title: Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration.
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.
authors.affiliation: Institute for Medical Physics and Biophysics, Medical Department, Universität Leipzig, Härtelstrasse 16-18, D-04107 Leipzig, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Witte
authors.forename: Anette
authors.initials: A
authors.lastname: Kaiser
authors.forename: Peter
authors.initials: P
authors.lastname: Schmidt
authors.forename: Victoria
authors.initials: V
authors.lastname: Splith
authors.forename: Lars
authors.initials: L
authors.lastname: Thomas
authors.forename: Sandra
authors.initials: S
authors.lastname: Berndt
authors.forename: Daniel
authors.initials: D
authors.lastname: Huster
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
mesh_terms: D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017354:Point Mutation; D017510:Protein Folding; D055550:Protein Stability; D017476:Receptors, Neuropeptide Y / Q000737:chemistry* / Q000235:genetics
chemical_list: D017476:Receptors, Neuropeptide Y; C095889:neuropeptide Y2 receptor; D003545:Cysteine