Journal of Membrane Biology

, Volume 223, Issue 1, pp 49–57 | Cite as

The Full-Length Mu-Opioid Receptor: A Conformational Study by Circular Dichroism in Trifluoroethanol and Membrane-Mimetic Environments

  • Isabelle Muller
  • Valérie Sarramégna
  • Marie Renault
  • Vincent Lafaquière
  • Sarra Sebai
  • Alain Milon
  • Franck Talmont


The secondary structure content of the recombinant human mu-opioid receptor (HuMOR) solubilized in trifluoroethanol (TFE) and in detergent micelles was investigated by circular dichroism. In both conditions, this G protein–coupled receptor adopts a characteristic α-helical structure, with minima at 208 and 222 nm as observed in the circular dichroism spectra. After deconvolution of spectra, the α-helix contents were estimated to be in the range of 50% in TFE and in sodium dodecyl sulfate at pH 6. These values are in accordance with the predicted secondary structure content determined for the mu-opioid receptor. A pH-dependent effect was observed on the secondary structure of the receptor solubilized in detergents, which demonstrates the essential role of ionic and hydrophobic interactions on the secondary structure. Circular dichroism spectra of EGFP–HuMOR, a fusion protein between the enhanced green fluorescent protein (EGFP) and the mu-opioid receptor, and EGFP solubilized in TFE were also analyzed as part of this study.


G protein–coupled receptor Mu-opioid receptor Circular dichroism Detergent Trifluoroethanol Folding 



We are grateful to Dr. Monique Erard and Dr. Magali Blaud for their help in the circular dichroism experiments and to Dr. Virginie Gervais for the utilization of the PROMOTIF program. This work was supported by the Centre National de la Recherche Scientifique and by the University Paul Sabatier (Toulouse III).


