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

这项研究的目的是了解膜蛋白在体外的生物合成。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结构域组成，而功能性寡聚通道是四聚体。通过这项工作，我们的目标是了解自发插入，折叠和寡聚化的α-螺旋膜蛋白的一般机制。与通过移位酶或插入酶如SecYEG或YidC插入蛋白质相反，我们的研究集中在物理化学参数，如静电相互作用，两亲性或疏水性或蛋白质本身，以及其周围。本研究分为三个部分：第一部分是离子通道的插入实验，第二部分是单体在脂质膜中的折叠实验。在第三部分中，我们研究了单体与功能性寡聚体的缔合。由于这些过程不能同步，我们采用单分子技术。一方面，我们使用荧光相关光谱（FCS）。利用FCS和基于FCS的Förster-Resonance-Energy-Transfer（FRET）技术，我们研究了脂质囊泡中的插入、折叠和寡聚化。另一方面，我们采用人工脂质双层系统，即所谓的液滴界面双层（DIB）。通过使用DIB，我们有可能检测功能通道的形成，因为除了光学测量之外，我们还可以进行电学测量。

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.