Abstract
Nanodiscs are self-assembled discoidal fragments of lipid bilayers 8–16 nm in diameter, stabilized in solution by two amphipathic helical scaffold proteins. As stable and highly soluble membrane mimetics with controlled lipid composition and ability to add affinity tags to the scaffold protein, nanodiscs represent an attractive model system for solubilization, isolation, purification, and biophysical and biochemical studies of membrane proteins. In this chapter we overview various approaches to structural and functional studies of different classes of integral membrane proteins such as ion channels, transporters, GPCR and other receptors, membrane enzymes, and blood coagulation cascade proteins which have been incorporated into nanodiscs. We outline the advantages provided by homogeneity, ability to control oligomerization state of the target protein and lipid composition of the bilayer. Special attention is paid to the opportunities afforded by nanodisc system for the detailed studies of the role of different lipid properties and protein–lipid interactions in the functional behavior of membrane proteins.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Jonas A, Kezdy KE, Wald JH (1989) Defined apolipoprotein A-I conformations in reconstituted high density lipoprotein discs. J Biol Chem 264:4818–4824
Wald JH, Krul ES, Jonas A (1990) Structure of apolipoprotein A-I in three homogeneous, reconstituted high density lipoprotein particles. J Biol Chem 265:20037–20043
Carlson JW, Jonas A, Sligar SG (1997) Imaging and manipulation of high-density lipoproteins. Biophys J 73:1184–1189
Beck von Bodman S et al (1986) Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5. Proc Natl Acad Sci U S A 83:9443–9447
Bayburt TH, Carlson JW, Sligar SG (2000) Single molecule height measurements on a membrane protein in nanometer-scale phospholipid bilayer disks. Langmuir 16:5993–5997
Bayburt TH, Sligar SG (2002) Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks. Proc Natl Acad Sci U S A 99:6725–6730
Bayburt TH, Grinkova YV, Sligar SG (2002) Self-assembly of discoidal phospholipid bilayer nanoparticles with membrane scaffold proteins. Nano Lett 2:853–856
Bayburt TH, Sligar SG (2003) Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers. Protein Sci 12:2476–2481
Civjan NR et al (2003) Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers. Biotechniques 35:556–558, 560, 562–563
Denisov IG et al (2004) Directed Self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size. J Am Chem Soc 126:3477–3487
Li Y et al (2006) Structural analysis of nanoscale self-assembled discoidal lipid bilayers by solid-state NMR spectroscopy. Biophys J 91:3819–3828
Phillips JC et al (1997) Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks. Biophys J 73:2337–2346
Wlodawer A et al (1979) High-density lipoprotein recombinants: evidence for a bicycle tire micelle structure obtained by neutron scattering and electron microscopy. FEBS Lett 104:231–235
Shih AY et al (2005) Molecular dynamics simulations of discoidal bilayers assembled from truncated human lipoproteins. Biophys J 88:548–556
Grinkova YV, Denisov IG, Sligar SG (2010) Engineering extended membrane scaffold proteins for self-assembly of soluble nanoscale lipid bilayers. Protein Eng Des Sel 23:843–848
Mendelsohn R et al (1989) Quantitative determination of conformational disorder in the acyl chains of phospholipid bilayers by infrared spectroscopy. Biochemistry 28:8934–8939
Lins L et al (1993) Structure of the apolipoprotein A-IV/lipid discoidal complexes: an attenuated total reflection polarized Fourier transform infrared spectroscopy study. Biochim Biophys Acta 1149:267–277
Nagle JF, Tristram-Nagle S (2000) Structure of lipid bilayers. Biochim Biophys Acta 1469:159–195
Tristram-Nagle S, Nagle JF (2004) Lipid bilayers: thermodynamics, structure, fluctuations, and interactions. Chem Phys Lipids 127:3–14
Denisov IG et al (2005) Thermotropic phase transition in soluble nanoscale lipid bilayers. J Phys Chem B 109:15580–15588
Shaw AW, McLean MA, Sligar SG (2004) Phospholipid phase transitions in homogeneous nanometer scale bilayer discs. FEBS Lett 556:260–264
Gennis RB (1989) Biomembranes. Molecular structure and function. Springer, New York
Marsh D (2008) Protein modulation of lipids, and vice-versa, in membranes. Biochim Biophys Acta 1778:1545–1575
Marsh D (2010) Electron spin resonance in membrane research: protein-lipid interactions from challenging beginnings to state of the art. Eur Biophys J 39:513–525
Simons K, Gerl MJ (2010) Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 11:688–699
