Understanding the Structural Pathways for Lipid Nanodisc Formation: How Styrene Maleic Acid Copolymers Induce Membrane Fracture and Disc Formation

[Image: see text] Lipid nanodiscs formed by mixtures of styrene maleic acid (SMA) copolymers and lipid membranes are important tools for studying membrane proteins in many biotechnological applications. However, molecular interactions leading up to their formation are not well understood. Here, we elucidate the nanodisc formation pathways for SMA/lipid vesicle mixtures using small-angle X-ray scattering (SAXS) that allows detailed in situ nanostructural information. SMA copolymer that is initially aggregated in solution inserts its styrene units into the lipid bilayer hydrocarbon region, leading to fractures in the membrane. The initial copolymer–lipid interactions observed in the vesicles are also present in the formed discs, with excess copolymer distributed along the normal of the bilayer. The size and SMA distribution in the resulting discs strongly depend on the temperature, lipid/copolymer ratio, and lipid type. We find that the solubilization limit increases for membranes above the melting point, suggesting that defects in gel-like lipid membranes play a significant role in membrane fracturing and nanodisc formation. These findings provide unique insights into the formation of nanodiscs as well as into the microscopic mechanism of solubilization, which plays an important role in many applications and products ranging from household goods to biotechnology and medicine.