Purification and Characterization of the Tetrameric Potassium Channel Kcsa in “Native Nanodiscs”

Abstract

We explored a new discovery in membrane research of amphipathic copolymers of styrene and maleic acid (SMA) that have the unique potential to directly extract proteins from membranes in the form of “native nanodiscs” without the need for detergent [1,2]. E-coli cells overexpressing a His-tagged version of the potassium channel KcsA were incubated with SMA and the conditions were optimized for extraction of the protein. After solubilization of the membrane, we found that KcsA indeed could be purified on a Ni2+-NTA column in the form of nano-sized discs, as was confirmed by negative stain transmission electron microscopy (TEM) experiments. SDS-PAGE analysis showed that the protein in these nanodiscs is present as a tetramer, running at the same position as when subjected to SDS-PAGE from its native membrane or after purification in detergent. Presently we are comparing the stability of this tetramer in the nanodiscs with that of purified KcsA in n-dodecyl β-D-maltoside (DDM) micelles and KcsA in native membranes. This is done by comparing the effects of heat-incubation and exposure to small alcohols using gel shift assays. Furthermore, to gain information about possible preferential lipid interactions of KcsA, the lipid composition of the purified, KcsA containing nanodiscs is being analyzed and compared to that of the total lipid composition in E-coli. Results of these studies will be presented. 1) Knowles TJ, Finka R, Smith C, Lin Y-P, Dafforn T, Overduin M (2009) J Am Chem Soc 131, 7484-7485 2) Orwich-Rydmark M, Lovett JE, Lindholm L, Graziadei A, Hicks M, Watts A (2012) Nanoletters 12, 4687-4692