Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

Abstract

0.04% sodium cholate. For the measurements, MsbA-loaded nanodiscs were enriched based on the affinity of the His-tagged MsbA for Ni 2+. The supernatant from the solubilization of proteoliposomes was mixed with Ni-NTA beads (Thermo Fisher Scientific) at a ratio of 100 µL resin/mL solubilized protein, and after incubation at 4 °C overnight with gentle rotation the samples were transferred to a gravity flow column. The resin was washed with 10 column volumes of 100 mM NaCl and 20 mM Tris/HCl, with 0.1 mM TCEP and 20 mM imidazole, pH 7.4, and elution was achieved by increasing imidazole to 200 mM. Eluted fractions were analyzed on gels (16% SDS-PAGE) stained with Instant Blue (Expedeon) and used for the ATPase measurements. Estimation of nanodiscs size by dynamic light scattering (DLS). Measurements were performed at 22 °C on a Zetasizer Nano ZSP (Malvern Instruments, Westborough, MA) using 40-µL disposable microcuvettes. Size-number distributions were generated using the Zetasizer software version 7.11 and were analyzed using the protein analysis distribution. Statistics. Statistical comparisons were performed by the Student's t test for paired or unpaired data, or one-way analysis of variance, as appropriate. P < 0.05 in a two-tailed analysis was considered significant. The number of experiments given in the main text and figure legends corresponds to independent measurements.

Polymer-Encased Nanodiscs and Polymer Nanodiscs: New Platforms for Membrane Protein Research and Applications

“…The maleic acid groups of SMA are easily accessible and amenable to modification, enabling the addition of different chemical groups to create new polymers. One of the promising SMA-like copolymers that we developed to address the buffer incompatibilities of SMAs and the size limitations of SMALPs are the zwitterionic styrene maleic amides (zSMAs) (Fiori et al, 2017a(Fiori et al, , 2020. In these copolymers, the anionic carboxyl acids are replaced with zwitterionic phosphatidylcholine groups.…”

“…Second, the size of SMALPs may be too small to contain “large” MPs. The average diameter of SMALPs is 6–9 nm, which can restrict the proteins that can be studied ( Fiori et al, 2017a , 2020 ; Ravula et al, 2017b , 2019 ). Third, SMA polymers can bind divalent cations, such as Mg 2+ and Ca 2+ , reducing the charge of the maleic acid units, which results in precipitation of the copolymers out of solution ( Fiori et al, 2017a , 2020 ; Ravula et al, 2017a ).…”

“…The average diameter of SMALPs is 6–9 nm, which can restrict the proteins that can be studied ( Fiori et al, 2017a , 2020 ; Ravula et al, 2017b , 2019 ). Third, SMA polymers can bind divalent cations, such as Mg 2+ and Ca 2+ , reducing the charge of the maleic acid units, which results in precipitation of the copolymers out of solution ( Fiori et al, 2017a , 2020 ; Ravula et al, 2017a ). The sensitivity to divalent cations can be restrictive to downstream analyses that require their presence, such as measurements of ATPase activities ( Fiori et al, 2017a ).…”

“…The latter is important when studies are aimed to reproduce native-like conditions as closely as possible. In addition, purified MPs reconstituted in liposomes can be re-solubilized to form traditional or novel SMALPs where the lipid composition of the bilayer can be controlled through the composition of the liposomes ( Figure 1A ; Ravula et al, 2017b ; Fiori et al, 2020 ).…”

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

“…Immobilization of biomacromolecular motion by chaperone-like confinement in polymers has emerged as a way to stabilize soluble proteins, but the accessibility of addressable functional groups of transmembrane proteins is reduced from confinement in a lipid matrix and the poor stability of membrane-bound proteins further complicates bioconjugation. 7 Recently, polymeric excipients designed to match the natural distribution of polar residues on proteins 7 or stabilization in amphipathic copolymer-based membrane nanodiscs 8 have advanced the state-of-the-art; however, these coatings are neither removable nor scalable to protect a functional catalytic proteoliposome system. Specifically, a "sheddable" coating that avoids excipients or chemical functionalization of either protein or lipid would help advance work on these systems tremendously.…”