The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes

Abstract

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (< 30 kDa weight average molecular weight). The effectiveness of 10 kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane.

Membrane Solubilization by Styrene-Maleic Acid Copolymers: Delineating the Role of Polymer Length

“…A broad range of SMA polymers differing in composition and in length are commercially available. It has been shown that the composition of the SMA polymers strongly affects the membrane solubilization properties in model membranes (11) as well as in biological membranes (12,13) and that it also affects the properties of the resulting nanodisks (9). Recently, also the effect of polymer length in membrane solubilization has been investigated.…”

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

“…Eight SMA types have been surveyed for their abilities to solubilize monomeric, dimeric, trimeric and tetrameric forms of the Rhodobacter sphaeroides reaction centre [16]. The most effective are those with lower molecular weights in the 10 kDa range and having 2:1 and 3:1 ratios of styrene to maleic acid.…”

Methods of reconstitution to investigate membrane protein function

“…This will convert the anhydride units to their acid form containing two deprotonated carboxyl groups to increase the water solubility. The amount of base to be added can be controlled in this step in order to make the pH of the precipitated polymer close to 8.0 (59) iii) Once the solution cools down to room temperature, the pH is lowered to <5 with the addition of concentrated HCl. This causes the polymer to precipitate iv) The polymer is washed with diluted HCl and then resuspended in 0.6M NaOH/KOH and the pH adjusted to 8.0…”

“…This can be a problem for proteins that are stable or active in acidic environments such as lysosomal PfCRT (pH 6.0) which is responsible for resistance against antimalarial drugs (67). (59). However, some studies have suggested that the size of the disc is flexible (70) and can be exploited for large proteins by targeting the protein to lipid ratio (45,74) and the ratio of the polymer (75).…”