Synthesis and Evaluation of a Library of Alternating Amphipathic Copolymers to Solubilize and Study Membrane Proteins

Abstract

Amphipathic copolymers such as poly(styrene-maleic acid) (SMA) are promising tools for the facile extraction of membrane proteins (MPs) into native nanodiscs. Here, we designed and synthesized a library of well-defined alternating copolymers of SMA analogues in order to elucidate polymer properties that are important for MP solubilization and stability. MP extraction efficiency was determined using KcsA from E. coli membranes, and general solubilization efficiency was investigated via turbidimetry experiments on membranes of E. coli , yeast mitochondria, and synthetic lipids. Remarkably, halogenation of SMA copolymers dramatically improved solubilization efficiency in all systems, while substituents on the copolymer backbone improved resistance to Ca 2+ . Relevant polymer properties were found to include hydrophobic balance, size and positioning of substituents, rigidity, and electronic effects. The library thus contributes to the rational design of copolymers for the study of MPs.

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight

“…[40] In addition, in a very recent comprehensive study, Kopf et al presented a number of SMA derivatives with diverse substitution mainly at the aromatic ring, as well as acrylic acid copolymers with substituted styrenes. [41] Finally, new amphiphilic small molecules (nonpolymeric but of higher MW) for membrane protein solubilization have been introduced recently, further blurring the boundary between the originally used detergents and the newly developed (mostly low-MW) polymers. [42,43] Despite the considerable progress made, the new copolymers still share some of the drawbacks of SMA mentioned above, e.g., they incorporate the potentially problematic styrenic or maleic acid moieties or show broad MW distribution.…”

“…[14,17] For this reason, reversible additionfragmentation chain-transfer (RAFT) polymerization, that allows the preparation of low-dispersity polymers, has been applied in several recent studies to control the MW. [12,14,19,31,41] Another key property of the successful amphiphilic membrane-solubilizing copolymers is the proper balance between their hydrophilic and hydrophobic parts, where even small changes to the copolymer composition can impact significantly on the material performance in solubilization assays. [14] Moreover, the distribution of the different monomeric units within the polymeric chain also plays an important role, with compositional drift shown to be detrimental in some cases.…”

Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids

“…A systematic and quantitative analysis of MP extraction was recently undertaken with SMA derivatives 41 but was limited to polymers carrying aromatic cycles. In our case, two sets of polymers bearing either aromatic (ArylAPols) or cycloalkane groups (CyclAPols) were screened for their ability to extract MPs.…”

Characterization of Divalent Cation Interactions with AASTY Nanodiscs

“…We previously showed that poly­(acrylic acid- co -styrene) (AASTY) copolymers (Figure B) are effective at solubilizing small, unilameller vesicles (SUVs) as well as extracting a model membrane protein from HEK293 cells . AASTY can be synthesized by reversible addition–fragmentation chain-transfer (RAFT) polymerizations, which allow the control of copolymer length and the dispersity of size, compared with free-radical polymerization. − …”