title: SMA-SH: Modified Styrene-Maleic Acid Copolymer for Functionalization of Lipid Nanodiscs.
abstract: Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene-maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology . Lorentzweg 1, Delft 2628 CJ, The Netherlands.
authors.forename: Simon
authors.initials: S
authors.lastname: Lindhoud
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology . Lorentzweg 1, Delft 2628 CJ, The Netherlands.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Carvalho
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology . Lorentzweg 1, Delft 2628 CJ, The Netherlands.
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Pronk
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology . Lorentzweg 1, Delft 2628 CJ, The Netherlands.
authors.forename: Marie-Eve
authors.initials: ME
authors.lastname: Aubin-Tam
mesh_terms: D002850:Chromatography, Gel; D003543:Cysteamine / Q000737:chemistry; D000067493:Dynamic Light Scattering; D008051:Lipid Bilayers / Q000378:metabolism*; D008298:Maleates / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis*; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D011510:Proteolipids / Q000378:metabolism*; D020058:Styrene / Q000737:chemistry*
chemical_list: D008051:Lipid Bilayers; D008298:Maleates; D008565:Membrane Proteins; D011108:Polymers; D011510:Proteolipids; C020485:proteoliposomes; D020058:Styrene; D003543:Cysteamine; C030272:maleic acid