Reversible stacking of lipid nanodiscs for structural studies of clotting factors

Nanodiscs (ND) are discoidal phospholipid bilayers stabilized by a pair of membrane-scaffolding proteins (MSP). The macromolecular composition and size of ND are ideal for structural and functional studies of membrane and membrane-associated proteins. In this work, we investigate the assembly of ND from a galactosylceramide and dioleoyl phosphatidylserine (PS) lipid mixture with two different MSP and at four MSP-to-lipid ratios. This lipid composition has been optimized for structural and biophysical studies of membrane-bound blood clotting factors that require Ca²⁺ ions for function. We have demonstrated that CaCl₂ induces reversible stacking of the ND that depends on the ND size and Ca²⁺ concentrations. Our biophysical and electron microscopy (EM) studies show a predominant ND population of ∼12 nm in diameter for both the ND assembled from MSP1D1 to lipids ratio of 1:40 and from MSP1E3D1-to-lipids ratio of 1:80. Approximately half of the ND population assembled at MSP1E3D1-to-lipids ratio of 1:150 has a diameter of ∼16 nm. These larger ND form ordered stacks at 5-mm Ca²⁺ concentrations, as shown by cryo-EM. The number and length of the ND stacks increase with the increasing in Ca²⁺ concentration. Adding millimolar concentrations of EDTA reverses the stacking of the ND.