Covalently Circularized Nanodiscs : EM and NMR Applications

Abstract

A traditional nanodisc is composed of a nanometer-sized phospholipid bilayer patch encircled by two copies of membrane scaffold protein (MSP). To date, the utility of this system for structural studies has been limited by the heterogeneity in size and only small nanodiscs could be assembled with the currently available MSP. Here, we present novel nanodiscs encircled by DNA scaffold and covalently circularized MSP variants (cMSPs). The use of cMSPs enhanced the homogeneity, thermal stability and proteolytic resistance of the nanodiscs. Moreover, the use of cMSPs and DNA scaffold enabled us to extend the size of nanodiscs up to 100 nm in diameter. Interestingly, we were able to manipulate the shape of the new nanodiscs and produce polygonal as well as elliptical shaped nanodiscs.In this study, we show that reconstitution into cNDs enhanced the quality of NMR spectra for both VDAC-1, a beta-barrel membrane protein, and the G protein-coupled receptor NTR1, an alpha-helical membrane protein.Using our method, we have created 50 nm nanodiscs and used them to study poliovirus entry and RNA translocation. A 50 nm nanodisc is sufficiently large to accommodate multiple copies of the poliovirus receptor, and has enough surface area to act as a surrogate membrane for the RNA translocation complex during viral uncoating. Virus binding to nanodisc-CD155 complex and subsequent insertion of viral components into and across the membrane were confirmed by EM. To obtain a high-resolution structure for the RNA translocation complex we conducted single-particle cryo-EM studies.The method for making these novel nanodiscs as well as the NMR and Cryo-EM data will be will be presented and discussed.