  1. Arevalo E, Estephan R, Madeo J, Arshava B, Dumont M, Becker JM, Naider F (2003) Biosynthesis and biophysical analysis of domains of a yeast G protein–coupled receptor. Biopolymers 71:516–531PubMedCrossRefGoogle Scholar
  2. Arunkumar AI, Kumar TK, Jayaraman G, Samuel D, Yu C (1996) Induction of helical conformation in all beta-sheet proteins by trifluoroethanol. J Biomol Struct Dyn 14:381–385PubMedGoogle Scholar
  3. Bane SE, Velasquez JE, Robinson AS (2007) Expression and purification of milligram levels of inactive G protein–coupled receptors in E. coli. Protein Expr Purif 52:348–355CrossRefGoogle Scholar
  4. Baneres JL, Martin A, Hullot P, Girard JP, Rossi JC, Parello J (2003) Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1. J Mol Biol 329:801–814PubMedCrossRefGoogle Scholar
  5. Baneres JL, Mesnier D, Martin A, Joubert L, Dumuis A, Bockaert J (2005) Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy. J Biol Chem 280:20253–20260PubMedCrossRefGoogle Scholar
  6. Booth PJ (2003) The trials and tribulations of membrane protein folding in vitro. Biochim Biophys Acta 1610:51–56PubMedCrossRefGoogle Scholar
  7. Buck M (1998) Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q Rev Biophys 31:297–355PubMedCrossRefGoogle Scholar
  8. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein–coupled receptor. Science 318:1258–1265PubMedCrossRefGoogle Scholar
  9. Choi G, Guo J, Makriyannis A (2005) The conformation of the cytoplasmic helix 8 of the CB1 cannabinoid receptor using NMR and circular dichroism. Biochim Biophys Acta 1668:1–9PubMedCrossRefGoogle Scholar
  10. Choi G, Landin J, Xie XQ (2002) The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and 1H NMR spectroscopy in aqueous and membrane-like environments. J Pept Res 60:169–177PubMedCrossRefGoogle Scholar
  11. Ding FX, Schreiber D, VerBerkmoes NC, Becker JM, Naider F (2002) The chain length dependence of helix formation of the second transmembrane domain of a G protein–coupled receptor of Saccharomyces cerevisiae. J Biol Chem 277:14483–14492PubMedCrossRefGoogle Scholar
  12. Feng GJ, Kellett E, Scorer CA, Wilde J, White JH, Milligan G (2003) Selective interactions between helix VIII of the human mu-opioid receptors and the C terminus of periplakin disrupt G protein activation. J Biol Chem 278:33400–33407PubMedCrossRefGoogle Scholar
  13. Fowler CB, Pogozheva ID, LeVine H 3rd, Mosberg HI (2004a) Refinement of a homology model of the mu-opioid receptor using distance constraints from intrinsic and engineered zinc-binding sites. Biochemistry 43:8700–8710PubMedCrossRefGoogle Scholar
  14. Fowler CB, Pogozheva ID, Lomize AL, LeVine H 3rd, Mosberg HI (2004b) Complex of an active mu-opioid receptor with a cyclic peptide agonist modeled from experimental constraints. Biochemistry 43:15796–15810PubMedCrossRefGoogle Scholar
  15. Fraser NJ (2006) Expression and functional purification of a glycosylation deficient version of the human adenosine 2a receptor for structural studies. Protein Expr Purif 49:129–137PubMedCrossRefGoogle Scholar
  16. Hamed MM, Robinson RM, Mattice WL (1983) Helix formation upon acidification of protein-dodecyl sulfate complexes. Biochim Biophys Acta 743:260–267PubMedGoogle Scholar
  17. Hutchinson EG, Thornton JM (1996) PROMOTIF—a program to identify and analyze structural motifs in proteins. Protein Sci 5:212–220PubMedCrossRefGoogle Scholar
  18. Johnson WC (1999) Analyzing protein circular dichroism spectra for accurate secondary structures. Proteins 35:307–312PubMedCrossRefGoogle Scholar
  19. Kerman A, Ananthanarayanan VS (2005) Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor. Biochim Biophys Acta 1747:133–140PubMedGoogle Scholar
  20. Kerman A, Ananthanarayanan VS (2007) Conformation of a double-membrane-spanning fragment of a G protein–coupled receptor: effects of hydrophobic environment and pH. Biochim Biophys Acta 1768:1199–1210PubMedCrossRefGoogle Scholar
  21. Kiefer H (2003) In vitro folding of alpha-helical membrane proteins. Biochim Biophys Acta 1610:57–62PubMedCrossRefGoogle Scholar
  22. Kiefer H, Krieger J, Olszewski JD, Von Heijne G, Prestwich GD, Breer H (1996) Expression of an olfactory receptor in Escherichia coli: purification, reconstitution, and ligand binding. Biochemistry 35:16077–16084PubMedCrossRefGoogle Scholar
  23. Lazarova T, Brewin KA, Stoeber K, Robinson CR (2004) Characterization of peptides corresponding to the seven transmembrane domains of human adenosine A2a receptor. Biochemistry 43:12945–12954PubMedCrossRefGoogle Scholar
  24. Lin S, Gether U, Kobilka BK (1996) Ligand stabilization of the beta2 adrenergic receptor: effect of DTT on receptor conformation monitored by circular dichroism and fluorescence spectroscopy. Biochemistry 35:14445–14451PubMedCrossRefGoogle Scholar
  25. Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383:819–823PubMedCrossRefGoogle Scholar
  26. Molina PE (2006) Opioids and opiates: analgesia with cardiovascular, haemodynamic and immune implications in critical illness. J Intern Med 259:138–154PubMedCrossRefGoogle Scholar
  27. Montserret R, McLeish MJ, Bockmann A, Geourjon C, Penin F (2000) Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding. Biochemistry 39:8362–8373PubMedCrossRefGoogle Scholar
  28. O’Malley MA, Lazarova T, Britton ZT, Robinson AS (2007) High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor. J Struct Biol 159:166–178PubMedCrossRefGoogle Scholar
  29. Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395PubMedCrossRefGoogle Scholar
  30. Palchevskyy SS, Posokhov YO, Olivier B, Popot JL, Pucci B, Ladokhin AS (2006) Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin. Biochemistry 45:2629–2635PubMedCrossRefGoogle Scholar
  31. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M (2000) Crystal structure of rhodopsin: A G protein–coupled receptor. Science 289:739–745PubMedCrossRefGoogle Scholar