He HT, Marguet D (2011) Detecting nanodomains in living cell membrane by fluorescence correlation spectroscopy. Annu Rev Phys Chem 62:417–436
Needham D, Evans E (1988) Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 degrees C below to 10 degrees C above the liquid crystal-crystalline phase transition at 24 degrees C. Biochemistry 27:8261–8269
Cevc G (ed) (1993) Phospholipids handbook. Marcel Dekker, Inc, New York
Bayburt TH, Sligar SG (2010) Membrane protein assembly into nanodisks. FEBS Lett 584:1721–1727
Leitz AJ et al (2006) Functional reconstitution of β2-adrenergic receptors utilizing self-assembling nanodisc technology. Biotechniques 40:601–602, 604, 606, 608, 610, 612
Whorton MR et al (2007) A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc Natl Acad Sci U S A 104:7682–7687
Whorton MR et al (2008) Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer. J Biol Chem 283:4387–4394
Bayburt TH et al (2011) Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding. J Biol Chem 286:1420–1428
Knepp AM et al (2011) Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands. Biochemistry 50:502–511
Goldsmith BR et al (2011) Biomimetic chemical sensors using nanoelectronic readout of olfactory receptor proteins. ACS Nano 5:5408–5416
Raschle T et al (2009) Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer Nanodiscs. J Am Chem Soc 131:17777–17779
Yu T-Y et al (2012) Solution NMR spectroscopic characterization of human VDAC-2 in detergent micelles and lipid bilayer nanodiscs. Biochim Biophys Acta. doi:10.1016/j.bbamem.2011.11.012
Shenkarev ZO et al (2010) Lipid-protein nanodiscs as reference medium in detergent screening for high-resolution NMR studies of integral membrane proteins. J Am Chem Soc 132:5628–5629
Shenkarev ZO et al (2010) NMR structural and dynamical investigation of the isolated voltage-sensing domain of the potassium channel KvAP: implications for voltage gating. J Am Chem Soc 132:5630–5637
Alami M et al (2007) Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA. EMBO J 26:1995–2004
Dalal K, Duong F (2010) Reconstitution of the SecY translocon in nanodiscs. Methods Mol Biol 619:145–156
Dalal K et al (2009) Structure, binding, and activity of Syd, a SecY-interacting protein. J Biol Chem 284:7897–7902
Kawai T et al (2011) Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer. FEBS Lett 585:3533–3537
Ritchie TK, Kwon H, Atkins WM (2011) Conformational analysis of human ATP-binding cassette transporter ABCB1 in lipid nanodiscs and inhibition by the antibodies MRK16 and UIC2. J Biol Chem 286:39489–39496
Alvarez FJ, Orelle C, Davidson AL (2010) Functional reconstitution of an ABC transporter in nanodiscs for use in electron paramagnetic resonance spectroscopy. J Am Chem Soc 132:9513–9515
Dalal K, Duong F (2011) The SecY complex: conducting the orchestra of protein translocation. Trends Cell Biol 21:506–514
Katayama H et al (2010) Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles. Proc Natl Acad Sci U S A 107:3453–3457, S3453/3451-S3453/3453
Boldog T et al (2006) Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties. Proc Natl Acad Sci U S A 103:11509–11514
Li M, Hazelbauer GL (2011) Core unit of chemotaxis signaling complexes. Proc Natl Acad Sci U S A 108:9390–9395
Glueck JM, Koenig BW, Willbold D (2011) Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies. Anal Biochem 408:46–52
Mi L-Z et al (2008) Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs. Biochemistry 47:10314–10323
Näsvik Öjemyr L et al (2012) Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer. FEBS Lett 586(5):640–645
Bayburt TH et al (2007) Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins. J Biol Chem 282:14875–14881
Ranaghan MJ et al (2011) Green proteorhodopsin reconstituted into nanoscale phospholipid bilayers (nanodiscs) as photoactive monomers. J Am Chem Soc 133:18318–18327
Boldog T, Li M, Hazelbauer GL (2007) Using nanodiscs to create water-soluble transmembrane chemoreceptors inserted in lipid bilayers. Methods Enzymol 423:317–335
Zhang XX et al (2012) Nanodiscs and SILAC-based mass spectrometry to identify a membrane protein interactome. J Proteome Res 11:1454–1459
Baas BJ, Denisov IG, Sligar SG (2004) Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment. Arch Biochem Biophys 430:218–228
Denisov IG, Sligar SG (2011) Cytochromes P450 in nanodiscs. Biochim Biophys Acta 1814:223–229
Duan H et al (2004) Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers. Arch Biochem Biophys 424:141–153
