The Full-Length Mu-Opioid Receptor: A Conformational Study by Circular Dichroism in Trifluoroethanol and Membrane-Mimetic Environments
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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.
KeywordsG 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).
- 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
- Phillips GNJ (2006) The three-dimensional structure of green fluorescent protein and its implication for function and design. John Wiley, Hoboken, NJGoogle Scholar
- 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
- Venyaminov S, Yang J (1996) Determination of protein secondary structure. Plenum Press, New YorkGoogle Scholar