Formation of Lipid-Bilayer Nanodiscs by Diisobutylene/Maleic Acid (DIBMA) Copolymer.

Membrane proteins usually need to be extracted from their native environment and separated from other membrane components for in-depth in vitro characterization. The use of styrene/maleic acid (SMA) copolymers to solubilize membrane proteins and their surrounding lipids into bilayer nanodiscs is an attractive approach toward this goal. We have recently shown that a diisobutylene/maleic acid (DIBMA) copolymer similarly solubilizes model and cellular membranes but, unlike SMA(3:1), has a mild impact on lipid acyl-chain order and thermotropic phase behavior. Here, we used fluorescence spectroscopy, small-angle X-ray scattering, size-exclusion chromatography, dynamic light scattering, and 31P nuclear magnetic resonance spectroscopy to examine the self-association of DIBMA and its membrane-solubilization properties against lipids differing in acyl-chain length and saturation. Although DIBMA is less hydrophobic than commonly used SMA(3:1) and SMA(2:1) copolymers, it efficiently formed lipid-bilayer nanodiscs that decreased in size with increasing polymer/lipid ratio while maintaining the overall thickness of the membrane. DIBMA fractions of different molar masses were similarly efficient in solubilizing a saturated lipid. Coulomb screening at elevated ionic strength or reduced charge density on the polymer at low pH enhanced the solubilization efficiency of DIBMA. The free-energy penalty for transferring phospholipids from vesicular bilayers into nanodiscs became more unfavorable with increasing acyl-chain length and unsaturation. Altogether, these findings provide a rational framework for using DIBMA in membrane-protein research by shedding light on the effects of polymer and lipid properties as well as experimental conditions on membrane solubilization.

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

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.

Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer.

Once removed from their natural environment, membrane proteins depend on membrane-mimetic systems to retain their native structures and functions. To this end, lipid-bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane-protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl-chain order, does not interfere with optical spectroscopy in the far-UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations.