Carpenter, Byron and Tate, Christopher G. (2016) Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation. Protein Engineering Design and Selection, 29 (12). pp. 583-594. doi:10.1093/protein/gzw049 ISSN 1741-0126.

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Abstract

G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs. Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.

Item Type:	Journal Article
Subjects:	Q Science > QP Physiology R Medicine > RS Pharmacy and materia medica
Divisions:	Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH):	G proteins , Cell receptors, Drugs -- Design
Journal or Publication Title:	Protein Engineering Design and Selection
Publisher:	Oxford University Press
ISSN:	1741-0126
Official Date:	28 December 2016
Dates:	Date Event 28 December 2016 Published 28 November 2016 Available 23 August 2016 Accepted
Volume:	29
Number:	12
Page Range:	pp. 583-594
DOI:	10.1093/protein/gzw049
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access (Creative Commons)
Date of first compliant deposit:	12 October 2016
Date of first compliant Open Access:	12 October 2016
Funder:	Heptares Therapeutics, Medical Research Council (Great Britain) (MRC)
Grant number:	MRC U105197215

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