Characterization of Divalent Cation Interactions with AASTY Nanodiscs

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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.

OriginalsprogEngelsk
TidsskriftACS Applied Polymer Materials
Vol/bind4
Udgave nummer2
Sider (fra-til)1071-1083
DOI
StatusUdgivet - 2022

Bibliografisk note

Funding Information:
The authors thank the Novo Nordisk Foundation (NNF20OC0060692) and the Carlsberg Foundation (CF20-0533) for the support of the equipment and infrastructure. A.A.A.A. was funded by grants NNF18OC0030896 from the Novo Nordisk Foundation and the Stanford Bio-X Program and 0171-00081B from Independent Research Fund Denmark. H.E.A. was funded by grant R265-2017-4015 from the Lundbeck Foundation.

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