Thema der Dissertation:
Fluoreszierende Membranspannungssensoren zur Quantifizierung des Membranpotentials der Cytochrom-c- Oxidase und der Analyse von essenziellen Aminosäuren zum Aufbau des Membranpotentials
Fluoreszierende Membranspannungssensoren zur Quantifizierung des Membranpotentials der Cytochrom-c- Oxidase und der Analyse von essenziellen Aminosäuren zum Aufbau des Membranpotentials
Abstract: The ability of cells to generate energy and communicate with other cells is highly dependable of their ability to generate a membrane potential. In mammalian cells the proton motif force created by pumping protons across the inner membrane of the mitochondria is used to provide the cells with energy. A large number of proteins are involved in the pumping. Due to the complexity of the processes involved, the intrinsic work of the proteins and the pathways of the protons across the membrane are yet not fully understood. The investigation of the proteins in vitro may lack important restrictions placed by the membrane and ultimately hiding the correct proton pathways. In this thesis, a proteoliposome membrane voltage-sensitive assay is developed. This assay aims to directly enable the investigation of membrane potential development of membrane-bound ion pumps, such as cytochrome c oxidase. As part of this thesis, several fluorescence setups had to be reconceptualized to facilitate the application of these methods. To first understand the functionality of the assay at the molecular level, a suitable membrane-bound voltage sensor was identified. The voltage sensor protein Archon 1 is used, and its physical coupling of the voltage-sensitive protein group and its fluorescence group were examined. Subsequently, its integration and functionality were examined in the proteoliposome assay. Based on the characterization, the assay was utilized to investigate the establishment of membrane voltage generation through the active proton transport of cytochrome c oxidase. The assay is applied to mutants of cytochrome c oxidase to investigate potential effects on proton uptake and release. Additionally, the evaluation of surface mutants reveals a significant impact on the generation of required membrane potential. Furthermore, the interaction of cytochrome c oxidase with an inhibitor, which is used as a medical drug, is identified.
Zeit & Ort
27.02.2025 | 10:00
Hörsaal B (0.1.01)
Fachbereich Physik, Arnimallee 14, 14195 Berlin