Nanodisc-solubilized membrane protein library reflects the membrane proteome

Abstract

The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

“…We expect that this should provide sufficient lipids, since the lipid to protein ratio in natural membranes is close to 1:1 (w/w) and large proteins and complexes will not easily incorporate into nanodiscs. In fact, it has been recently demonstrated that nanodisc solubilization does represent the entire membrane proteome (Marty et al, 2013). Our approach is schematically explained in Figure 1B.…”

“…For the generation of nanodiscs we opted for the membrane scaffold protein zebrafish Apo A-1 (ZAP1), shown before to form functional nanodiscs with purified rhodopsin (Banerjee et al, 2008). To estimate the minimal quantity of ZAP1 required for proper nanodisc formation of the membrane protein population of Sf 9 cells, we used a membrane protein content of 225±25 μg per 10 6 cells (DeCaluwé et al, 1993) and estimated the average mass of the membrane protein population at 55±5 kDa (Prior et al, 2011;Marty et al, 2013).…”

“…Over the last decade, elegant technology has been developed reconstituting integral membrane proteins into a soluble discoidal phospholipid bilayer environment confined by two copies of a genetically engineered MSP, termed nanodiscs (Bayburt et al, 2002Ritchie et al, 2009;Marty et al, 2013). These nanodiscs have diameters ranging from 9.5 nm (nanodiscs) to 30 nm (macrodiscs) (Chromy et al, 2007;Park et al, 2011) and provide a native-like environment surrounding the hydrophobic domains of the membrane proteins.…”

Nanoyeast and Other Cell Envelope Compositions for Protein Studies and Biosensor Applications

“…Nanodiscs: discoidal nanoparticles with a membrane protein assembled with phospholipids and surrounded by membrane scaffold proteins. Reprinted with permission from ref (32). Copyright 2013 Springer Science+Business Media.…”

Chromatographic and electrophoretic methods for nanodisc purification and analysis

“…Only recently, a progress in native mass spectrometry might allow in the near future a mass spectral analysis of intact nanodisc complexes at single lipid resolution (Hopper et al 2013, Marty et al 2013, 2014. Several excellent reviews have surveyed methods used for analyzing and purifying nanodiscs and nanodisc-reconstituted membrane proteins (Nath et al 2007, Ritchie et al 2009a, Bayburt and Sligar 2010, Inagaki et al 2013.…”