A crucial step during apoptosis is the accumulation of the pro-apoptotic protein Bax on the mitochondria where it triggers permeabilization of the outer membrane. This causes the release of cytochrome c into the cytosol and is considered a point of no return in programmed cell death. Endophilin B1, also known as Bax-interacting factor 1 (Bif-1) stimulates mitochondrial recruitment of Bax during apoptosis and loss of endophilin B1 is noted in many cancer types. Despite the importance of their interaction its role and function during cell death remains unclear. To examine the molecular mechanism behind their interaction this project aimed at solving the structure of endophilin B1-Bax complexes when bound to membrane mimicking platforms known as nanodiscs (NDs). NDs are composed of a lipid bilayer held together by a membrane scaffolding protein (MSP) that encircles the bilayer creating a disc-shaped structure. By designing NDs that resembles the mitochondrial outer membrane (MOM), this study intended to stimulate complex formation and stable binding to nanodiscs with the ambition of visualising their interaction using Cryo-EM. Due to difficulties of expressing and purifying Bax as well as time consuming optimization of ND assembly the final goal could not be reached. By establishing an optimized protocol for NDs using the MSP variant MSP2N2 and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipids as well as identifying challenges of expressing and purifying Bax this study lays ground for future structural studies that aims at elucidating the molecular mechanism behind the interaction.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-475248 |
| Date | January 2022 |
| Creators | Kollberg Hedström, Tobias |
| Publisher | Uppsala universitet, Institutionen för biologisk grundutbildning |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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