Abstract
Integral membrane proteins have historically been challenging targets for biophysical research due to their low solubility in aqueous solution. Their importance for chemical and electrical signaling between cells, however, makes them fascinating targets for investigators interested in the regulation of cellular and physiological processes. Since membrane proteins shunt the barrier imposed by the cell membrane, they also serve as entry points for drugs, adding pharmaceutical research and development to the interests. In recent years, detailed understanding of membrane protein function has significantly increased due to high-resolution structural information obtained from single-particle cryo-EM, X-ray crystallography, and NMR. In order to further advance our mechanistic understanding on membrane proteins as well as foster drug development, it is crucial to generate more biophysical and functional data on these proteins under defined conditions. To that end, different techniques have been developed to stabilize integral membrane proteins in native-like environments that allow both structural and biophysical investigations—amphipols, lipid bicelles, and lipid nanodiscs. In this chapter, we provide detailed protocols for the reconstitution of membrane proteins according to these three techniques. We also outline some of the possible applications of each technique and discuss their advantages and possible caveats.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fagerberg L, Jonasson K, von Heijne G, Uhlén M, Berglund L (2010) Prediction of the human membrane proteome. Proteomics 10(6):1141–1149. https://doi.org/10.1002/pmic.200900258
KĂĽhlbrandt W (2014) Cryo-EM enters a new era. Elife 3:e03678. https://doi.org/10.7554/eLife.03678
Cheng Y (2015) Single-particle cryo-EM at crystallographic resolution. Cell 161(3):450–457. https://doi.org/10.1016/j.cell.2015.03.049
Cherezov V (2011) Lipidic cubic phase technologies for membrane protein structural studies. Curr Opin Struct Biol 21(4):559–566. https://doi.org/10.1016/j.sbi.2011.06.007
Liang B, Tamm LK (2016) NMR as a tool to investigate the structure, dynamics and function of membrane proteins. Nat Struct Mol Biol 23(6):468–474. https://doi.org/10.1038/nsmb.3226
Warschawski DE, Arnold AA, Beaugrand M, Gravel A, Chartrand É, Marcotte I (2011) Choosing membrane mimetics for NMR structural studies of transmembrane proteins. Biochim Biophys Acta 1808(8):1957–1974. https://doi.org/10.1016/j.bbamem.2011.03.016
Hunte C, Richers S (2008) Lipids and membrane protein structures. Curr Opin Struct Biol 18(4):406–411. https://doi.org/10.1016/j.sbi.2008.03.008
McCoy JG, Rusinova R, Kim DM, Kowal J, Banerjee S, Jaramillo Cartagena A, Thompson AN, Kolmakova-Partensky L, Stahlberg H, Andersen OS, Nimigean CM (2014) A KcsA/MloK1 chimeric ion channel has lipid-dependent ligand-binding energetics. J Biol Chem 289(14):9535–9546. https://doi.org/10.1074/jbc.M113.543389
Quick M, Winther AM, Shi L, Nissen P, Weinstein H, Javitch JA (2009) Binding of an octylglucoside detergent molecule in the second substrate (S2) site of LeuT establishes an inhibitor-bound conformation. Proc Natl Acad Sci U S A 106(14):5563–5568. https://doi.org/10.1073/pnas.0811322106
Quick M, Shi L, Zehnpfennig B, Weinstein H, Javitch JA (2012) Experimental conditions can obscure the second high-affinity site in LeuT. Nat Struct Mol Biol 19(2):207–211. https://doi.org/10.1038/nsmb.2197
