Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor.
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
A
Department of Biochemistry, HSC 4H25, McMaster University, Hamilton, Ontario, Canada L8N 3Z5.
Ananthanarayanan
Vettai S
VS
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D017450:Receptors, Opioid, mu / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D010446:Peptide Fragments; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase