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.
Highlights
We present the first account of the equilibrium solubilisation properties of Styrene/maleic acid copolymers (SMA)(2:1) against large unilamellar vesicles (LUVs) composed of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as monitored by 31P nuclear magnetic resonance (NMR) spectroscopy, dynamic light scattering (DLS), and differential scanning calorimetry (DSC)
We have recently shown that refractometry is a useful tool for determining the concentrations of SMA(3:1)[16, 17] and DIBMA18 in aqueous solutions
While various types of SMA have been used over the past few years, there is a clear trend in the field to focus on SMA(2:1)[14]
Summary
R b,SAT S and RSm,SOL denote the polymer/lipid molar ratios in vesicular bilayers and nanodiscs at which the vesicles become saturated with polymer and at which solubilisation is complete, respectively Both lines meet at a common ordinate intercept, csaq,o, which corresponds to the concentration of “free” polymer in the aqueous phase within the coexistence range. Pairs of cSSAT and cSSOL values at a given lipid concentration were obtained by analysing the areas derived from the corresponding 31P NMR signals in terms of equations 9–1116–18. In addition to such local fits considering only one lipid concentration at a time, peak areas measured at four different lipid concentrations were globally fitted with equations 9–11 in order to obtain the best-fit RSb,SAT and RSm,SOL values. In addition to such local fits considering only one lipid concentration at a time, peak areas measured at four different lipid concentrations were globally fitted with equations 9–11 in order to obtain the best-fit RSb,SAT and RSm,SOL values. 95% confidence intervals were derived by nonlinear least-squares fitting in Excel spreadsheets, as detailed elsewhere[22]
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