Lipid Curvature and Fluidity Influence Lipid Incorporation Disparities in Nanodiscs

Nanodiscs have become a popular membrane mimetic system offering a well-defined bilayer environment to stabilize membrane proteins for in vitro analyses using a range of analytical methods; however, lipid compositions common to their deployment are simplistic and often fail to model native membrane complexity. Furthermore, there has been a general lack of rigorous analytical and biophysical characterization of nanodiscs comprising more than one lipid. To address these challenges, we coupled a nanodisc formation and purification workflow with targeted LC-MS/MS analysis to quantify lipids in nanodiscs made with different compositions. We screened lipids with a variety of headgroups and acyl chains and found that lipids did not always incorporate into nanodiscs at expected levels. Disparities in lipid incorporation were found to increase upon the addition of lipids known to induce curvature or rigidity to the membrane. Additionally, we found that adding just one additional type of lipid to nanodiscs changes the particle diameter and dispersity compared to nanodiscs containing a single lipid. We also formed and characterized nanodiscs using a complex starting composition inspired by the endoplasmic reticulum membrane and observed native-like cholesterol dynamics that modulated the lipid fluidity in the model bilayer system. Taken together, this work serves as a foundation for understanding nonstoichiometric lipid incorporation into nanodiscs and provides a basis for more thorough nanodisc characterization and quality control, which is critical to ensure multilipid nanodiscs synthesized accurately model the biological system of interest, enabling robust characterization of how the lipid landscape affects membrane protein structure and activity.