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Studying insertion, folding and assembly of ion channels at the single molecule level.

研究单分子水平上离子通道的插入、折叠和组装。

基本信息

批准号：
282187439
负责人：
Privatdozent Dr. Sebastian Leptihn
金额：
--
依托单位：
Department of Biological Sciences
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/282187439?language=en
关键词：
Studying insertion folding assembly ion

项目摘要

Goal of the proposed research is to understand membrane protein biogenesis in vitro. In this work, the prototypical potassium channel KcsA serves as a model protein. Monomers of the protein consist of two transmembrane domains whereas the functional, oligomeric channel is tetrameric. With this work we aim to understand general mechanisms of spontaneous insertion, folding and oligomerization of alpha-helical membrane proteins. In contrast to the insertion of proteins by translocases or insertases such as SecYEG or YidC, our research focusses on physico-chemical parameters such as electrostatic interactions, amphipathity or hydrophobicity or the protein itself, but also of its surrounding. One focus is to understand the influence of lipids on insertion, folding and oligomerization of the model proteins.The research project is divided in three parts: In the first part we conduct experiments on the insertion of ion channels, in the second part the folding of monomers in a lipid membrane. In the third part we investigate the association of monomers to a functional oligomer. Since these processes cannot be synchronized we employ single molecule techniques. On the one hand we use fluorescence correlation spectroscopy (FCS). Using FCS and FCS-based Förster-Resonance-Energy-Transfer (FRET), we investigate the insertion, folding and oligomerization in lipid vesicles. On the other hand, we employ an artificial lipid bilayer system, the so-called Droplet-Interface-Bilayers (DIBs). By using DIBs we have the possibly to detect the formation of functional channels, since we can perform electrical measurements in addition to optical ones.

这项研究的目标是了解膜蛋白的体外生物发生。在这项工作中，原型钾通道KCSA作为模型蛋白。蛋白质的单体由两个跨膜区组成，而功能性的寡聚体通道是四聚体。通过这项工作，我们旨在了解α-螺旋膜蛋白自发插入、折叠和寡聚的一般机制。与通过转位酶或插入酶如SecYEG或YidC插入蛋白质不同，我们的研究集中在物理化学参数上，如静电相互作用、两亲性或疏水性或蛋白质本身，但也包括其周围的参数。一个重点是了解脂质对模型蛋白插入、折叠和齐聚的影响。研究项目分为三个部分：第一部分是离子通道的插入实验，第二部分是单体在脂膜中的折叠实验。在第三部分中，我们研究了单体与功能齐聚物的缔合作用。由于这些过程不能同步，我们采用了单分子技术。一方面，我们使用了荧光相关光谱(FCS)。利用FCS和基于FCS的Förster-Resonance-Energy Transfer(FRET)方法，我们研究了脂泡的插入、折叠和寡聚作用。另一方面，我们使用了一种人工脂质双层系统，即所谓的液滴-界面-双层(DIBS)。通过使用DIBS，我们有可能检测到功能通道的形成，因为除了光学测量之外，我们还可以进行电测量。