Contents lists available at ScienceDirect European Polymer Journal journal homepage: www.elsevier.com/locate/europolj Evaluation of commercially available styrene-co-maleic acid polymers for the extraction of membrane proteins from spinach chloroplast thylakoids Olena Korotych a,b, ⁎ , Jyotirmoy Mondal a , Kerim M. Gattás-Asfura c , Jessica Hendricks a , Barry D. Bruce a,d, ⁎ a Department of Biochemistry, and Cellular, and Molecular Biology, University of Tennessee at Knoxville, 1311 Cumberland Avenue, Knoxville, TN 37996-1939, United States b Department of Chemical Engineering, University of Florida, 1030 Center Drive, Gainesville, FL 32611-6131, United States c Department of Biomedical Engineering, University of Florida, 1275 Center Drive, Gainesville, FL 32611-6131, United States d Department of Microbiology, University of Tennessee at Knoxville, 1311 Cumberland Avenue, Knoxville, TN 37996-1937, United States ARTICLEINFO Keywords: styrene-co-maleic acid lipid particle (SMALP) styrene-maleic acid (SMA) copolymer poly(styrene-co-maleic acid) salt (pSMA-S) membrane protein thylakoid solubilization efcacy ABSTRACT Solubilizationofmembraneproteinsbypoly(styrene-co-maleicacid)salts(pSMA-S)hassignifcantpotential for membraneproteinstudies.Thisapproachprovidesanopportunitytoovercomemanydisadvantagesassociated withatraditionaldetergent-basedtechniqueincludingproteindenaturationanddisplacementofboundarylipids which may ofer both structural and functional stability to membrane proteins. Thylakoid membranes (TMs) fromphotosyntheticorganismsarewellstudiedprotein-richmembranesthathostseveralmulti-subunitprotein complexes associated with oxygenic photosynthesis. These protein complexes are important for applied pho- tosynthesisandbybeingextractedandpurifedtheycanbeusedinthenear futurefordirectenergyconversion. In this study, we used spinach TMs isolated from purifed intact chloroplasts to systematically test the solubi- lizationefcacyof12commerciallyavailablestyrene-maleicacid(SMA)copolymersthatvaryinsize,styrene- to-maleicacidmolarratio,andtypeofestergroup.TheefcacyofthesepSMA-Stosolubilizeprotein-containing biomembranes was evaluated via quantifcation of protein and chlorophyll content in the resulting SMA Lipid Particles(SMALPs).Inaddition,theextractedpolymer-lipid-proteincomplexeswerestudiedbylowtemperature fuorescence,sodiumdodecylsulfateandclearnativepolyacrylamidegelelectrophoresis(SDS-andCN-PAGE), and immunoblot analysis. Our results indicate considerable variability in the solubilization efcacy of com- mercially available pSMA-S with at least 5 polymer formulations being able to efciently extract membrane proteins from TMs. These 5 SMA copolymers may also be efective in extraction of membrane proteins from other biomembranes. 1. Introduction Poly(styrene-co-maleic anhydride) (pSMAnh) is a polymer that is synthesized from styrene and maleic anhydride monomers and can be convertedintowater-solublesubstancessuchassaltsandestersthrough alkaline hydrolysis and esterifcation, respectively, (Fig. 1) or via chemical modifcation, for example, yielding poly(styrene-co-mal- eimide quaternary ammonium) [1]. Salts of SMA copolymers, poly (styrene-co-maleicacid)salts(pSMA-S),representaclassofsubstances known as polymeric surfactants which have been used as emulsifying anddispersingagentsforatleasthalfacentury [2],andjustadecade ago researchers started to utilize these polymers for solubilization of https://doi.org/10.1016/j.eurpolymj.2018.10.035 Received26June2018;Receivedinrevisedform17October2018;Accepted22October2018 Abbreviations: APS, ammonium persulfate; ATPase, adenosine triphosphatase; ATR, attenuated total refection; BCA, bicinchoninic acid; BSA, bovine serum al- bumin;CAB,chlorophyll a/b binding;CBB,coomassiebrilliantblue;CN-PAGE,clearnative–polyacrylamidegelelectrophoresis;DMF,dimethylformamide;DDM,n- dodecyl-β-D-maltoside; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; FTIR, Fourier transform infrared; IF, insoluble fraction; HRP, horseradish per- oxidase;LHC,light-harvestingcomplex;LT,lowtemperature;NIST,theNationalInstituteofStandardsandTechnology;PS,photosystem;pSMAnh,poly(styrene-co- maleicanhydride);pSMA-S,poly(styrene-co-maleicacid)salt;s/mratio,styrene-to-maleicacidmolarratio;SDS-PAGE,sodiumdodecylsulfate–polyacrylamidegel electrophoresis; SE, solubilization efcacy; SF, soluble fraction; SI, supportive information; SMA, styrene-co-maleic acid; SMALP, styrene-co-maleic acid lipid par- ticle; TEMED, tetramethylethylenediamine, N, N, N', N'-; TM, thylakoid membrane; UV, ultraviolet ⁎ Corresponding authors at: Department of Biochemistry, and Cellular, and Molecular Biology, University of Tennessee at Knoxville, 1311 Cumberland Avenue, Knoxville, TN 37996-1937, United States. E-mail addresses: o.i.korotych@gmail.com (O. Korotych), bbruce@utk.edu (B.D. Bruce). European Polymer Journal 114 (2019) 485–500 Available online 23 October 2018 0014-3057/ © 2018 Published by Elsevier Ltd. T