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Beta-Barrel scaffolds for the grafting of extracellular loops from G- protein coupled receptors


Walser, R; Kleinschmidt, J H; Skerra, A; Zerbe, O (2012). Beta-Barrel scaffolds for the grafting of extracellular loops from G- protein coupled receptors. Biological Chemistry, 393(11):1341-1355.

Abstract

Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) β-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural systems.

Abstract

Owing to the difficulties in production and purification of G-protein-coupled receptors (GPCRs), relatively little structural information is available about this class of receptors. Here we aim at developing small chimeric proteins, displaying the extracellular ligand-binding motifs of a human GPCR, the Y receptor. This allows the study of ligand-receptor interactions in simplified systems. We present comprehensive information on the use of transmembrane (OmpA) and soluble (Blc) β-barrel scaffolds. Whereas Blc appeared to be not fully compatible with our approach, owing to problems with refolding of the hybrid constructs, loop-grafted versions of OmpA delivered encouraging results. Previously, we described a chimeric construct based on OmpA displaying all three extracellular Y1 receptor loops in different topologies and showing moderate affinity to one of the natural ligands. Now, we present detailed data on the interaction of these constructs with several Y receptor ligands along with data on new constructs. Our findings suggest a common binding mode for all ligands, which is mediated through the C-terminal residues of the peptide ligand, supporting the functional validity of these hybrid receptors. The observed binding affinities, however, are well below those observed for the natural receptors, clearly indicating limitations in mimicking the natural systems.

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Additional indexing

Other titles:β-Barrel scaffolds for the grafting of extracellular loops from G-protein-coupled receptors
Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:November 2012
Deposited On:11 Oct 2012 11:41
Last Modified:05 Apr 2016 15:59
Publisher:De Gruyter
ISSN:1431-6730
Funders:Forschungskredit Uni Zürich
Publisher DOI:https://doi.org/10.1515/hsz-2012-0234
Official URL:http://www.degruyter.com/view/j/bchm.2012.393.issue-11/hsz-2012-0234/hsz-2012-0234.xml?format=INT

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