Dowhan W (1997) Molecular basis for membrane phospholipid diversity: why are there so many lipids? Annu Rev Biochem 66:199–232. https://doi.org/10.1146/annurev.biochem.66.1.199
Yu CA, Yu L (1980) Structural role of phospholipids in ubiquinol-cytochrome c reductase. Biochemistry 19(25):5715–5720
Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666(1–2):105–117. https://doi.org/10.1016/j.bbamem.2004.04.011
Raschle T, Hiller S, Etzkorn M, Wagner G (2010) Nonmicellar systems for solution NMR spectroscopy of membrane proteins. Curr Opin Struct Biol 20(4):471–479. https://doi.org/10.1016/j.sbi.2010.05.006
Popot JL, Althoff T, Bagnard D, Banères JL, Bazzacco P, Billon-Denis E, Catoire LJ, Champeil P, Charvolin D, Cocco MJ, Crémel G, Dahmane T, de la Maza LM, Ebel C, Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kühlbrandt W, Le Bon C, Martinez KL, Picard M, Pucci B, Sachs JN, Tribet C, van Heijenoort C, Wien F, Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408. https://doi.org/10.1146/annurev-biophys-042910-155219
Tribet C, Audebert R, Popot JL (1996) Amphipols: polymers that keep membrane proteins soluble in aqueous solutions. Proc Natl Acad Sci U S A 93(26):15047–15050
Tehei M, Perlmutter JD, Giusti F, Sachs JN, Zaccai G, Popot JL (2014) Thermal fluctuations in amphipol A8-35 particles: a neutron scattering and molecular dynamics study. J Membr Biol 247(9–10):897–908. https://doi.org/10.1007/s00232-014-9725-1
Le Bon C, Marconnet A, Masscheleyn S, Popot JL, Zoonens M (2018) Folding and stabilizing membrane proteins in amphipol A8-35. Methods 147:95–105. https://doi.org/10.1016/j.ymeth.2018.04.012
Gohon Y, Giusti F, Prata C, Charvolin D, Timmins P, Ebel C, Tribet C, Popot JL (2006) Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35. Langmuir 22(3):1281–1290. https://doi.org/10.1021/la052243g
Perlmutter JD, Drasler WJ, Xie W, Gao J, Popot JL, Sachs JN (2011) All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing polymer. Langmuir 27(17):10523–10537. https://doi.org/10.1021/la202103v
Barrera NP, Zhou M, Robinson CV (2013) The role of lipids in defining membrane protein interactions: insights from mass spectrometry. Trends Cell Biol 23(1):1–8. https://doi.org/10.1016/j.tcb.2012.08.007
Schmidpeter PAM, Gao X, Uphadyay V, Rheinberger J, Nimigean CM (2018) Ligand binding and activation properties of the purified bacterial cyclic nucleotide-gated channel SthK. J Gen Physiol 150(6):821–834. https://doi.org/10.1085/jgp.201812023
Martinez KL, Gohon Y, Corringer PJ, Tribet C, Mérola F, Changeux JP, Popot JL (2002) Allosteric transitions of Torpedo acetylcholine receptor in lipids, detergent and amphipols: molecular interactions vs. physical constraints. FEBS Lett 528(1–3):251–256
Catoire LJ, Damian M, Giusti F, Martin A, van Heijenoort C, Popot JL, Guittet E, Banères JL (2010) Structure of a GPCR ligand in its receptor-bound state: leukotriene B4 adopts a highly constrained conformation when associated to human BLT2. J Am Chem Soc 132(26):9049–9057. https://doi.org/10.1021/ja101868c
Charvolin D, Perez JB, Rouvière F, Giusti F, Bazzacco P, Abdine A, Rappaport F, Martinez KL, Popot JL (2009) The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports. Proc Natl Acad Sci U S A 106(2):405–410. https://doi.org/10.1073/pnas.0807132106
Polovinkin V, Gushchin I, Sintsov M, Round E, Balandin T, Chervakov P, Shevchenko V, Utrobin P, Popov A, Borshchevskiy V, Mishin A, Kuklin A, Willbold D, Chupin V, Popot JL, Gordeliy V (2014) High-resolution structure of a membrane protein transferred from amphipol to a lipidic mesophase. J Membr Biol 247(9–10):997–1004. https://doi.org/10.1007/s00232-014-9700-x
