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Mini-G proteins : novel tools for studying GPCRs in their active conformation
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Nehmé, Rony, Carpenter, Byron, Singhal, Ankita, Strege, Annette, Edwards, Patricia C., White, Courtney F., Du, Haijuan, Grisshammer, Reinhard and Tate, Christopher G. (2017) Mini-G proteins : novel tools for studying GPCRs in their active conformation. PLoS One, 12 (4). e0175642. doi:10.1371/journal.pone.0175642 ISSN 1932-6203.
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Official URL: https://doi.org/10.1371/journal.pone.0175642
Abstract
Mini-G proteins are the engineered GTPase domains of Gα subunits. They couple to GPCRs and recapitulate the increase in agonist affinity observed upon coupling of a native heterotrimeric G protein. Given the small size and stability of mini-G proteins, and their ease of expression and purification, they are ideal for biophysical studies of GPCRs in their fully active state. The first mini-G protein developed was mini-Gs. Here we extend the family of mini-G proteins to include mini-Golf, mini-Gi1, mini-Go1 and the chimeras mini-Gs/q and mini-Gs/i. The mini-G proteins were shown to couple to relevant GPCRs and to form stable complexes with purified receptors that could be purified by size exclusion chromatography. Agonist-bound GPCRs coupled to a mini-G protein showed higher thermal stability compared to the agonist-bound receptor alone. Fusion of GFP at the N-terminus of mini-G proteins allowed receptor coupling to be monitored by fluorescence-detection size exclusion chromatography (FSEC) and, in a separate assay, the affinity of mini-G protein binding to detergent-solubilised receptors was determined. This work provides the foundation for the development of any mini-G protein and, ultimately, for the structure determination of GPCRs in a fully active state.
Item Type: | Journal Article | |||||||||||||||
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Subjects: | Q Science > QP Physiology | |||||||||||||||
Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | |||||||||||||||
Library of Congress Subject Headings (LCSH): | G proteins, Cell receptors, Chromatographic analysis | |||||||||||||||
Journal or Publication Title: | PLoS One | |||||||||||||||
Publisher: | Public Library of Science | |||||||||||||||
ISSN: | 1932-6203 | |||||||||||||||
Official Date: | 20 April 2017 | |||||||||||||||
Dates: |
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Volume: | 12 | |||||||||||||||
Number: | 4 | |||||||||||||||
Article Number: | e0175642 | |||||||||||||||
DOI: | 10.1371/journal.pone.0175642 | |||||||||||||||
Status: | Peer Reviewed | |||||||||||||||
Publication Status: | Published | |||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | |||||||||||||||
Copyright Holders: | Plos One | |||||||||||||||
Date of first compliant deposit: | 24 April 2017 | |||||||||||||||
Date of first compliant Open Access: | 25 April 2017 | |||||||||||||||
Funder: | European Research Council (ERC), Heptares Therapeutics (Firm), Medical Research Council (Great Britain) (MRC), National Institute of Neurological Disorders and Stroke (U.S.) (NINDS) | |||||||||||||||
Grant number: | EMPSI 339995 (ERC), MC_U1051 97215 (MRC), ZIA NS003016-11 (NINDS) | |||||||||||||||
RIOXX Funder/Project Grant: |
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