Characterization of Divalent Cation Interactions with AASTY Nanodiscs

Amphiphilic copolymers show promise in extracting membrane proteins directly from lipid bilayers into native nanodiscs. However, many such copolymers are polyanionic and sensitive to divalent cations, limiting their applicability. We characterize the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers with analytical UV and fluorescent size exclusion chromatography, enabling us to separate signals from nanodiscs, copolymers, and soluble aggregates. We find that divalent cations promote aggregation and precipitation of both free and lipid bound copolymers. We see that excess, free copolymer acts as a cation sink that protects nanodiscs from Ca2+ induced aggregation. Removal of the free copolymer through dialysis induces aggregation that can be mitigated by KCl. Finally, we find that the nanodisc size is dynamic and dependent on lipid concentration. Our results offer insight into nanodisc behavior and can help guide experimental design aimed at mitigating the shortcomings inherent in negatively charged nanodisc forming copolymers.

Automated summary

Amphiphilic copolymers have the potential to extract membrane proteins directly from lipid bilayers into native nanodiscs. However, many such copolymers are polyanionic and sensitive to divalent cations. In this study, we characterized the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers and found that divalent cations promote aggregation and precipitation of both free and lipid-bound copolymers. Excess, free copolymer acts as a cation sink that protects nanodiscs from Ca2+-induced aggregation. Removal of the free copolymer induces aggregation, which can be mitigated by KCl. Our findings also revealed that the size of the nanodiscs is dynamic and dependent on lipid concentration.