van Pee K, Neuhaus A, D’Imprima E, Mills DJ, Kühlbrandt W, Yildiz Ö (2017) CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin. Elife 6. https://doi.org/10.7554/eLife.23644
Liao M, Cao E, Julius D, Cheng Y (2013) Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504(7478):107–112. https://doi.org/10.1038/nature12822
Hirschi M, Herzik MA, Wie J, Suo Y, Borschel WF, Ren D, Lander GC, Lee SY (2017) Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3. Nature 550(7676):411–414. https://doi.org/10.1038/nature24055
Li M, Zhou X, Wang S, Michailidis I, Gong Y, Su D, Li H, Li X, Yang J (2017) Structure of a eukaryotic cyclic-nucleotide-gated channel. Nature 542(7639):60–65. https://doi.org/10.1038/nature20819
Sanders CR, Landis GC (1995) Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies. Biochemistry 34(12):4030–4040
Sanders CR, Schwonek JP (1992) Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR. Biochemistry 31(37):8898–8905
Ujwal R, Bowie JU (2011) Crystallizing membrane proteins using lipidic bicelles. Methods 55(4):337–341. https://doi.org/10.1016/j.ymeth.2011.09.020
Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, Kobilka BK (2007) Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450(7168):383–387. https://doi.org/10.1038/nature06325
Ujwal R, Cascio D, Colletier JP, Faham S, Zhang J, Toro L, Ping P, Abramson J (2008) The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating. Proc Natl Acad Sci U S A 105(46):17742–17747. https://doi.org/10.1073/pnas.0809634105
Faham S, Bowie JU (2002) Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J Mol Biol 316(1):1–6. https://doi.org/10.1006/jmbi.2001.5295
Kim DM, Dikiy I, Upadhyay V, Posson DJ, Eliezer D, Nimigean CM (2016) Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles. J Gen Physiol 148(2):119–132. https://doi.org/10.1085/jgp.201611602
Dürr UH, Gildenberg M, Ramamoorthy A (2012) The magic of bicelles lights up membrane protein structure. Chem Rev 112(11):6054–6074. https://doi.org/10.1021/cr300061w
De Angelis AA, Opella SJ (2007) Bicelle samples for solid-state NMR of membrane proteins. Nat Protoc 2(10):2332–2338. https://doi.org/10.1038/nprot.2007.329
Opella SJ, Marassi FM (2004) Structure determination of membrane proteins by NMR spectroscopy. Chem Rev 104(8):3587–3606. https://doi.org/10.1021/cr0304121
Morrison EA, Henzler-Wildman KA (2012) Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile. Biochim Biophys Acta 1818(3):814–820. https://doi.org/10.1016/j.bbamem.2011.12.020
Bibow S, Hiller S (2018) A guide to quantifying membrane protein dynamics in lipids and other native-like environments by solution-state NMR spectroscopy. FEBS J 286:1610. https://doi.org/10.1111/febs.14639
Denisov IG, Grinkova YV, Lazarides AA, Sligar SG (2004) Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J Am Chem Soc 126(11):3477–3487. https://doi.org/10.1021/ja0393574
Banerjee S, Huber T, Sakmar TP (2008) Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles. J Mol Biol 377(4):1067–1081. https://doi.org/10.1016/j.jmb.2008.01.066
Jonas A, Kézdy KE, Wald JH (1989) Defined apolipoprotein A-I conformations in reconstituted high density lipoprotein discs. J Biol Chem 264(9):4818–4824
Davidson WS, Thompson TB (2007) The structure of apolipoprotein A-I in high density lipoproteins. J Biol Chem 282(31):22249–22253. https://doi.org/10.1074/jbc.R700014200
