Dokument: SecYEG/YidC-Mediated Membrane Protein Biogenesis
| Titel: | SecYEG/YidC-Mediated Membrane Protein Biogenesis | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=64470 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20240130-083746-0 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Kamel, Michael [Autor] | |||||||
| Dateien: |
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| Beitragende: | Jun.-Prof. Dr. Kedrov, Alexej [Gutachter] Prof. Dr. Lutz Schmitt [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibung: | Proteins destined to leave the cytosol of the cell are transported to and across the cytoplasmic
membrane in prokaryotes or the endoplasmic reticulum in eukaryotes using the universal Sec machinery. In a simple view, proteins destined to cross the cytoplasmic membrane in prokaryotes are delivered post-translationally to the SecYEG: SecA translocon where they get transported through the SecYEG channel using ATP hydrolysis of the motor protein SecA. On the other hand, proteins destined to be transported to the cytoplasmic membrane are delivered co-translationally to SecYEG or the membrane protein insertase, YidC, where they get inserted and properly folded into the membrane. Advances in membrane mimetic systems allowed for better studying and characterization of key processes associated with the membrane. Here, the advances in membrane mimetic systems were employed to study key steps in protein transport to and across the cytoplasmic membrane of bacteria. Combining the use of membrane vesicles and nanodisc technology with biochemical and biophysical methods (fluorescence spectroscopy, SPR, and QCM), it was shown that monounsaturated fatty acids (UFAs) stimulate protein transport via the SecYEG: SecA translocon. Mono-UFAs induce lipid packing defects into the membrane that might promote SecA binding to the membrane via its N-terminal amphipathic helix. The N-terminal helix of SecA is split into helices, a small and a long helix as confirmed by the AlphaFold2 model of the E.coli SecA and the crystal structure of B. subtilis SecA. The results suggest that the long helix promotes electrostatic interactions with the anionic lipids of the membrane via its positively charged face to recruit SecA to the membrane. On the other hand, the small helix promotes hydrophobic interactions with the membrane, where it gets inserted. This step primes SecA for the functional assembly with SecYEG and activates its translocating ATPase activity. Additionally, the N- terminal helix regulates the binding of SecA homologs to the diverse membranes across different bacterial species. Using supported lipid bilayers with single-molecule fluorescence microscopy, the oligomeric state of the Sec translocon, and the assembly of the SecYEG: SecA and SecYEG: ribosomes nascent chain complexes (RNCs) were addressed. The results showed that SecYEG exists mainly as a monomer in the membrane, and the dimer fraction increases once bound to SecA. Moreover, the lateral mobility of translocon was measured and has been shown to be slower once bound to RNCs. Finally, combining the use of cell-free protein synthesis (CFPS) and nanodisc technology, the co-translational assembly of the bacterial membrane protein insertion pathway was established. SecYEG and/or YidC reconstituted in MSP-based nanodiscs or directly isolated using maleic acid copolymers were supplied to CFPS reactions, where they formed complexes with SecM-stalled RNCs. The assembled complexes were purified and can be further used for structural studies using cryo-EM. Nascent chains with different lengths were designed that can be used with the established platform to provide different snapshots of the insertion pathway and provide more details about the path of the nascent chain from the ribosome exit tunnel to the membrane. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Biochemie | |||||||
| Dokument erstellt am: | 30.01.2024 | |||||||
| Dateien geändert am: | 30.01.2024 | |||||||
| Promotionsantrag am: | 03.08.2023 | |||||||
| Datum der Promotion: | 11.12.2023 |
