膜环境中的动态结构测定，而且还可以转运蛋白的生化表征和体外系统的建立，以研究配体-蛋白质相互作用。在分子水平上的运输机制的理解可能使新的抗生素的开发对人类病原体，这是依赖于ECF转运蛋白的维生素需求，由于缺乏生物合成途径。