Detergent-Free Extraction of Membrane Proteins into Native Nanodiscs. Application to the Reaction center of Rhodobacter Sphaeroides

Abstract

Recently, it has been discovered that styrene-maleic acid (SMA) copolymers are able to solubilize membrane proteins in the form of nanodiscs (SMALPs), also called ‘native nanodiscs’, directly from their native membrane [1,2]. To understand what physical properties of membranes and SMA modulate this unique property of SMA, we first studied the solubilization of vesicles of synthetic phospholipids by SMA using transmission experiments. We found that SMA is an excellent membrane solubilizer, able to solubilize different types of membranes below, at and above the gel to crystalline-liquid phase temperature. Based on the kinetics of solubilization under different experimental conditions we developed a model for the mode of action of SMA that will also explain why SMA is an efficient solubilizer, whereas membrane scaffold proteins (MSPs) and amphipols are not.Next we used the SMA technology to purify and characterize reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides in the form of native nanodiscs. Monitoring of heat stability and recombination kinetics of P+QB- in photo-excited RCs in native nanodiscs showed that (1) RCs are much more stable in native nanodiscs than in detergent, and (2) the charge recombination kinetics display native-membrane like behavior.Our study contributes fundamental knowledge about the mode of action of SMA that is essential for optimizing methods to extract membrane proteins from different organisms and it promotes the general applicability of SMALPs as host for membrane proteins in studies on interactions of proteins with native lipids and in protein structure determination studies.1. Knowles et al., 2009, JACS, 131, 7484-7485.2. Long et al., 2013, BMC Biotechnology, 13:41.