Formation of pH‐Resistant Monodispersed Polymer–Lipid Nanodiscs

Abstract

Polymer lipid nanodiscs are an invaluable system for structural and functional studies of membrane proteins in their near-native environment. Despite the recent advances in the development and usage of polymer lipid nanodisc systems,lack of control over sizea nd poor tolerance to pH and divalent metal ions are major limitations for further applications.A facile modification of al ow-molecular-weight styrene maleic acid copolymer is demonstrated to form monodispersed lipid bilayer nanodiscs that show ultra-stability towardsd ivalent metal ion concentration over ap Hr ange of 2.5 to 10. The macro-nanodiscs (> 20 nm diameter) show magnetic alignment properties that can be exploited for high-resolution structural studies of membrane proteins and amyloid proteins using solid-state NMR techniques.T he new polymer,S MA-QA, nanodisc is ar obust membrane mimetic tool that offers significant advantages over currently reported nanodisc systems.Controlled molecular self-assembly in the formation of soft nanomaterials has been achallenge in bio-nanotechnology. [1,2] Nanodiscs,l ipid bilayers surrounded by an amphiphilic belt, are engineered soft nanomaterials that have been inspired from biological systems such as high-density lipo-particles (HDL). [3] These nanodiscs provide al ipid bilayer environment that is nearly like an ative membrane,a nd they have been used to study the structure and function membrane proteins. [3][4][5] Recent developments have expanded the formation of nanodiscs using different types of amphiphilic systems such as proteins, [6][7][8][9][10] peptides, [11] and polymers. [12][13][14][15] Polymer nanodiscs exhibit significant advantages over conventional protein-based nanodiscs,s uch as detergent-free membrane protein extraction, [16] and they are devoid of interferences from the belt-forming protein or peptide. [17] Currently,n o polymer nanodisc systems have been able to demonstrate precise control of size and morphology over aw ide range of sizes or tolerance towards ab road range of pH and divalent metal ions. [16] These unique properties are needed to greatly expand the applicability of nanodisc technology.H erein we report the directed self-assembly of covalently modified styrene maleic acid copolymer with lipid bilayers to form monodispersed nanodiscs that show ultra-stability towards abroad range of pH and divalent metal ion concentration. We also demonstrate the ability to control the size of the selfassembled nanodiscs and size-dependent unique magnetic alignment properties.Synthesis of SMA-QA (styrene maleimide quaternary ammonium) was achieved by the treatment of al ow-molecular-weight SMA (ca. 1.6 kDa) with (2-aminoethyl)trimethylammonium chloride hydrochloride in anhydrous dimethylformamide while heating in the presence of excess triethylamine.Maleimide formation was accomplished by adehydration reaction using acetic anhydride,s odium acetate,a nd triethylamine (Figure 1a). Then ewly synthesized SMA-QA polymer was characterized using FTIR spectropscopy and 13 CPMAS (cross-polarization mag...

Characterizing the structure of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for membrane protein spectroscopic studies

“…However, there are advantages and drawbacks to each of these mimetic systems 15 . Recent studies on the modified version of styrene-maleic acid copolymers have shown ultra-stability of monodispersed lipid bilayer nanodics 16 . Although liposomes are a widely accepted membrane mimetic system, they tend to form heterogeneous aggregates.…”

Magnetic-Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy

“…These results suggest that macro-nanodiscs possess a negative magnetic susceptibility and align in such a way that the lipid bilayer normal orients perpendicular to the magnetic field direction as shown in previous studies. 40,43,47 The magnetic susceptibility of these nanodiscs can be changed by the addition of paramagnetic metal ions such as YbCl3 which has a positive magnetic susceptibility. [48][49] To study the alignment behavior of macro-nanodiscs 31 P NMR spectra were acquired as a function of time by adding different concentrations of YbCl3 to the aligned macro-nanodiscs.…”

“…45 In spite of such unique magnetic-alignment properties of nanodiscs, a comprehensive study on understanding the factors affecting the alignment of nanodiscs is lacking. In this study, we undertook a comprehensive list of experiments to study the magnetic-alignment of polymer based nanodiscs made from a positively charged polymer SMA-QA 43 and DMPC lipids. Magnetic-alignment of SMA-QA+DMPC nanodiscs was studied using 31 P and 14 N solid-state NMR experiments.…”

Semen‐derived amyloidogenic peptides—Key players of HIV infection

“…Although this is a difficult task due to the challenges posed by the lipid membrane for high‐resolution structural studies, the recently developed nanodiscs can be used to accomplish this task . In particular, the recently developed polymers that vary in charge and can form lipid nanodiscs would be valuable to trap the intermediate structures of SEVI in different membrane compositions . In addition, the use of “styrene‐free” methacrylate polymer nanodiscs would enable high‐throughput screening of conditions by ThT fluorescence and CD experiments as demonstrated recently …”