A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs.

Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among the few commercial polymers that are capable of forming nanodiscs, alternating diisobutylene/maleic acid (DIBMA) copolymers have gained considerable popularity as gentle and UV-transparent alternatives to aromatic polymers. However, their moderate hydrophobicities and high electric charge densities render all existing aliphatic copolymers rather inefficient under near-physiological conditions. Here, we introduce Glyco-DIBMA, a bioinspired glycopolymer that possesses increased hydrophobicity and reduced charge density but nevertheless retains excellent solubility in aqueous solutions. Glyco-DIBMA outperforms established aliphatic copolymers in that it solubilizes lipid vesicles of various compositions much more efficiently, thereby furnishing smaller, more narrowly distributed nanodiscs that preserve a bilayer architecture and exhibit rapid lipid exchange. We demonstrate the superior performance of Glyco-DIBMA in preparative and analytical applications by extracting a broad range of integral membrane proteins from cellular membranes and further by purifying a membrane-embedded voltage-gated K+ channel, which was fluorescently labeled and analyzed with the aid of microfluidic diffusional sizing (MDS) directly within native-like lipid-bilayer nanodiscs.

Lipid exchange among polymer-encapsulated nanodiscs by time-resolved Förster resonance energy transfer.

Some amphiphilic copolymers such as diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) copolymers are able to directly extract cellular membranes into nanosized polymer-bounded lipid-bilayer patches. These polymer-encapsulated nanodiscs offer the possibility to investigate delicate membrane proteins along with their surrounding lipids and, thus, protein/lipid interactions, in a near-native bilayer environment. By dissecting the kinetics of lipid exchange among DIBMA- and SMA-bounded nanodiscs, we have recently shown that the encapsulated lipid bilayer does not represent a static snapshot of the membrane at the time point of solubilisation. Instead, nanoscale lipid-bilayer patches remain in equilibrium with one another through lipid exchange enabled by nanodisc collisions. This finding is important for correctly interpreting any attempts at studying protein/lipid interactions with the aid of polymer-based nanodiscs and will be relevant to characterising the rapidly growing repertoire of new amphiphilic copolymers for membrane extraction. A highly sensitive and efficient technique for measuring the kinetics of lipid transfer among various kinds of nanosized membrane mimics consists in time-resolved Förster resonance energy transfer (TR-FRET) monitored in a stopped-flow apparatus. Here, we provide detailed instructions on how to measure the kinetics and unravel the underlying mechanisms of lipid exchange among lipid-bilayer nanodiscs under various solution conditions.

Influence of Mg2+ and Ca2+ on nanodisc formation by diisobutylene/maleic acid (DIBMA) copolymer.

Most membrane-solubilising amphiphilic copolymers such as diisobutylene/maleic acid (DIBMA) and styrene/maleic acid (SMA) carry high negative charge densities. Their polyanionic character results in strong Coulombic repulsion, both between polymer chains and lipid membranes during the solubilisation process as well as among polymer-encapsulated nanodiscs after solubilisation. Coulombic repulsion is attenuated by charge screening and, more efficiently, by counterion association, which is particularly strong for multivalent cations binding to polyanionic copolymers. Here, we investigated the effects of the two common alkaline earth metal ions Mg2+ and Ca2+ on the solubilisation properties of and the nanodiscs formed by DIBMA. By quantifying the kinetics and the equilibrium efficiency of lipid solubilisation by static and dynamic light scattering, respectively, we found that millimolar concentrations of Mg2+ or Ca2+ accelerated DIBMA-mediated lipid solubilisation several-fold and resulted in considerably smaller nanodiscs than without divalent cations. Time-resolved Förster resonance energy transfer spectroscopy revealed that collisional transfer of phospholipids among nanodiscs was up to ∼20 and ∼25 times faster in the presence of 10 mM Mg2+ or 7.5 mM Ca2+ than in the absence of divalent cations. These major effects of Mg2+ and Ca2+ contrasted with a moderate influence on the morphology and the thermotropic phase behaviour of the nanodiscs. Finally, we compared the yields of membrane-protein extraction from Escherichia coli membranes, which increased by up to two-fold upon addition of Mg2+ or Ca2+. None of these effects could be explained by Coulombic screening alone, as the change in ionic strength resulting from low millimolar concentrations of divalent cations was minor. Thus, we conclude that Mg2+ and Ca2+ specifically associated with DIBMA to neutralise part of the polymer's carboxylate groups.

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.

Fast Collisional Lipid Transfer Among Polymer-Bounded Nanodiscs.

Some styrene/maleic acid (SMA) copolymers solubilise membrane lipids and proteins to form polymer-bounded nanodiscs termed SMA/lipid particles (SMALPs). Although SMALPs preserve a lipid-bilayer core, they appear to be more dynamic than other membrane mimics. We used time-resolved Förster resonance energy transfer and small-angle neutron scattering to determine the kinetics and the mechanisms of phospholipid transfer among SMALPs. In contrast with vesicles or protein-bounded nanodiscs, SMALPs exchange lipids not only by monomer diffusion but also by fast collisional transfer. Under typical experimental conditions, lipid exchange occurs within seconds in the case of SMALPs but takes minutes to days in the other bilayer particles. The diffusional and second-order collisional exchange rate constants for SMALPs at 30 °C are kdif = 0.287 s-1 and kcol = 222 M-1s-1, respectively. Together with the fast kinetics, the observed invariability of the rate constants with probe hydrophobicity and the moderate activation enthalpy of ~70 kJ mol-1 imply that lipids exchange through a "hydrocarbon continuum" enabled by the flexible nature of the SMA belt surrounding the lipid-bilayer core. Owing to their fast lipid-exchange kinetics, SMALPs represent highly dynamic equilibrium rather than kinetically trapped membrane mimics, which has important implications for studying protein/lipid interactions in polymer-bounded nanodiscs.

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.

Modulating bilayer mechanical properties to promote the coupled folding and insertion of an integral membrane protein.

Bilayer mechanical properties are not only of crucial importance to the mechanism of action of mechanosensation in lipid membranes but also affect preparative laboratory tasks such as membrane-protein refolding. We report this for coupled refolding and bilayer insertion of outer membrane phospholipase A (OmpLA), an integral membrane enzyme that catalyses the hydrolytic cleavage of glycerophospholipids. OmpLA can be refolded into a variety of detergent micelles and unilamellar vesicles composed of short-chain phospholipids but, in the absence of chemical or molecular chaperones, not into thicker membranes. Controlled modulation of bilayer mechanical properties by judicious use of subsolubilising concentrations of detergents induces monolayer curvature strain, acyl chain fluidisation, membrane thinning, and transient aqueous bilayer defects. This enables quantitative and functional refolding of OmpLA even into bilayer membranes composed of long-chain phospholipids to yield enzymatically active proteoliposomes without requiring membrane solubilisation.

A fluorinated detergent for membrane-protein applications.

Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon-based detergents for the solubilization and handling of integral membrane proteins. However, their inertness towards lipid bilayer membranes has limited the usefulness of fluorinated surfactants in situations where detergent-like activity is required. We demonstrate that fluorination does not necessarily preclude detergency, as exemplified by a fluorinated octyl maltoside derivative termed F6 OM. This nonionic compound readily interacts with and completely solubilizes phospholipid vesicles in a manner reminiscent of conventional detergents without, however, compromising membrane order at subsolubilizing concentrations. Owing to this mild and unusual mode of detergency, F6 OM outperforms a lipophobic fluorinated surfactant in chaperoning the functional refolding of an integral membrane enzyme by promoting bilayer insertion in the absence of micelles.