UBC Theses and Dissertations
The study of the P4-ATPase, ATP8A2, investigating disease-causing mutants and methods for high throughput production and purification Choi, Hanbin
Human diseases have been linked to genetic mutations, causing the inability of proteins to function properly. Of these proteins, membrane proteins are crucial for the function of a cell; thus, a mutation affecting these classes of proteins causes extensive harm. This study was focused on the phospholipid transporter P₄-ATPase, ATP8A2, investigating the effects of mutations associated with human diseases. Six associated mutants have been studied: I376M, K429M, K429N, A544P, R625W, and W702R. The study allowed us to differentiate between two classes of mutants: proteins which fold in a native-like conformation allowing export from endoplasmic reticulum (ER), but lacking activity; and proteins that are highly misfolded, retained in the ER, and are rapidly degraded by proteasomes. The well-studied P2-ATPases transport small ion substrates, whereas P4-ATPases transport much larger substrates across the plasma membrane (PM). ATP8A2 was further studied with its β-subunit, CDC50A, to develop a high expression system for protein structural studies. The use of 20 mM CHAPS and 0.04% GDN was the best combination of detergents to solubilize HEK 293F cells with sonication and stirring at 4°C for 45 minutes for maximal purification. Samples analyzed under a transmission electron microscope (TEM) were monodispersed and non-aggregated; however, the same peptidisc-reconstituted sample was aggregated by cryo-electron microscopy (cryo-EM). Further studies are required to prevent aggregation during freezing of ATP8A2-CDC50A for analysis by cryo-EM.
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