Leitz AJ, Bayburt TH, Barnakov AN, Springer BA, Sligar SG (2006) Functional reconstitution of Beta2-adrenergic receptors utilizing self-assembling Nanodisc technology. Biotechniques 40(5):601–602, 604, 606, passim. https://doi.org/10.2144/000112169
Grinkova YV, Denisov IG, Sligar SG (2010) Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Eng Des Sel 23(11):843–848. https://doi.org/10.1093/protein/gzq060
Ritchie TK, Grinkova YV, Bayburt TH, Denisov IG, Zolnerciks JK, Atkins WM, Sligar SG (2009) Chapter 11: Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464:211–231. https://doi.org/10.1016/S0076-6879(09)64011-8
Efremov RG, Gatsogiannis C, Raunser S (2017) Lipid nanodiscs as a tool for high-resolution structure determination of membrane proteins by single-particle cryo-EM. Methods Enzymol 594:1–30. https://doi.org/10.1016/bs.mie.2017.05.007
McLean MA, Gregory MC, Sligar SG (2018) Nanodiscs: a controlled bilayer surface for the study of membrane proteins. Annu Rev Biophys 47:107. https://doi.org/10.1146/annurev-biophys-070816-033620
Bibow S, Polyhach Y, Eichmann C, Chi CN, Kowal J, Albiez S, McLeod RA, Stahlberg H, Jeschke G, Güntert P, Riek R (2017) Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I. Nat Struct Mol Biol 24(2):187–193. https://doi.org/10.1038/nsmb.3345
Morgan CR, Hebling CM, Rand KD, Stafford DW, Jorgenson JW, Engen JR (2011) Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs. Mol Cell Proteomics 10(9):M111.010876. https://doi.org/10.1074/mcp.M111.010876
Schuler MA, Denisov IG, Sligar SG (2013) Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol 974:415–433. https://doi.org/10.1007/978-1-62703-275-9_18
Bayburt TH, Sligar SG (2010) Membrane protein assembly into Nanodiscs. FEBS Lett 584(9):1721–1727. https://doi.org/10.1016/j.febslet.2009.10.024
Koehl A, Hu H, Feng D, Sun B, Zhang Y, Robertson MJ, Chu M, Kobilka TS, Laermans T, Steyaert J, Tarrasch J, Dutta S, Fonseca R, Weis WI, Mathiesen JM, Skiniotis G, Kobilka BK (2019) Structural insights into the activation of metabotropic glutamate receptors. Nature 566(7742):79–84. https://doi.org/10.1038/s41586-019-0881-4
Rheinberger J, Gao X, Schmidpeter PA, Nimigean CM (2018) Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures. Elife 7. https://doi.org/10.7554/eLife.39775
Kalienkova V, Clerico Mosina V, Bryner L, Oostergetel GT, Dutzler R, Paulino C (2019) Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM. Elife 8. https://doi.org/10.7554/eLife.44364
Quentin D, Ahmad S, Shanthamoorthy P, Mougous JD, Whitney JC, Raunser S (2018) Mechanism of loading and translocation of type VI secretion system effector Tse6. Nat Microbiol 3(10):1142–1152. https://doi.org/10.1038/s41564-018-0238-z
Falzone ME, Rheinberger J, Lee BC, Peyear T, Sasset L, Raczkowski AM, Eng ET, Di Lorenzo A, Andersen OS, Nimigean CM, Accardi A (2019) Structural basis of Ca2+-dependent activation and lipid transport by a TMEM16 scramblase. Elife 8. https://doi.org/10.7554/eLife.43229
Bayburt TH, Sligar SG (2003) Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci 12(11):2476–2481. https://doi.org/10.1110/ps.03267503
Boldog T, Grimme S, Li M, Sligar SG, Hazelbauer GL (2006) Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties. Proc Natl Acad Sci U S A 103(31):11509–11514. https://doi.org/10.1073/pnas.0604988103
Li Y, Soubias O, Li J, Sun S, Randazzo PA, Byrd RA (2019) Functional expression and characterization of human myristoylated-Arf1 in nanodisc membrane mimetics. Biochemistry 58:1423. https://doi.org/10.1021/acs.biochem.8b01323
