Krepel, Sabrina (2021) Directing towards structural insights into the α2β2 isoform of the Na,K-ATPase and the effect of FXYD7. Master's Research Project 2, Biomolecular Sciences.
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Abstract
The Na,K-ATPase establishes an electrochemical gradient of Na- and K-ions over the plasma membrane of a large number of tissues, including the heart, kidney and brain. The properties of the Na,K-ATPase are adjusted accordingly by varying the isoform assembly of its subunits alpha, beta and accessory subunit FXYD. The combination of α1β1 is most common and present in all tissues where α2β2 is found predominantly in muscle cells and astrocytes. The disfunction of α2β2 has been connected to brain-related conditions such as Familial Hemiplegic Migraine (FHM) and Rapid-onset Dystonia Parkinsonism (RDP). In this study, α2β2 Na,K-ATPase was expressed in Pichia pastoris, purified and reconstituted in Saposin A nanodiscs and stabilized with MgFx and ATP in an E2 conformation. Structural analysis by negative stain electron microscopy resulted in a promising dataset with visible subunit domains, but cryo-EM of the same sample did not extend this into a high-resolution map.Additionally, due to the stabilizing nature of FXYD proteins and reported cross-linking of α2β2 to FXYD7, FXYD1 and FXYD7 were compared in terms of stabilization and inhibition by MgFx. FXYD7 stabilized α2β2 during purification in the same manner as FXYD1 but decrease in activity after purification was virtually identical between α2β2 without or with any FXYD, reflecting the general instability of the isoform.
| Item Type: | Thesis (Master's Research Project 2) |
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| Supervisor name: | Slotboom, D.J. |
| Degree programme: | Biomolecular Sciences |
| Thesis type: | Master's Research Project 2 |
| Language: | English |
| Date Deposited: | 06 May 2021 09:00 |
| Last Modified: | 06 May 2021 09:00 |
| URI: | https://fse.studenttheses.ub.rug.nl/id/eprint/24329 |
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