Structural characterization of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using EPR spectroscopy

Abstract

Spectroscopic studies of membrane proteins (MPs) are challenging due to difficulties in preparing homogenous and functional lipid membrane mimetic systems into which membrane proteins can properly fold and function. It has recently been shown that styrene-maleic acid (SMA) copolymers act as a macromolecular surfactant and therefore facilitate the formation of disk-shaped lipid bilayer nanoparticles (styrene-maleic acid copolymer-lipid nanoparticles (SMALPs)) that retain structural characteristics of native lipid membranes. We have previously reported controlled synthesis of SMA block copolymers using reversible addition-fragmentation chain transfer (RAFT) polymerization, and that alteration of the weight ratio of styrene to maleic acid affects nanoparticle size. RAFT-synthesis offers superior control over SMA polymer architecture compared to conventional radical polymerization techniques used for commercially available SMA. However, the interactions between the lipid bilayer and the solubilized RAFT-synthesized SMA polymer are currently not fully understood. In this study, EPR spectroscopy was used to detect the perturbation on the acyl chain upon introduction of the RAFT-synthesized SMA polymer by attaching PC-based nitroxide spin labels to the 5^th, 12^th, and 16^th positions along the acyl chain of the lipid bilayer. EPR spectra showed high rigidity at the 12^th position compared to the other two regions, displaying similar qualities to commercially available polymers synthesized via conventional methods. In addition, central EPR linewidths and correlation time data were obtained that are consistent with previous findings.

Keywords: CW-EPR; Electron paramagnetic resonance; Lipid bilayer; POPC vesicles; SMALPs; Styrene-maleic acid copolymers.

Fig. 1.

(A) Chemical structure of the DOXYL lipid spin label probe, (B) Cartoon representation of the addition of a SMA polymer to an intact vesicle and the structural implications of the addition to form the membrane RAFT SMALP.

Fig. 2.

CW-EPR spectral data for each spin labelled carbon position: 5 (A), 12 (B), and 16 (C) DOXYL stearic acid at 296 K (23 °C) (middle panel) and 318 K (45 °C) (right panel). Left panel shows the cartoon representation of the spin labels attached to the 5^th, 12^th, and 16^th carbon of the acyl chain.

Fig. 3.

Central linewidth (ΔH) of EPR spectra as a function of spin labeled carbon position in vesicles and with the addition of SMA at temperatures Room Temperature (296 K) (23 °C) (A) 318K (45 °C) (B). The error bars represent uncertainties (standard deviation) arising from the triple batch of sample preparations and data analysis.

Fig. 4.

Spectral width (2A_zz) of EPR spectra as a function of spin labeled carbon position in vesicles and with the addition of SMA at temperatures 296 K (23 °C) (A) 318 K (45 °C) (B). The error bars represent uncertainties (standard deviation) arising from the triple batch of sample preparations and data analysis.

Fig. 5.

Order parameter (S) of EPR spectra as a function of spin labeled carbon position in vesicles and with the addition of SMA at temperatures 296 K (23 °C) (A) 318 K (45 °C) (B). The error bars represent uncertainties (standard deviation) arising from the triple batch of sample preparations and data analysis.

Fig. 6.

EPR spectral simulations for spin labeled acyl chain at carbon position 12 in vesicles (A) and with the addition of SMA (B) at temperatures 296 K (23 °C) (left panel) and 318 K (45 °C) (right panel).