Enhancing the stability and homogeneity of non-ionic polymer nanodiscs by tuning electrostatic interactions

The nanodisc technology is increasingly used for structural studies on membrane proteins and drug delivery. The development of synthetic polymer nanodiscs and the recent discovery of non-ionic inulin-based polymers have significantly broadened the scope of nanodiscs. While the lipid exchange and size flexibility properties of the self-assembled polymer-based nanodiscs are valuable for various applications, the non-ionic polymer nanodiscs are remarkably unique in that they enable the reconstitution of any protein, protein-protein complexes, or drugs irrespective of their charge. However, the non-ionic nature of the belt could influence the stability and size homogeneity of inulin-based polymer nanodiscs. In this study, we investigate the size stability and homogeneity of nanodiscs formed by non-ionic lipid-solubilizing polymers using different biophysical methods. Polymer nanodiscs containing zwitterionic DMPC and different ratios of DMPC:DMPG lipids were made using anionic SMA-EA or non-ionic pentyl-inulin polymers. Non-ionic polymer nanodiscs made using zwitterionic DMPC lipids produced a very broad elution profile on SEC due to their instability in the column, thus affecting sample monodispersity which was confirmed by DLS experiments that showed multiple peaks. However, the inclusion of anionic DMPG lipids improved the stability as observed from SEC and DLS profiles, which was further confirmed by TEM images. Whereas, anionic SMA-EA-based DMPC-nanodiscs showed excellent stability and size homogeneity when solubilizing zwitterionic lipids. The stability of DMPC:DMPG non-ionic polymer nanodiscs is attributed to the inter-nanodisc repulsion by the anionic-DMPG that prevents the uncontrolled collision and fusion of nanodiscs. Thus, the reported results demonstrate the use of electrostatic interactions to tune the solubility, stability, and size homogeneity of non-ionic polymer nanodiscs which are important features for enabling functional and atomic-resolution structural studies of membrane proteins, other lipid-binding molecules, and water-soluble biomolecules including cytosolic proteins, nucleic acids and metabolites.