Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration
-
Kristina Witte
, Anette Kaiser , Peter Schmidt, Victoria Splith
, Lars Thomas , Sandra Berndt , Daniel Huster und Annette G. Beck-Sickinger
Abstract
In vitro folding of G protein-coupled receptors into a detergent environment represents a promising strategy for obtaining sufficient amounts of functional receptor molecules for structural studies. Typically, these preparations exhibit a poor long-term stability especially at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequently folded neuropeptide Y receptor type 2. We identified the free cysteines in the receptor as one major reason for intermolecular protein aggregation. Therefore, six out of the eight cysteine residues were mutated to alanine or serine without any significant loss of functionality of the receptor as demonstrated in cell culture models. Furthermore, the disulfide bond between the remaining two cysteines was irreversibly formed by applying oxidative in vitro folding. Applying this strategy, the stability of the functionally folded Y2 receptor could be increased to 20 days at a concentration of 15 μm in a micelle environment consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine and n-dodecyl-ß-D-maltoside.
The authors thank Stefanie Babilon for assistance with receptor solubilization and analysis from eukaryotic cells. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 610, Projects A1 and A14) and by the ‘Nachwuchsförderung’ of the Medical Department, University of Leipzig.
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Artikel in diesem Heft
- Masthead
- Masthead
- Guest Editorial
- Highlight: Protein states with cell biological and medicinal relevance
- HIGHLIGHT: PROTEIN STATES WITH CELL BIOLOGICAL AND MEDICAL RELEVANCE
- Towards improved receptor targeting: anterograde transport, internalization and postendocytic trafficking of neuropeptide Y receptors
- Progress in demystification of adhesion G protein-coupled receptors
- The unresolved puzzle why alanine extensions cause disease
- Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD
- Structure and allosteric regulation of eukaryotic 6-phosphofructokinases
- Polyionic and cysteine-containing fusion peptides as versatile protein tags
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- Lysine-specific histone demethylase LSD1 and the dynamic control of chromatin
- Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why
- Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration
- Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts
- In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain
- Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains
- High level expression of the Drosophila Toll receptor ectodomain and crystallization of its complex with the morphogen Spätzle
Artikel in diesem Heft
- Masthead
- Masthead
- Guest Editorial
- Highlight: Protein states with cell biological and medicinal relevance
- HIGHLIGHT: PROTEIN STATES WITH CELL BIOLOGICAL AND MEDICAL RELEVANCE
- Towards improved receptor targeting: anterograde transport, internalization and postendocytic trafficking of neuropeptide Y receptors
- Progress in demystification of adhesion G protein-coupled receptors
- The unresolved puzzle why alanine extensions cause disease
- Molecular function of the prolyl cis/trans isomerase and metallochaperone SlyD
- Structure and allosteric regulation of eukaryotic 6-phosphofructokinases
- Polyionic and cysteine-containing fusion peptides as versatile protein tags
- p0071/PKP4, a multifunctional protein coordinating cell adhesion with cytoskeletal organization
- Lysine-specific histone demethylase LSD1 and the dynamic control of chromatin
- Methylation of the nuclear poly(A)-binding protein by type I protein arginine methyltransferases – how and why
- Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration
- Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts
- In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain
- Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains
- High level expression of the Drosophila Toll receptor ectodomain and crystallization of its complex with the morphogen Spätzle