Benchmarks of SMA-Copolymer Derivatives and Nanodisc Integrity

Abstract

Poly­(styrene-co-maleic acid) or SMA and its derivatives, a family of synthetic amphipathic copolymers, are increasingly used to directly solubilize cell membranes to functionally reconstitute membrane proteins in native-like copolymer–lipid nanodiscs. Although these copolymers act, de facto, like a “macromolecular detergent”, the polymer-based lipid-nanodiscs has been demonstrated to be an excellent membrane mimetic for structural and functional studies of membrane proteins and their complexes by a variety of biophysical and biochemical approaches. In many studies reported in the literature, the choice of the right SMA formulation can depend on a number of factors, and the experimental conditions are typically developed according to a trial-and-error process since each studied system requires adapted protocols. While increasing number of nanodisc-forming copolymers are reported to be useful and they provide flexibilities in optimizing the sample preparation conditions, it is important to develop a systematic protocol that can be used for various applications. In this context, there is a vital necessity of benchmarking the performances of existing copolymer formulations, assessing crucial parameters for the successful extraction, isolation, and stabilization of membrane proteins. In this study, we compare both copolymers and copolymer–lipid nanodiscs obtained by SMA-EA with a set of anionic XIRAN copolymer formulations commercially available under the names of SL25010 P, SL30010 P, and SL40005 P. The reported results show how the critical micellar concentration (c.m.c.) of each copolymer is significantly altered in the presence of lipids and confirms the existence of an equilibrium between nanodisc-bound and “free” or “micellar” copolymer chains in the solution. We believe that these findings can be exploited to optimize studies that involve the necessity of special copolymers, which would not only simplify the applications but also broaden the scope of polymer-based nanodiscs.

Characterization of Divalent Cation Interactions with AASTY Nanodiscs

“…This fits with the altered lipid/polymer ratio of these samples, which has also been observed for SMA . Our findings corroborate the study by Di Mauro et al, who showed that removing excess polymer resulted in an increased critical micelle concentration for DOPC copolymer mixtures …”

Solid-State NMR Study to Probe the Effects of Divalent Metal Ions (Ca2+ and Mg2+) on the Magnetic Alignment of Polymer-Based Lipid Nanodiscs

“…The SEC profile shown in Figure shows the elution of nanodiscs at 20 mL, which are estimated to have a size of ∼25 ± 4 nm diameter as reported in our previous publication, and the free polymers elute around 40 mL. We have previously reported the effect of critical micellar concentration (CMC) on the equilibrium between free- and nanodisc-bound polymers . After concentrating the nanodisc-containing solution to 200 μL, the solution was kept at 4 °C.…”

“…We have previously reported the effect of critical micellar concentration (CMC) on the equilibrium between free-and nanodisc-bound polymers. 66 After concentrating the nanodisc-containing solution to 200 μL, the solution was kept at 4 °C.…”

Characterization of divalent cation interactions with AASTY native nanodiscs

“…36 Our findings corroborate the study by Mauro et al who showed that removing excess polymer resulted in an increased critical micelle concentration for DOPC copolymer mixtures. 37 A comparison of the nanodisc sample prior to dialysis (Supp. Figure S8A and S9A) with the dialyzed sample (Supp.…”