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. 2016 Nov 1;111(9):1974-1986.
doi: 10.1016/j.bpj.2016.09.025.

Effect of Polymer Composition and pH on Membrane Solubilization by Styrene-Maleic Acid Copolymers

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Effect of Polymer Composition and pH on Membrane Solubilization by Styrene-Maleic Acid Copolymers

Stefan Scheidelaar et al. Biophys J. .
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Abstract

The styrene-maleic acid (SMA) copolymer is rapidly gaining attention as a tool in membrane research, due to its ability to directly solubilize lipid membranes into nanodisk particles without the requirement of conventional detergents. Although many variants of SMA are commercially available, so far only SMA variants with a 2:1 and 3:1 styrene-to-maleic acid ratio have been used in lipid membrane studies. It is not known how SMA composition affects the solubilization behavior of SMA. Here, we systematically investigated the effect of varying the styrene/maleic acid on the properties of SMA in solution and on its interaction with membranes. Also the effect of pH was studied, because the proton concentration in the solution will affect the charge density and thereby may modulate the properties of the polymers. Using model membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipids at pH > pHagg, we found that membrane solubilization is promoted by a low charge density and by a relatively high fraction of maleic acid units in the polymer. Furthermore, it was found that a collapsed conformation of the polymer is required to ensure efficient insertion into the lipid membrane and that efficient solubilization may be improved by a more homogenous distribution of the maleic acid monomer units along the polymer chain. Altogether, the results show large differences in behavior between the SMA variants tested in the various steps of solubilization. The main conclusion is that the variant with a 2:1 styrene-to-maleic acid ratio is the most efficient membrane solubilizer in a wide pH range.

Figures

Figure 1
Figure 1
Chemical structure of the SMA polymer at 50% ionization. In this study, polymers with four different average styrene/maleic acid (n/m) = 1.4:1, 2:1, 3:1, and 4:1 have been used.
Figure 2
Figure 2
(A) pH dependence of optical density values of SMA solutions in standard BR buffer, which represent the solubility of the polymers. (B) pH-dependent optical density of solutions of the SMA 2:1 variant in BR buffer at varying ionic strength. All measurements were performed at a polymer concentration of 0.1% w/v. The optical density was measured at λ = 350 nm. Solid lines were added to guide the eye. To see this figure in color, go online.
Figure 3
Figure 3
Influence of ionization state on aqueous solubility of SMA. (A) Protonation state (left axis) and corresponding ionization state (right axis) of the monomol of three SMA variants. The monomol is defined as the smallest unit of the polymer that represents its overall monomer composition. Titrations were performed in triplicate and gave very similar results. From the three repeated experiments the maximum error in pKa values is estimated to be ±0.2. For clarity, only a single representative ionization curve is shown for each SMA variant. (B) Aqueous solubility of the SMA variants as function of the linear charge density, which is given as the number of charges per monomer unit where a monomer unit represents either maleic acid or styrene. This graph was prepared by combining the results that are shown in Figs. 2A and 3A. Solid lines were added to guide the eye. To see this figure in color, go online.
Figure 4
Figure 4
Fluorescence of NR in SMA solutions to probe polymer conformation. (A) Emission spectra of NR in 0.1% w/v SMA solutions at pH 8.0 excited at 490 nm. (B) Maximum emission wavelength as function of pH in 0.1% w/v SMA solutions (λex = 490 nm). (C) Maximum emission wavelength as function of SMA concentration (λex = 550 nm). The CAC values for the 2:1, 3:1, and SMA 4:1 variants found to be 5.8, 5.9, and 5.9 μg/mL, respectively. For SMA 1.4:1, a CAC could not be determined in this concentration range. In (B), only the pH range is shown where the SMA variants were found to be water soluble according to Fig. 2A. The maximum emission wavelengths were estimated to have a maximum error of ∼±1 nm (n = 3), except for the SMA 1.4:1 variant in (C), where the error is larger (±3 nm) due to very low emission intensities. Solid lines were added to guide the eye. To see this figure in color, go online.
Figure 5
Figure 5
Effect of SMA composition and pH on the insertion into a lipid monolayer. (A) Insertion of the SMA variants into a di-14:0 PC lipid monolayer at pH 8.0. (B) pH-dependent insertion of the SMA variants into a di-14:0 PC monolayer. (C) pH-dependent insertion of the SMA variants into a di-14:0 PC/PG (1:1) monolayer. In (B) and (C), only the data points are shown where no polymer precipitation was observed during the time course of the experiment. The maximum increase in surface pressure was determined from the time point at which the signal was found to be stable, mostly at ∼45 min. In all experiments the initial surface pressure was 25 mN/m, a standard BR-buffer was used, and the SMA concentration was 0.005% (w/v). Subsequent addition of more SMA did not increase the observed surface pressure any further, demonstrating that the experiments were performed under conditions of excess SMA. The maximum error in surface pressure increase for each experiment is estimated to be ±1 mN/m as determined from repeated experiments (n = 2 or n = 3). Solid lines were added to guide the eye. To see this figure in color go online.
Figure 6
Figure 6
Efficiency of different SMA variants in solubilizing lipid vesicles. (A) Time course solubilization of di-14:0 PC vesicles in standard BR-buffer at 15°C (gel phase) by the SMA variants at pH 8.0. The asterisks denote the time where the temperature was set to 23°C, which is the Tm of di-14:0 PC. (B) Normalized optical density values of the di-14:0 PC vesicle suspension 5 min after SMA addition. The open symbols in (B) indicate that the polymer is not water soluble in the absence of lipids according to Fig. 2 A. Experiments where the optical density increased above the value before SMA addition have been set to a value of 1 to enhance the clarity of the figure. The relative optical density values could be reproduced with a maximum error estimated to be ±0.05 A units (n = 3). All measurements were performed at a polymer concentration of 0.1% (w/v). Solid lines were added to guide the eye. To see this figure in color, go online.
Figure 7
Figure 7
Analysis of the monomer sequence of the SMA variants. The distribution of styrene monomer units is shown as a function of the length of the polystyrene segment they are found in. Each polystyrene segment connects two consecutive maleic acid units. Polymer models were generated according to the penultimate unit model with the assumption that neighboring maleic acid units are nonexistent (see Materials and Methods and references therein for details of this model). Solid lines were added to guide the eye. To see this figure in color, go online.
Figure 8
Figure 8
Schematic diagram that summarizes the effect of SMA composition and pH on the molecular conformation and solubilization efficiency of the SMA copolymer. The polymer conformation is shown as a cartoon in which the hydrophobic domains enriched in styrene units are shown in red, while the maleic acid-rich part of the polymer is shown in black. The efficiency of solubilization is depicted according to a color coding (see above). (Dark green) Complete and fast solubilization; (blue) solubilization is induced but remains incomplete; and (red) the polymer is not able to solubilize at all. To see this figure in color go online.

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