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

Abstract

Membrane proteins play an important role in maintaining the structure and physiology of an organism. Despite their significance, spectroscopic studies involving membrane proteins remain challenging due to the difficulties in mimicking their native lipid bilayer environment. Membrane mimetic systems such as detergent micelles, liposomes, bicelles, nanodiscs, lipodisqs have improved the solubility and folding properties of the membrane proteins for structural studies, however, each mimetic system suffers from its own limitations. In this study, using three different lipid environments, vesicles were titrated with styrene-maleic acid (StMA) copolymer leading to a homogeneous SMALP system (~10 nm) at a weight ratio of 1:1.5 (vesicle: StMA solution). A combination of Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) was used to characterize these SMALPs. We used a controlled synthesis mechanism to synthesize StMA based block copolymers called reversible addition-fragmentation chain transfer polymerization (RAFT) SMALPs. Incorporation of the Voltage sensor Domain of KCNQ1 (Q1-VSD) into RAFT SMALPs indicates that this is a promising application of this system to study membrane proteins using different biophysical techniques. V165C in Q1-VSD corresponding to the hydrophobic region was incorporated into the SMALP system. Continuous Wave-Electron Paramagnetic Resonance (CW-EPR) line shape analysis showed line shape broadening, exposing a lower rigid component and a faster component of the spin label.

Synthesis and characterization of block copolymers of styrene‐maleic acid with acrylamide and N,N‐dimethylacrylamide

“…These discoidal nanoparticles are often referred to in the literature as SMA-lipid particles (SMALPs). [9][10][11][12] In this behavior, they show a biomimetic resemblance to Surfactant Protein C (SPC), a pulmonary surfactant protein essential for lung function after birth. [7,8] An illustration of an SMA-lipid particle is shown in Figure 1, showing how the SMA wraps itself around a phospholipid bilayer containing the encapsulated membrane protein.…”

Development of Methodology to Investigate the Surface SMALPome of Mammalian Cells

“…The previous study employing proteomics on SMA extracted samples of bacterial membranes used acetone precipitation of protein prior to trypsin digestion, which may be another method of removing SMA from samples ( Carlson et al, 2019 ). It may, however, be possible to perform proteomics without removal of copolymer if extraction from membranes is with RAFT-synthesized copolymers ( Craig et al, 2016 ; Ravula et al, 2017 ; Hall et al, 2018 ; Harding et al, 2019 ; Cunningham et al, 2020 ) or copolymers with desired properties, such as acid compatibility ( Hall et al, 2018 ), that display much less heterogeneity or are less prone to aggregation. Such copolymers may not mask the signals from proteins so extensively and may themselves produce more discrete mass signals than the SMA copolymer used in this study.…”

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

“…However, no membrane mimetic systems are universally compatible to all membrane proteins requiring rigorous time-consuming optimization processes for their incorporation in a suitable membrane environment. Currently available and widely used membrane mimetic systems are detergent micelles, bicelles, liposomes, lipodiscs, and lipodisq nanoparticles/SMALPs (styrene maleic acid lipid particles) [12][13][14][15][16]. These membrane mimetic systems have their own benefits and limitations.…”