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Construction and characterization of a kappa opioid receptor devoid of all free cysteines


Ott, D; Frischknecht, R; Plückthun, A (2004). Construction and characterization of a kappa opioid receptor devoid of all free cysteines. Protein Engineering Design and Selection : PEDS, 17(1):37-48.

Abstract

We have constructed an optimized mutant of the kappa opioid receptor (KOR), which is devoid of its 10 free cysteines. It was necessary to test different amino acid replacements at various positions and we used a structural model and homology with other receptor family members as a guide. This mutant binds ligands and couples to the cognate G-proteins in a very similar fashion to wild-type KOR. The addition of the antagonist naloxone during cell growth greatly enhances heterogeneous expression of the mutant in mammalian cells, such that amounts similar to wild-type could be produced. We showed by fluorescence microscopy that naloxone stabilizes the mutant in the plasma membrane. This mutant, which now permits the insertion of single cysteines, was designed for use in spectroscopic studies of ligand-induced receptor conformational changes as well as to simplify folding studies.

Abstract

We have constructed an optimized mutant of the kappa opioid receptor (KOR), which is devoid of its 10 free cysteines. It was necessary to test different amino acid replacements at various positions and we used a structural model and homology with other receptor family members as a guide. This mutant binds ligands and couples to the cognate G-proteins in a very similar fashion to wild-type KOR. The addition of the antagonist naloxone during cell growth greatly enhances heterogeneous expression of the mutant in mammalian cells, such that amounts similar to wild-type could be produced. We showed by fluorescence microscopy that naloxone stabilizes the mutant in the plasma membrane. This mutant, which now permits the insertion of single cysteines, was designed for use in spectroscopic studies of ligand-induced receptor conformational changes as well as to simplify folding studies.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2004
Deposited On:22 Aug 2008 10:00
Last Modified:19 Feb 2018 20:29
Publisher:Oxford University Press
ISSN:1741-0126
OA Status:Closed
Publisher DOI:https://doi.org/10.1093/protein/gzh004
PubMed ID:14985536

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