Characterization of Divalent Cation Interactions with AASTY Nanodiscs

Abstract

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.

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight

“…[21][22][23][24][25][26][27][28] Apart from SMA and SMA-like copolymers, several other types of amphiphilic copolymers for membrane protein isolation were developed. [29] These included diisobutylene/maleic acid copolymers (DIBMA) and their derivatives, [16,[30][31][32][33] alkylamine-modified poly(acrylic acid) (APAA), [34] methacroylcholine chloride/butyl methacrylate copolymers (PMA), [35] acrylic acid/styrene copolymers (AASTY), [14,36] modified inulin, [37,38] methylstilbene/maleic acid copolymers (STMA), [39] and cycloalkylamine-modified poly(acrylic acid) (CyclAPol). [40] In addition, in a very recent comprehensive study, Kopf et al presented a number of SMA derivatives with diverse substitution mainly at the aromatic ring, as well as acrylic acid copolymers with substituted styrenes.…”

Cancer Immunotherapy through Tissue Adhering Polymers

“…Two AASTY polymers were tested to serve as the backbone for functional conjugations: AASTY 12.5 (12.5 kDa), developed and tested by Smith et al 26 and Timcenko et al, 27 and AASTY 9.9 -N 3 (9.9 kDa, with an Azide moiety), developed specifically for this experiment.…”

“…The synthesis of the AASTY copolymers was performed as in Smith et al 26 and Timcenko et al. 27 In short, the copolymers were synthesised using either the RAFT Agent 2-cyano-2-propyl dodecyl trithiocarbonate (1.10 g, 3.17 mmol; giving the copolymer AASTY 12.5 ) or 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester (0.052 g, 0.12 mmol; giving the copolymer AASTY 9.9 -N 3 ) in Schlenk flasks. The initiator azobisisobu-tyrunitrile (AIBN) and the monomers acrylic acid (AA) and styrene (STY) were added to the flasks (for AASTY 12.5 : AIBN: 104 mg, 0.634 mmol, AA: 13.0 g, 190 mmol, STY: 26.6 g, 232 mmol; for AASTY 9.9 -N 3 : AIBN: 3.8 mg, 0.023 mmol, AA: 0.324 g, 4.50 mmol, STY: 0.573 g, 5.50 mmol).…”

Determination of oligomeric organization of membrane proteins from native membranes at nanoscale-spatial and single-molecule resolution

“…Our approach uses commercially available amphipathic copolymers (SMA or AASTY) to isolate native nanodiscs (or circular patches) of unperturbed cell-membranes in a detergent-free extraction strategy (Fig. 1a-b) [27][28][29][30]39 . Such excision of isolated cell membranes to generate native nanodiscs enables isolation of membrane proteins in annular rings of neighboring native-lipids and proteins held together by the copolymers, preserving their corresponding proximal membrane environments.…”

“…Our approach isolates these native nanodiscs by detergent-free solubilization of cell membranes using amphipathic copolymers (such as styrene maleic acid (SMA) or styrene acrylic acid (AASTY), Fig. 1a) [27][28][29][30] . The solubilized GFP-tagged membrane proteins are surrounded by an annular ring of native lipids and neighboring proteins held together by these amphipathic copolymers.…”