Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support
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Le résumé fourni par la source
The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40-70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.
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Le contrôle bibliographique ouvert
DOI retrouvé dans Crossref DOI retrouvé ; titre concordant.
- Titre Crossref
- Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support
- Date Crossref
- 16/03/2016
- Éditeur
- Public Library of Science (PLoS)
- Type
- journal-article
Ce recoupement confirme des métadonnées liées au DOI. Il ne confirme ni la méthode ni les conclusions de l’étude, et il ne compte pas comme une seconde source scientifique indépendante.
Où se fait cette recherche
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University of Bristol pays non établi dans la noticeUniversité ou école supérieure
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University of Strathclyde Organisational and Staff Development Unit pays non établi dans la noticeUniversité ou école supérieure
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MRC Laboratory of Molecular Biology pays non établi dans la noticeStructure de recherche
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Paul Scherrer Institute pays non établi dans la noticeStructure de recherche
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School of Biochemistry pays non établi dans la noticeUniversité ou école supérieure
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Laboratory of Biomolecular Research pays non établi dans la noticeStructure de recherche
University of Bristol, Organisational and Staff Development Unit — University of Strathclyde et MRC Laboratory of Molecular Biology, avec 3 autres affiliations.
Une affiliation ne permet pas de déduire la nationalité d’un auteur.