Tsukamoto H, Szundi I, Lewis JW, Farrens DL, Kliger DS (2011) Rhodopsin in nanodiscs has native membrane-like photointermediates. Biochemistry 50(22):5086–5091. https://doi.org/10.1021/bi200391a
Ranaghan MJ, Schwall CT, Alder NN, Birge RR (2011) Green proteorhodopsin reconstituted into nanoscale phospholipid bilayers (nanodiscs) as photoactive monomers. J Am Chem Soc 133(45):18318–18327. https://doi.org/10.1021/ja2070957
Mörs K, Roos C, Scholz F, Wachtveitl J, Dötsch V, Bernhard F, Glaubitz C (2013) Modified lipid and protein dynamics in nanodiscs. Biochim Biophys Acta 1828(4):1222–1229. https://doi.org/10.1016/j.bbamem.2012.12.011
Inagaki S, Ghirlando R, Grisshammer R (2013) Biophysical characterization of membrane proteins in nanodiscs. Methods 59(3):287–300. https://doi.org/10.1016/j.ymeth.2012.11.006
Bayburt TH, Grinkova YV, Sligar SG (2002) Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett 2(8):853–856. https://doi.org/10.1021/nl025623k
Hagn F, Nasr ML, Wagner G (2018) Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nat Protoc 13(1):79–98. https://doi.org/10.1038/nprot.2017.094
Mäler L, Gräslund A (2009) Artificial membrane models for the study of macromolecular delivery. Methods Mol Biol 480:129–139. https://doi.org/10.1007/978-1-59745-429-2_9
Hagn F, Etzkorn M, Raschle T, Wagner G (2013) Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. J Am Chem Soc 135(5):1919–1925. https://doi.org/10.1021/ja310901f
Nasr ML, Baptista D, Strauss M, Sun ZJ, Grigoriu S, Huser S, Plückthun A, Hagn F, Walz T, Hogle JM, Wagner G (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14(1):49–52. https://doi.org/10.1038/nmeth.4079
Yusuf Y, Massiot J, Chang YT, Wu PH, Yeh V, Kuo PC, Shiue J, Yu TY (2018) Optimization of the production of covalently circularized nanodiscs and their characterization in physiological conditions. Langmuir 34(11):3525–3532. https://doi.org/10.1021/acs.langmuir.8b00025
Miehling J, Goricanec D, Hagn F (2018) A split-intein-based method for the efficient production of circularized nanodiscs for structural studies of membrane proteins. Chembiochem 19(18):1927–1933. https://doi.org/10.1002/cbic.201800345
Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280(5360):69–77
Mazhab-Jafari MT, Marshall CB, Smith MJ, Gasmi-Seabrook GM, Stathopulos PB, Inagaki F, Kay LE, Neel BG, Ikura M (2015) Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site. Proc Natl Acad Sci U S A 112(21):6625–6630. https://doi.org/10.1073/pnas.1419895112
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612. https://doi.org/10.1002/jcc.20084
Acknowledgments
This work was funded by the National Institutes of Health (GM088352 and GM124451 to C.N.) and the American Heart Association (18POST33960309 to P.S.). We thank J. Rheinberger for providing the gel filtration profiles and the negative stain EM image in Fig. 3a, b.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Schmidpeter, P.A.M., Sukomon, N., Nimigean, C.M. (2020). Reconstitution of Membrane Proteins into Platforms Suitable for Biophysical and Structural Analyses. In: Perez, C., Maier, T. (eds) Expression, Purification, and Structural Biology of Membrane Proteins. Methods in Molecular Biology, vol 2127. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0373-4_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0373-4_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0372-7
Online ISBN: 978-1-0716-0373-4
eBook Packages: Springer Protocols