Detergent-Free Extraction of the Reaction Center from Rhodobacter sphaeroides into Native Nanodiscs. Nanodisc Size Matters!

Abstract

It has been discovered that styrene-maleic acid (SMA) copolymers are able to solubilize membrane proteins directly from their native membrane in the form of nanodiscs [1]. Using the SMA technology, we purified and characterized reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides [2]. Our most significant findings were (i) that the SMA copolymer efficiently solubilizes membranes of this bacterium, (ii) that it allows preservation of the local lipid environment of the solubilized RCs, (iii) that the protein is even more stable in these “native nanodiscs” than in the native membrane, and (iv) that the size of the RC containing nanodiscs is significantly larger than that of protein-free nanodiscs.We next investigated what physical properties of the polymer and lipids determine the efficiency and kinetics of solubilization and how they affect the size of the nanodiscs. In particular, we tested the effect of SMA copolymer length, hydrophobicity, and charge state (varying pH) using different lipid compositions. In complementary assays, we determined how the cross-sectional diameter of a protein affects the size of a nanodisc by using purified constructs of covalently linked oligomeric RCs of different size.Our study contributes to the fundamental knowledge about the mode of action of SMA and thereby to the general applicability of native nanodiscs as host for membrane proteins and protein complexes of different size.1. Knowles et al., 2009, JACS, 131, 7484-74852. Swainsbury & Scheidelaar et al. 2014, Angewandte Chemie, 53, http://dx.doi.org/10.1002/anie.201406412