Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here we describe a technique to obtain purified, homogeneous membrane protein dimers in nanodiscs using a split GFP tether. Complementary split GFP tags a... More
Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here we describe a technique to obtain purified, homogeneous membrane protein dimers in nanodiscs using a split GFP tether. Complementary split GFP tags associate to tether the co-expressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative western blotting and negative stain electron microscopy. The split GFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody-conjugated resin. We used this method to purify a Frizzled-4 (Fzd4) homodimer and a Fzd4/low-density lipoprotein receptor-related protein 6 (LRP6) heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs.
