Expression and spectroscopic characterization of a large fragment of the μ-opioid receptor Academic Article
We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human mu-opioid receptor. The fragment ('TM2-3'), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2-3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2-3 in lysophosphatidylcholine micelles showed that TM2-3 adopts approximately 50% alpha-helical structure in this environment, with the remainder consisting of disordered and/or beta-structure. This is consistent with the assumption of an alpha-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2-3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the mu-opioid receptor and, possibly, other G-protein-coupled receptors as well.
Kerman, Aaron
Ananthanarayanan, Vettai
Amino Acid Sequence (MeSH)
Biochemistry & Molecular Biology (Science Metrix)
Biophysics (Science Metrix)
Circular Dichroism (MeSH)
Escherichia coli (MeSH)
Glutathione Transferase (MeSH)
Inclusion Bodies (MeSH)
Peptide Fragments (MeSH)
Protein Structure, Secondary (MeSH)
Receptors, Opioid, mu (MeSH)
Recombinant Fusion Proteins (MeSH)
Biochimica et Biophysica Acta - Proteins and Proteomics Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology Journal
Circular Dichroism
Glutathione Transferase
Inclusion Bodies
Protein Structure, Secondary
Receptors, Opioid, mu
Recombinant Fusion Proteins
10.1016/j.bbapap.2004.10.009