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

• Applicant: Privatdozent Dr. Sebastian Leptihn
• Subject Area: Biophysics
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 282187439

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.

• DFG Programme: Research Grants