K.P. Das, Z. Zhao, M. Zhang, W. Shih, G. Wagner, M.L. Nasr
Brigham and Women's Hospital -Harvard Medical School,
Keywords: DNA-corralled nanodisc, membrane proteins, viral entry, nanodisc
Summary:We will present a modular method to produce stable nanodiscs with sizes up to ~90 nm in diameter using DNA-origami barrel as scaffold. Site-specific functionalization of DNA-origami provides a tool to precisely control membrane protein insertion, with precisely controlled stoichiometry and transmembrane orientation. Therefore, our platform provides an excellent tool for producing homogenous membrane protein complexes in native-like environment with designed composition, stoichiometry, and orientation. We will present some applications for this model system including the reconstitution of membrane protein arrays for structure determination and the application to study viral entry through host membranes. Using our method, we have created 70 nm DNA-corralled nanodiscs (inner diameter) and used them to study poliovirus entry. A 70 nm nanodisc is sufficiently large to accommodate multiple copies of the poliovirus receptor (PVR), and has enough surface area to act as a surrogate membrane for the RNA translocation complex during viral uncoating. Virus binding to PVR-containing nanodisc complex and subsequent pore formation were confirmed by EM. The method for making these novel nanodiscs as well as the EM data will be presented and discussed.