Solubilization of lipids and membrane proteins into nanodiscs : Mode of action and applications of SMA copolymers

Abstract

Cell membranes separate the inside and outside of cells. Membrane proteins in the cell membrane control the traffic of molecules across the membrane and are therefore targets for a lot of drugs: about 50 % of all approved drugs target a membrane protein! Unfortunately, scientists only know little about membrane proteins as compared to, for example, water soluble proteins. That is because membrane proteins are hard to study since they reside in the hydrophobic patch of the membrane. To study membrane proteins, they need to be isolated and detergents (soap) are required to do so. The problem is: soap molecules wash away and replace the native membrane environment. In that way, many membrane proteins are destabilized by detergents. In this thesis, a novel method is described that overcomes the issues that detergents bring to the study of lipid membranes and membrane proteins. And help comes unexpectedly. It is the synthetic industrial polymer called styrene-maleic acid, which is often used in the car industry, that is able to isolate membrane protein in a very soft manner. Upon the addition of SMA to membranes, the membranes solubilize in nanodisc particles in which a membrane protein is captured along with a small piece of its native lipid environment. This thesis uses a photosynthetic protein named ‘reaction center’ (RC) form the purple bacterium Rhodobacter sphaeroides as model to validate SMA polymers as alternative to soap molecules. The results show that the RC in SMA nanodiscs maintains the native lipid environment, which is in contrast to soaps such as LDAO and DDM. Furthermore, RCs in a nanodisc are more heat and light stable than RCs in soap and in the native membrane. That opens options to use RCs in SMA nanodiscs in bio-solar cells for which protein stability is key. More results give insight in the molecular action of membrane solubilization by SMA polymers. For example, SMA is a very efficient solubilizing agent, much more than for example membrane scaffold proteins (MSPS, which is the conventional way to produce nanodiscs. Also, the SMA variant with a 2:1 styrene-to-maleic acid ratio is the best SMA variant to use. That is because this polymer has the optimal balance in hydrophobic and hydrophilic groups to attack and solubilize lipid membranes, while stabilizing nanodiscs in water. SMA was also found to solubilize the exact membrane lipid composition in nanodiscs, i.e. SMA has no preference to solubilize certain lipid species. This is a very important result, because thanks to this, SMA is the first way to study lipid-lipid and lipid-protein interactions in a direct and biochemical way. The SMA technique is still young (since 2009 reported in the scientific literature) and many concepts need still to be explored. But due to the potential of SMA to solubilize and stabilize membrane proteins, it is possible that purification of membrane proteins using SMA could become the standard tool for biophysical studies of membrane proteins, and that the polymer will greatly facilitate the wider use of SMA nanodiscs in biohybrid devices