Detergent-free isolation, characterization, and functional reconstitution of a tetrameric K+ channel: the power of native nanodiscs.

ABSTRACT: A major obstacle in the study of membrane proteins is their solubilization in a stable and active conformation when using detergents. Here, we explored a detergent-free approach to isolating the tetrameric potassium channel KcsA directly from the membrane of Escherichia coli, using a styrene-maleic acid copolymer. This polymer self-inserts into membranes and is capable of extracting membrane patches in the form of nanosize discoidal proteolipid particles or "native nanodiscs." Using circular dichroism and tryptophan fluorescence spectroscopy, we show that the conformation of KcsA in native nanodiscs is very similar to that in detergent micelles, but that the thermal stability of the protein is higher in the nanodiscs. Furthermore, as a promising new application, we show that quantitative a

SUBMITTER: Dorr JM 

PROVIDER: S-EPMC4284610 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature