Thermotropic properties of phosphatidylcholine nanodiscs bounded by styrene-maleic acid copolymers

Abstract

Styrene-maleic acid copolymers (SMA) have been gaining interest in the field of membrane research due to their ability to solubilize membranes into nanodics. The SMA molecules act as an amphipathic belt that surrounds the nanodiscs, whereby the hydrophobic styrene moieties can insert in between the lipid acyl chains. Here we used SMA variants with different styrene-to-maleic acid ratio (i.e. 2:1, 3:1 and 4:1) to investigate how lipid packing in the nanodiscs is affected by the presence of the polymers and how it depends on polymer composition. This was done by analyzing the thermotropic properties of a series of saturated phosphatidylcholines in nanodiscs using laurdan fluorescence and differential scanning calorimetry. In all cases it was found that the temperature of the main phase transition (T) of the lipids in the nanodiscs is downshifted and that its cooperativity is strongly reduced as compared to the situation in vesicles. These effects were least pronounced for lipids in nanodiscs bounded by SMA 2:1. Unexpected trends were observed for the calorimetric enthalpy of the transition, suggesting that the polymer itself contributes, possibly by rearranging around the nanodiscs when the lipids adopt the fluid phase. Finally, distinct differences in morphology were observed for nanodiscs at relatively high polymer concentrations, depending on the SMA variant used. Overall, the results suggest that the extent of preservation of native thermodynamic properties of the lipids as well as the stability of the nanodiscs at high polymer concentrations is better for SMA 2:1 than for the other SMA variants.

Membrane Solubilization by Styrene-Maleic Acid Copolymers: Delineating the Role of Polymer Length

“…Overall, lipids encircled by nanodisks bound by low-M n SMA retain the native thermotropic properties the least. Nanodisks bounded by SMA 3:1 P96 polymers seem to undergo an additional transition, which has recently been ascribed to an energetic contribution of the SMA polymer during the melting transition of the lipids, possibly related to the poor thermodynamic stability of the nanodisk particles (9). Overall, these results suggest that nanodisks are more stable when bounded by long SMA polymers.…”

“…Styrene-maleic acid (SMA) copolymers are in the spotlight of membrane protein research because of their ability to solubilize lipid membranes into nanodisks (for reviews, see (1,2)). SMA-mediated solubilization offers many advantages over conventional solubilization methods based on the use of detergents, including retention of the membrane architecture (i.e., lipid bilayer conformation) in nanodisks (3,4) and the suitability of nanodisk particles for analysis by biophysical techniques such as NMR (5,6), turbidimetry (7,8), and calorimetry (3,9,10).…”

“…A broad range of SMA polymers differing in composition and in length are commercially available. It has been shown that the composition of the SMA polymers strongly affects the membrane solubilization properties in model membranes (11) as well as in biological membranes (12,13) and that it also affects the properties of the resulting nanodisks (9). Recently, also the effect of polymer length in membrane solubilization has been investigated.…”

Mass Photometry of Membrane Proteins

“…In addition to the main SMALP peak, 40% of all observed particles were polydisperse and at higher molecular weight, approximately 500-2000 kDa, indicating some degree of ND oligomerisation. In line with this, it has been reported that SMALPs can form oligomeric 'rouleaux' stacks 15 , although this was attributed to a transmission election microscopy (TEM) artefact. This inhomogeneity can also result from an unoptimised SMALP assembly and purification process.…”

“…By contrast, a KcsA:MSP1D1:DMPC lipid ratio of 1:20:800 exhibited a slightly poorer size exclusion profile (Figure 4b, blue), but a much more homogeneous MP profile (Figure 4c, blue), with a mass of 154 kDa corresponding to a single KcsA tetramer embedded in a nanodisc consisting of 2 MSP1D1 proteins and approximately 25 lipids. The latter preparation exhibited 15 NH4 + ion binding upon addition of 50 mM 15 NH4 + , as revealed by the presence of 2 strong and 2 very weak NH4 + cross peaks in a 2D [ 15 N, 1 H]-HMQC NMR spectrum assigned to the 4 ion binding sites in the selectivity filter of KcsA (Figure 4d). By contrast, we could not detect any signatures of ion binding in the former sample preparation shown to be highly heterogeneous and lacking KcsA tetramers by MP, demonstrating the lack of functional KcsA (Figure 4d, inset).…”