An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles.

Stephen C L Hall,Pooja Sridhar,Mark Wheatley,Jack Charlton,Tim R Dafforn,Éilís C Bragginton,Alice J Rothnie,Timothy J Knowles,Thomas Arnold,Cecilia Tognoloni,Karen J Edler

doi:10.1039/c8nr01322e

Abstract

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form "SMA Lipid Particles" (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. "SMILPs" thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.

Highlights

The recent development of SMALP nanodiscs provides benefits over other alternative solubilization strategies by being able to solubilize membrane proteins directly from the host cell membrane whilst keeping the annular lipids present within the nanodisc to maintain the native environment of the membrane protein.[29]
SMALPs are limited by two predominant factors: insolubility at low pH and precipitation in the presence of divalent cations
We have presented data showing that the positively charged SMI polymer can self-assemble in the presence of phospholipids in acidic conditions to form SMI lipid particle (SMILP) nanodiscs which are both thermally stable and stable in the presence of divalent cations

Summary

Introduction

With an increasing interest in membrane proteins due to their physiological and pharmacological significance,[1,2,3,4] recent developments have yielded alternative solutions to the solubilization bottleneck often limiting purification and characterization.[5,6,7,8,9,10] A commonly adopted method involves the use of the amphipathic, helical membrane scaffold proteins (MSP),[11] or peptides inspired by the amino acid residue sequence of the MSP helix,[12,13,14] to solubilise phospholipidAn alternate strategy is the use of poly(styrene-co-maleic acid) (SMA) (Fig. 1a) to extract nanodiscs containing a segment of native cell bilayer, encapsulated by the SMA polymer (termed SMA lipid particles, SMALPs).[19,20,21,22] Since the first report of SMA-mediated solubilization[19] the method has been successfully employed to solubilize a wide variety of targets directly from a range of biological membranes.[23,24,25,26,27,28] SMALPsPaper have proven useful in downstream functional[23,25,29] and structural characterization.[22,24,30,31,32]The commonly used variants of the SMA polymer have been investigated thoroughly[21,33,34,35,36,37,38,39] showing that the first used polymer (SMA2000) is the best performing of the polymers so far studied. The styrene moiety shows significant absorption of UV light,[40] which overlaps with the absorption from aromatic residues within proteins This interferes with spectroscopic techniques to study solubilized proteins. SMALPs have been shown to be unstable in the presence of divalent cations such as Mg2+, with precipitation of the polymer occurring under such conditions This can be a useful property under certain circumstances, it can be a limitation for many potential downstream applications where divalent cations are necessary for membrane protein function.[41,42] SMA is pH sensitive; at acidic pH values, maleic acid groups become protonated and the polymer becomes insoluble.[39] This limits the SMALP method to proteins that are stable at basic pHs

Results

Discussion

Conclusion