Thermodynamics of nanodisc formation mediated by styrene/maleic acid (2:1) copolymer

Abstract

Styrene/maleic acid copolymers (SMA) have recently attracted great interest for in vitro studies of membrane proteins, as they self-insert into and fragment biological membranes to form polymer-bounded nanodiscs that provide a native-like lipid-bilayer environment. SMA copolymers are available in different styrene/maleic acid ratios and chain lengths and, thus, possess different charge densities, hydrophobicities, and solubilisation properties. Here, we studied the equilibrium solubilisation properties of the most commonly used copolymer, SMA(2:1), by monitoring the formation of nanodiscs from phospholipid vesicles using 31P nuclear magnetic resonance spectroscopy, dynamic light scattering, and differential scanning calorimetry. Comparison of SMA(2:1) phase diagrams with those of SMA(3:1) and diisobutylene/maleic acid (DIBMA) revealed that, on a mass concentration scale, SMA(2:1) is the most efficient membrane solubiliser, despite its relatively mild effects on the thermotropic phase behaviour of solubilised lipids. In contrast with previous kinetic studies, our equilibrium experiments demonstrate that the solubilisation of phospholipid bilayers by SMA(2:1) is most efficient at moderately alkaline pH values. This pH dependence was also observed for the solubilisation of native Escherichia coli membranes, for which SMA(2:1) again turned out to be the most powerful solubiliser in terms of the total amounts of membrane proteins extracted.

Examining the stability of membrane proteins within SMALPs

“…In addition we found that SZ30010 also affords the improved purity observed with SMA2000. Despite having small differences in average size and ratio of styrene:maleic acid, the particles produced using SMA2000 and SZ30010 were very similar in size at just under 10 nm diameter, which agrees well with previous reports using these two polymers [3,17,24,25].…”

“…Previous studies on the thermodynamics of phospholipid solubilisation by SMA2000 [27] and SZ30010 [24] indicate that these polymers have similar thermodynamic efficiencies. SMA2000 exhibits a larger negative free energy change associated with the polymer, indicating it is more efficient at solubilising phospholipids.…”

Putative interaction site for membrane phospholipids controls activation of TRPA1 channel at physiological membrane potentials

“…To ensure complete solubilization of the MLVs, a ratio of 0.5 ([SMA]/[lipid]) was used that is above the reported Rsol value (ratio above which the liposome solubilization is complete) for POPC liposomes [44]. To ensure complete solubilization of the MLVs, a ratio of 0.5 ([SMA]/[lipid]) was used that is above the reported Rsol value (ratio above which the liposome solubilization is complete) for POPC liposomes [44].…”

“…Styrene maleic acid lipid particles were prepared by incubating MLVs (1.3 mM) with SMA solution (6.5 mM) both in PBS. To ensure complete solubilization of the MLVs, a ratio of 0.5 ([SMA]/[lipid]) was used that is above the reported Rsol value (ratio above which the liposome solubilization is complete) for POPC liposomes [44]. Formation of SMALPs was followed by Dynamic Light Scattering (Vasco, Cordouan) and also by MDS (microfluidic diffusional sizing; in this case, we have included 10% palmitoyl-oleoyl-phosphatidylserine in the lipid system in order to have a reactive primary amine for fluorescent labeling).…”

Methods of reconstitution to investigate membrane protein function

“…The most hydrophilic SMA is 1.4:1 and the most hydrophobic is 4:1. The potential of these polymers to solubilise different membrane proteins has been assessed with the most promising SMA variant bearing a styrene to maleic acid ratio of 2:1 (50). SMA with a 3:1 ratio has comparable efficiency to insert into membranes and both co-polymers display average M w in the range of 7.5-10kDa.…”