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Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

Wiktor, Maciej and Morin, Sébastien and Sass, Hans-Jürgen and Kebbel, Fabian and Grzesiek, Stephan. (2013) Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor. Journal of Biomolecular NMR, 55 (1). pp. 79-95.

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Official URL: http://edoc.unibas.ch/dok/A6070334

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Abstract

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled ((2)H/(15)N/(13)C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K(D) values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Grzesiek)
UniBasel Contributors:Grzesiek, Stephan
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Springer
ISSN:0925-2738
e-ISSN:1573-5001
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:23 Nov 2017 15:02
Deposited On:01 Mar 2013 11:09

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