Solubilization, Purification and Characterization of the Potassium Channel Kcsa in its Native Lipid Environment: The Power of Native Nanodiscs

Abstract

The common use of detergents in solubilizing integral membrane proteins to allow for their purification and biophysical study involves the complete disruption of the lipid bilayer, which can result in a decrease of protein stability and function. We report here the detergent-free solubilization and affinity purification of the tetrameric potassium channel KcsA in native nanodiscs stabilized by a styrene-maleic acid (SMA) copolymer. The polymer self-inserts into the membrane and stabilizes and isolates discoidal proteolipid particles, thus conserving the native lipid environment of KcsA. Analysis of the lipids isolated from native nanodiscs by thin layer chromatography and mass spectrometry revealed an enrichment of the anionic lipids cardiolipin and phosphatidylglycerol in close proximity to the channel. Using an SDS-PAGE assay as well as circular dichroism and fluorescence spectroscopy, we found the thermal stability of the KcsA tetramer to be higher in native nanodiscs as compared to detergent micelles. Together, these findings highlight the potential of the use of native nanodiscs as a general tool in the study of membrane proteins.Key words:lipid-protein interactions, styrene-maleic acid copolymer, nanodisc, membrane-protein solubilization, KcsA.