  32. Palm GJ, Zdanov A, Gaitanaris GA, Stauber R, Pavlakis GN, Wlodawer A (1997) The structural basis for spectral variations in green fluorescent protein. Nat Struct Biol 4:361–365PubMedCrossRefGoogle Scholar
  33. Park K, Perczel A, Fasman GD (1992) Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins. Protein Sci 1:1032–1049PubMedCrossRefGoogle Scholar
  34. Peterson GL, Toumadje A, Johnson WC Jr, Schimerlik MI (1995) Purification of recombinant porcine m2 muscarinic acetylcholine receptor from Chinese hamster ovary cells. Circular dichroism spectra and ligand binding properties. J Biol Chem 270:17808–17814PubMedCrossRefGoogle Scholar
  35. Phillips GNJ (2006) The three-dimensional structure of green fluorescent protein and its implication for function and design. John Wiley, Hoboken, NJGoogle Scholar
  36. Popot JL, Berry EA, Charvolin D, Creuzenet C, Ebel C, Engelman DM, Flotenmeyer M, Giusti F, Gohon Y, Hong Q, Lakey JH, Leonard K, Shuman HA, Timmins P, Warschawski DE, Zito F, Zoonens M, Pucci B, Tribet C (2003) Amphipols: polymeric surfactants for membrane biology research. Cell Mol Life Sci 60:1559–1574PubMedCrossRefGoogle Scholar
  37. Popot JL, Engelman DM (1990) Membrane protein folding and oligomerization: the two-stage model. Biochemistry 29:4031–4037PubMedCrossRefGoogle Scholar
  38. Provencher SW, Glockner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20:33–37PubMedCrossRefGoogle Scholar
  39. 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 450:383–387PubMedCrossRefGoogle Scholar
  40. Rigby AC, Grant CW, Shaw GS (1998) Solution and solid state conformation of the human EGF receptor transmembrane region. Biochim Biophys Acta 1371:241–253PubMedCrossRefGoogle Scholar
  41. Sarramegna V, Demange P, Milon A, Talmont F (2002a) Optimizing functional versus total expression of the human mu-opioid receptor in Pichia pastoris. Protein Expr Purif 24:212–220PubMedCrossRefGoogle Scholar
  42. Sarramegna V, Talmont F, Seree de Roch M, Milon A, Demange P (2002b) Green fluorescent protein as a reporter of human mu-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris. J Biotechnol 99:23–39PubMedCrossRefGoogle Scholar
  43. Sarramegna V, Talmont F, Demange P, Milon A (2003) Heterologous expression of G-protein-coupled receptors: comparison of expression systems fron the standpoint of large-scale production and purification. Cell Mol Life Sci 60:1529–1546PubMedCrossRefGoogle Scholar
  44. Sarramegna V, Muller I, Mousseau G, Froment C, Monsarrat B, Milon A, Talmont F (2005) Solubilization, purification, and mass spectrometry analysis of the human mu-opioid receptor expressed in Pichia pastoris. Protein Expr Purif 43:85–93PubMedCrossRefGoogle Scholar
  45. Sarramegna V, Muller I, Milon A, Talmont F (2006) Recombinant G protein–coupled receptors from expression to renaturation: a challenge towards structure. Cell Mol Life Sci 63:1149–1164CrossRefGoogle Scholar
  46. Schievano E, Calisti T, Menegazzo I, Battistutta R, Peggion E, Mammi S, Palu G, Loregian A (2004) pH-dependent conformational changes and topology of a herpesvirus translocating peptide in a membrane-mimetic environment. Biochemistry 43:9343–9351PubMedCrossRefGoogle Scholar
  47. Schlyer S, Horuk R (2006) I want a new drug: G-protein-coupled receptors in drug development. Drug Discov Today 11:481–493PubMedCrossRefGoogle Scholar
  48. Sreerama N, Venyaminov SY, Woody RW (1999) Estimation of the number of alphahelical and betastrand segments in proteins using CD spectroscopy. Protein Sci 8:370–380PubMedGoogle Scholar
  49. Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287:252–260PubMedCrossRefGoogle Scholar
  50. Sreerama N, Woody RW (2004) On the analysis of membrane protein circular dichroism spectra. Protein Sci 13:100–112PubMedCrossRefGoogle Scholar
  51. Stevens TJ, Arkin IT (2000) Do more complex organisms have a greater proportion of membrane proteins in their genomes? Proteins 39:417–420PubMedCrossRefGoogle Scholar
  52. Thevenin D, Lazarova T, Roberts MF, Robinson CR (2005) Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor. Protein Sci 14:2177–2186PubMedCrossRefGoogle Scholar
  53. Tyndall JD, Sandilya R (2005) GPCR agonists and antagonists in the clinic. Med Chem 1:405–421PubMedCrossRefGoogle Scholar
  54. Venyaminov S, Yang J (1996) Determination of protein secondary structure. Plenum Press, New YorkGoogle Scholar
  55. Visser NV, Hink MA, Borst JW, van der Krogt GN, Visser AJ (2002) Circular dichroism spectroscopy of fluorescent proteins. FEBS Lett 521:31–35PubMedCrossRefGoogle Scholar
  56. Wu CS, Ikeda K, Yang JT (1981) Ordered conformation of polypeptides and proteins in acidic dodecyl sulfate solution. Biochemistry 20:566–570PubMedCrossRefGoogle Scholar
  57. Wu CS, Yang JT (1978) Conformation of naturally-occurring peptides in surfactant solution: its relation to the structure-forming potential of amino acid sequence. Biochem Biophys Res Commun 82:85–91PubMedCrossRefGoogle Scholar
  58. Xie XQ, Zhao J, Zheng H (2004) Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180–233). Protein Expr Purif 38:61–68PubMedCrossRefGoogle Scholar
  59. Yang F, Moss LG, Phillips GN Jr (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14:1246–1251PubMedCrossRefGoogle Scholar
  60. Zhang Y, Devries ME, Skolnick J (2006) Structure modeling of all identified G protein–coupled receptors in the human genome. PLoS Comput Biol 2:e13PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Isabelle Muller
    • 1
  • Valérie Sarramégna
    • 1
    • 2
  • Marie Renault
    • 1
  • Vincent Lafaquière
    • 1
  • Sarra Sebai
    • 1
  • Alain Milon
    • 1
  • Franck Talmont
    • 1
  1. 1.Institut de Pharmacologie et de Biologie Structurale (UMR 5089)Centre National de la Recherche Scientifique, Université de ToulouseCedex 4France
  2. 2.Unité de Recherche Mécanismes Adaptatifs et Biomolécules des plantes endémiques de Mélanésie (MABIOM)Université de la Nouvelle CalédonieCedexNew Caledonia

Personalised recommendations