Vinyl Ether Maleic Acid Polymers: Tunable Polymers for Self-Assembled Lipid Nanodiscs and Environments for Membrane Proteins

Abstract

Native lipid bilayer mimetics, including those that use amphiphilic polymers, are important for the effective study of membrane-bound peptides and proteins. Copolymers of vinyl ether monomers and maleic anhydride were developed with controlled molecular weights and hydrophobicity through reversible addition-fragmentation chain-transfer polymerization. After polymerization, the maleic anhydride units can be hydrolyzed, giving dicarboxylates. The vinyl ether and maleic anhydride copolymerized in a close to alternating manner, giving essentially alternating hydrophilic maleic acid units and hydrophobic vinyl ether units along the backbone after hydrolysis. The vinyl ether monomers and maleic acid polymers self-assembled with lipids, giving vinyl ether maleic acid lipid particles (VEMALPs) with tunable sizes controlled by either the vinyl ether hydrophobicity or the polymer molecular weight. These VEMALPs were able to support membrane-bound proteins and peptides, creating a new class of lipid bilayer mimetics.

Scheme 1:

Structure of VEMA copolymers and their proposed interactions with lipids to form nanodiscs containing membrane proteins.

Scheme 2:

A) RAFT copolymerization of vinyl ether and maleic anhydride monomers to give VEMA poylmers, including CTAs studied. B) RAFT end group replacement by reaction with lauroyl peroxide and AIBN. C) Hydrolysis of VEMA polymers.

Figure 1:

Molecular weight distributions for BVE MAn copolymerization using 4 different CTAs. Conditions: [MAn]:[BVE]:[CTA]:[AIBN] = 50:75:1:0.3 at 60 °C for 3h.

Figure 2:

a) Kinetics of polymerization, b) evolution of M_n and M_w/M_n vs M_n-th,. Polymerization under the conditions [MAn]:[BVE]:[iBADTC]:[AIBN] = 25:25:1:0.3, 50:50:1:0.3, 100:100:1:0.3 in 33 wt% solvent at 60 °C.

Figure 3:

Polymerization of MAn:BVE:DVE:PADTC = 50:53:13:1 and MAn:BVE:PADTC = 50:66:1 at 60 °C with PADTC as the CTA using PADTC:AIBN=1:0.3 in 33% solvent. A) Kinetics of copolymerization. B) Molecular weight distribution of end point polymer.

Figure 4:

A) DLS size (diameter) distribution of POPC vesicles (black), VEMALPs generated from self-assembly of POPC lipids with polymers of MA:BVE = 50:66 (blue), MA:BVE:DVE = 50:53:13 (orange) and MA:BVE:DVE = 50:44:22 (green). B) TEM image of POPC vesicles. C) VEMALPs generated via self-assembly of POPC lipids with MA:BVE:DVE = 50:53:13 polymers.

Figure 5:

A) DLS size distribution of POPC vesicles (black) and VEMALPs using lipid:BVE ratio of 1:2 v/v with different molecular weights of polymers with ratios [MA]:[BVE]:[DVE] = 50:53:13. B) Size distribution of POPC vesicles and VEMALPs from polymer of M_n = 13,000, [MA]:[BVE]:[DVE] = 50:53:13 with and without end-group removed. C) Size distribution of POPG vesicles and VEMALPs from polymer of M_n = 13,000, [MA]:[BVE]:[DVE] = 50:53:13 with end-group removed.

Figure 6:

Stability of VEMALPs (MA:BVE:DVE:PADTC = 50:53:13:1) and a SMALPs (MA:S=1:3) measured by UV-Vis spectroscopy. A) pH stability and B) Mg²⁺ stability.

Figure 7:

A) Structures of KNCE1, KCNE4 and gp28 including location of spin labels. B) CW-EPR spectra of KCNE1 T58C spin labelled system in different lipid bilayer mimetics. C) CW-EPR spectra of KCNE4 S9C spin labelled system in different lipid bilayer mimetics. D) CW-EPR spectra of gp28 T15C spin labelled system in different lipid bilayer mimetics. (EG-On refers to a polymers retaining the trithiocarbonate RAFT end group and EG-off refers to polymers where the RAFT end group was replaced with a dodecyl chain).