Membrane proteins, lipids and detergents: not just a soap operaIntroductionDifficulties in the study of membrane proteinsStrategies for studying membrane proteinsDetergentsTypes of detergentClassification of detergentsGeneral properties of detergentsRemoval of detergentsDialysisHydrophobic adsorptionGel chromatographyIon-exchange chromatographyNickel columns and His tagsAlternatives to traditional detergentsMixed lipid-detergent systemsDetergent-lipid micelles and bicellesReconstituting proteins into bilayersDetergent solubilization of liposomesOther reconstitution methodsThe effects of lipids on protein stability and foldingSpecific lipid interactionsCrystallization of membrane proteinsIn surfo methodIn cubo methodBicelle methodConclusionsAcknowledgementsReferencesReviewMembrane proteins, lipids and detergents: not just a soap operaAnnela M. Seddon*, Paul Curnow1, Paula J. Booth*Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UKReceived 27 February 2004; accepted 29 April 2004Available online 23 July 2004AbstractStudying membrane proteins represents a major challenge in protein biochemistry, with one of the major difficulties being the problemsencountered when working outside the natural lipid environment. In vitro studies such as crystallization are reliant on the successfulsolubilization or reconstitution of membrane proteins, which generally involves the careful selection of solubilizing detergents and mixedlipid/detergent systems. This review will concentrate on the methods currently available for efficient reconstitution and solubilization ofmembrane proteins through the use of detergent micelles, mixed lipid/detergent micelles and bicelles or liposomes. We focus on the relevantmolecular properties of the detergents and lipids that aid understanding of these processes. A significant barrier to membrane protein researchis retaining the stability and function of the protein during solubilization, reconstitution and crystallization. We highlight some of the lessonslearnt from studies of membrane protein folding in vitro and give an overview of the role that lipids can play in stabilizing the proteins.D 2004 Published by Elsevier B.V.Keywords: Membrane proteins; Lipid; Detergent; Bacteriorhodopsin; Reconstitution; FoldingContents1. Introduction ............................................................ 1061.1. Difficulties in the study of membrane proteins ...................................... 1061.2. Strategies for studying membrane proteins ........................................ 1062. Detergents ............................................................ 1072.1. Types of detergent .................................................... 1082.2. Classification of detergents ................................................ 1082.3. General properties of detergents ............................................. 1092.4. Removal of detergents .................................................. 1092.4.1. Dialysis ..................................................... 1092.4.2. Hydrophobic adsorption ............................................. 1090005-2736/$ - see front matter D 2004 Published by Elsevier B.V.doi:10.1016/j.bbamem.2004.04.011Abbreviations: bR, bacteriorhodopsin; LCHII, light harvesting complex; DAGK, diacylglycerol kinase; DAG, diacylglycerol; SDS, sodium dodecyl sulfate;OG, n-octyl-h-d-glucopyranoside; DDM, n-dodecyl-h-d-maltoside; DDAO, dodecyldimethyl-N-amineoxide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phos-phocholine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; MGDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacylgly-cerol; DPPG, l-a-dipalmitoylphosphatidylcholine; DOPG, l-a-dioleoylphosphatidylglycerol; DOPC, l-a-1,2-dioleylphospatidylcholine; DOPE, l-a-1,2-dioleylphospatidylethanolamine; DHPC, l-a-1,2-dihexanoylphosphatidylcholine; DAG, diacylglycerol; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine;CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate; hR, halorhodopsin; OmpA, E. coli outer membrane protein A; OmpF,E. coli outer membrane protein F* Corresponding authors. Paula J. Booth is to be contacted at Tel.: +44 117 9288976; fax: +44 117 9288274.E-mail address: [email protected] (P.J. Booth).1Current address: Marine Biotechnology Centre, University of California at Santa Barbara, Santa Barbara, CA 93106.Biochimica et Biophysica Acta 1666 (2004) 105 – 117http://www.elsevier.com/locate/bba2.4.3. Gel chromatography ............................................... 1102.4.4. Ion-exchange chromatography.......................................... 1102.4.5. Nickel columns and His tags .......................................... 1102.5. Alternatives to traditional detergents ........................................... 1103. Mixed lipid–detergent systems .................................................. 1103.1. Detergent–lipid micelles and bicelles ........................................... 1103.2. Reconstituting proteins into bilayers ........................................... 1113.3. Detergent solubilization of liposomes........................................... 1113.4. Other reconstitution methods ............................................... 1123.5. The effects of lipids on protein stability and folding ................................... 1123.6. Specific lipid interactions ................................................. 1134. Crystallization of membrane proteins .............................................. 1134.1. In surfo method...................................................... 1144.2. In cubo method ...................................................... 1144.3. Bicelle method ...................................................... 1145. Conclusions ........................................................... 114Acknowledgements .......................................................... 115References............................................................... 1151. IntroductionAnalysis of genomic sequence data predicts that 30% ofthe proteins produced by Homo sapiens, Escherichia coliand Saccharomyces cerevisae will be integral membraneproteins [1]. However, while the number of predicted genesequences for integral membrane proteins has increasedover the last few years, there is considerably less informa-tion about their three-dimensional structure and the nature oftheir behaviour within the membrane. There are severalreasons for this