Duan H, Schuler MA (2006) Heterologous expression and strategies for encapsulation of membrane-localized plant P450s. Phytochem Rev 5:507–523
Frank DJ, Denisov IG, Sligar SG (2011) Analysis of heterotropic cooperativity in cytochrome P450 3A4 using α-naphthoflavone and testosterone. J Biol Chem 286:5540–5545
Grinkova YV, Denisov IG, Sligar SG (2010) Functional reconstitution of monomeric CYP3A4 with multiple cytochrome P450 reductase molecules in nanodiscs. Biochem Biophys Res Commun 398:194–198
Grinkova YV et al (2008) The ferrous-oxy complex of human aromatase. Biochem Biophys Res Commun 372:379–382
Morrissey JH et al (2008) Blood clotting reactions on nanoscale phospholipid bilayers. Thromb Res 122:S23–S26
Jonas A, Krajnovich DJ (1978) Effect of cholesterol on the formation of micellar complexes between bovine A-I apolipoprotein and L-alpha-dimyristoyl-phosphatidylcholine. J Biol Chem 253:5758–5763
Shaw AW et al (2007) The local phospholipid environment modulates the activation of blood clotting. J Biol Chem 282:6556–6563
Jonas A, Maine GT (1979) Kinetics and mechanism of phosphatidylcholine and cholesterol exchange between single bilayer vesicles and bovine serum high-density lipoprotein. Biochemistry 18:1722–1728
Bayburt TH, Grinkova YV, Sligar SG (2006) Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs. Arch Biochem Biophys 450:215–222
Borch J, Roepstorff P, Moeller-Jensen J (2011) Nanodisc-based co-immunoprecipitation for mass spectrometric identification of membrane-interacting proteins. Mol Cell Proteomics 10:O110 006775, 006779
Borch J et al (2008) Nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor. Anal Chem 80:6245–6252
Jonas A (1976) Microviscosity of lipid domains in human serum lipoproteins. Biochim Biophys Acta 486:10–22
Shenkarev ZO et al (2009) Lipid-protein nanodiscs: possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides. Biochemistry (Mosc) 74:756–765
Zocher M et al (2012) Single-molecule force spectroscopy from nanodiscs: an assay to quantify folding, stability, and interactions of native membrane proteins. ACS Nano 6:961–971
Morrissey JH et al (2009) Protein-membrane interactions: blood clotting on nanoscale bilayers. J Thromb Haemost 7:169–172
Kobashigawa Y et al (2011) Phosphoinositide-incorporated lipid-protein nanodiscs: a tool for studying protein-lipid interactions. Anal Biochem 410:77–83
Boettcher JM et al (2011) Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers. Biochemistry 50:2264–2273
Morrissey JH et al (2010) Protein-phospholipid interactions in blood clotting. Thromb Res 125:S23–S25
Lambert MP et al (1998) Diffusible, nonfibrillar ligands derived from Abeta42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 95:6448–6453
Wilcox K et al (2011) Abeta oligomer-induced synapse degeneration in Alzheimer's disease. Cell Mol Neurobiol 31:939–948
Jonas A (1986) Reconstitution of high-density lipoproteins. Methods Enzymol 128:553–582
Jonas A, Jung RW (1975) Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements. Biochem Biophys Res Commun 66:651–657
Matz CE, Jonas A (1982) Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions. J Biol Chem 257:4535–4540
Marty MT, Das A, Sligar SG (2012) Ultra-thin layer MALDI mass spectrometry of membrane proteins in nanodiscs. Anal Bioanal Chem 402:721–729
Das A, Grinkova YV, Sligar SG (2007) Redox potential control by drug binding to cytochrome P 450 3A4. J Am Chem Soc 129:13778–13779
Das A, Sligar SG (2009) Modulation of the cytochrome P450 reductase redox potential by the phospholipid bilayer. Biochemistry 48:12104–12112
Jonas A, Hesterberg LK, Drengler SM (1978) Incorporation of excess cholesterol by high density serum lipoproteins. Biochim Biophys Acta 528:47–57
Raschle T et al (2010) Nonmicellar systems for solution NMR spectroscopy of membrane proteins. Curr Opin Struct Biol 20:471–479
Serebryany E, Zhu GA, Yan ECY (2012) Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors. Biochim Biophys Acta 1818:225–233
Acknowledgments
Our work is supported by grants from the National Institutes of Health GM33775 and GM31756.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Schuler, M.A., Denisov, I.G., Sligar, S.G. (2013). Nanodiscs as a New Tool to Examine Lipid–Protein Interactions. In: Kleinschmidt, J. (eds) Lipid-Protein Interactions. Methods in Molecular Biology, vol 974. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-275-9_18
Download citation
DOI: https://doi.org/10.1007/978-1-62703-275-9_18
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-274-2
Online ISBN: 978-1-62703-275-9
eBook Packages: Springer Protocols