The N-Terminal End Truncated Mu-Opioid Receptor: from Expression to Circular Dichroism Analysis

Abstract

In order to evaluate the biochemical, biophysical, and pharmacological implication of the N-terminal domain of the human mu-opioid receptor (HuMOR), deletion mutants lacking 64 amino acids from the amino terminus of HuMOR were constructed and expressed in the yeast Pichia pastoris. The recombinant proteins differed with respect to the presence of the Saccharomyces cerevisiae alpha-factor prepropeptide and the enhanced green fluorescent protein fused to the N terminus of the receptor. Pharmacological studies indicated that deletion of the N-terminal domain produced little effect on ligand affinities. The N-terminal end truncated and c-myc/6his-tagged receptor was subsequently purified to homogeneity and a yield of 5 mg/l was obtained after purification. The N-terminal end truncated receptor was further characterized by circular dichroism in trifluoroethanol and showed a characteristic pattern of alpha-helical structure. A pH effect on the structure of the receptor was observed when it was solubilized in sodium dodecyl sulfate micelles, with an increase of helicity at low pH.

A Computationally Designed Water-Soluble Variant of a G-Protein-Coupled Receptor: The Human Mu Opioid Receptor

“…Consistent with the secondary structure of the native human MUR expressed in yeast [48], [49], the CD spectra of the wsMUR-TM displayed a predominantly helical structure (48%) which is comparable with the native full length MUR [48]. Lower percentage of the helical content in the native full length MUR in the literature may be due to the inclusion of the N and C terminus and the higher concentration of the detergent (0.1% SDS).…”

Denatured G-Protein Coupled Receptors as Immunogens to Generate Highly Specific Antibodies

“…Contrasting with linear epitopes that are exposed on unfolded receptor, conformational antigenic determinants accessible on native receptor are usually lost upon denaturation. However, as we have previously shown for MOR [39], [40], SDS-solubilized GPCRs display true helical secondary structures. SDS-solubilized GPCRs are probably not fully unfolded, but rather partially pre-folded, at least as far as the secondary structure is considered [26].…”

Structure and dynamics changes induced by 2,2,2-trifluoro-ethanol (TFE) on the N-terminal half of hepatitis C virus core proteinThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

“…1C), and no structural changes were observed. Finally, to mimic a hydrophobic environment similar to the one found in the membrane (Muller et al 2009) or in proteinprotein interactions (Hua et al 1998;Moncoq et al 2004), we tested the ability of C82 to form secondary structures in the presence of TFE. Increasing amounts of TFE (from 0% to 90%) were tested to seek structural changes (Figs.…”

“…The relevance of TFE is multiple. First, TFE is a lipomimetic solvent (Muller et al 2009) and the HCV Core protein is known to interact with the ER membrane and lipid droplets (McLauchlan et al 2002). Second, TFE can mimic the environment found in protein-protein interactions (Hua et al 1998;Moncoq et al 2004) and HCV Core protein has several known protein partners.…”

“…Second, TFE can mimic the environment found in protein-protein interactions (Hua et al 1998;Moncoq et al 2004) and HCV Core protein has several known protein partners. Finally, TFE is widely used in structural studies to stabilize potent conformation (Muller et al 2009;Vamvakas et al 2009). In this context, we show that C82 can fold with the addition of TFE, going from a highly flexible unfolded form to a more rigid a-helical conformation.…”