No abstract

Styrene-maleic acid copolymers allow for solubilization and reconstitution of membrane proteins into nanodiscs. These polymer-encased nanodiscs are promising platforms for studies of membrane proteins in a near-physiologic environment without the use of detergents. However, current styrene-maleic acid copolymers display severe limitations in terms of buffer compatibility and ensued flexibility for various applications. Here, we present a new family of styrene-maleic acid copolymers that do not aggregate at low pH or in the presence of polyvalent cations, and can be used to solubilize membrane proteins and produce nanodiscs of controlled sizes.

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.