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UBC Theses and Dissertations

Pursuing structural characterization of membrane proteins in peptidisc Dhupar, Harveer Singh


Classical methods for reconstitution of membrane proteins in detergent-free buffer require extensive optimization and multiple purification steps. The recently developed membrane mimetic system - peptidisc - uses a short amphipathic peptide for fast and effective capture of membrane proteins in a single step. We demonstrate a new method to reconstitute proteins into the peptidisc, the on gradient reconstitution. This method works well for large and/or labile complexes. Using this method, we were able to capture a wide range of proteins from 100 kDa – 628 kDa. We have also been able to capture transient interactions with this method demonstrated by the reconstitution of the holo-translocon (HTL). Recent advances in cryo-EM have opened a whole new realm of membrane protein structural biology with a need for membrane protein preparations that are monodispersed and in a native membrane like state. Given its compositional homogeneity and packing density, the peptidisc may be advantageous for cryo-EM structural studies. Here we present the first sub-nm resolution structure of a membrane protein in peptidisc. With this, we demonstrate that the peptide conforms to the shape of the protein showing the versatility of the peptidisc. The peptidisc is a powerful tool on its own, but when combined with the advances in cryo-EM it can push the boundaries of membrane protein structural biology.

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