Tuning the size of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for biophysical studies

Abstract

Characterization of membrane proteins is challenging due to the difficulty in mimicking the native lipid bilayer with properly folded and functional membrane proteins. Recently, styrene-maleic acid (StMA) copolymers have been shown to facilitate the formation of disc-like lipid bilayer mimetics that maintain the structural and dynamic integrity of membrane proteins. Here we report the controlled synthesis and characterization of StMA containing block copolymers. StMA polymers with different compositions and molecular weights were synthesized and characterized by size exclusion chromatography (SEC). These polymers act as macromolecular surfactants for 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol (POPG) lipids, forming disc like structures of the lipids with the polymer wrapping around the hydrophobic lipid edge. A combination of dynamic light scattering (DLS), solid-state nuclear magnetic resonance (SSNMR) spectroscopy, and transmission electron microscopy (TEM) was used to characterize the size of the nanoparticles created using these StMA polymers. At a weight ratio of 1.25:1 StMA to lipid, the nanoparticle size created is 28+1nm for a 2:1 ratio, 10+1nm for a 3:1 StMA ratio and 32+1nm for a 4:1 StMA ratio independent of the molecular weight of the polymer. Due to the polymer acting as a surfactant that forms disc like nanoparticles, we term these StMA based block copolymers "RAFT SMALPs". RAFT SMALPs show promise as a new membrane mimetic with different nanoscale sizes, which can be used for a wide variety of biophysical studies of membrane proteins.

An Overview of the Top Ten Detergents Used for Membrane Protein Crystallization

“…The main feature of this class of detergents is the presence of quaternary carbon atom that allows incorporation of two hydrophilic head groups and two hydrophobic tails (Figure 10). LMNG has a low CMC value (0.001%/0.01 mM [33]), with the size of micelle of Mw~91 kDa (this work), which is in contrast with the recent result of~393 kDa obtained with MALDI-TOF MS [19]. It has been reported to be efficient both at protein extraction and stabilization of several delicate membrane proteins [37,61,62].…”

“…It has CMC value of 0.01%/0.2 mM, with the micelle size of 60 to 90 kDa (temperature-dependent) [63,64]. LMNG has a low CMC value (0.001%/0.01 mM [33]), with the size of micelle of Mw ~ 91 kDa (this work), which is in contrast with the recent result of ~ 393 kDa obtained with MALDI-TOF MS [19]. It has been reported to be efficient both at protein extraction and stabilization of several delicate membrane proteins [37,61,62].…”

“…Due to the micelle formation, a membrane protein becomes a part of the detergent (lipid)-protein complex, sometimes with the complete loss of surrounding lipids. In many cases, the importance of lipids for the function and/or getting crystals of membrane the cryo-Electron Microscopy (Cryo-EM) field [31,32] and with the future generations of SMAs, (e.g., with the various lengths and ratios of polymers [33]) they might become a very useful tool for studies on membrane proteins.…”

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

“…The stabilizing scaffold protein’s absorbance properties have also proven to interfere with the membrane protein to be studied 9,25 . Using polymers to form nanoparticles has proven to hold more advantages than scaffold protein nanodiscs 15 . Polymers such as styrene maleimide copolymer-lipid nanoparticles (SMILPs) and styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) can solubilize the protein of interest without the use of detergent.…”

Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers

“…Reversible addition-fragmentation chain transfer (RAFT) methods to produce 2:1 and 3:1 subunit ratio SMA polymers, which convert liposomes into nanodiscs with diameters of approximately 28 and 10 nm, respectively, as seen by dynamic light scattering (DLS) and EM methods [19]. The polymer length, on the other hand, has no apparent effect on particle size.…”