Mini-G proteins: Novel tools for studying GPCRs in their active conformation.
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,...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0175642&type=printable |
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| author | Rony Nehmé Byron Carpenter Ankita Singhal Annette Strege Patricia C Edwards Courtney F White Haijuan Du Reinhard Grisshammer Christopher G Tate |
| author_facet | Rony Nehmé Byron Carpenter Ankita Singhal Annette Strege Patricia C Edwards Courtney F White Haijuan Du Reinhard Grisshammer Christopher G Tate |
| author_sort | Rony Nehmé |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-a5fac57f40484242b84cb93eef39e9ee |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-a5fac57f40484242b84cb93eef39e9ee2025-08-20T03:24:27ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01124e017564210.1371/journal.pone.0175642Mini-G proteins: Novel tools for studying GPCRs in their active conformation.Rony NehméByron CarpenterAnkita SinghalAnnette StregePatricia C EdwardsCourtney F WhiteHaijuan DuReinhard GrisshammerChristopher G TateMini-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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0175642&type=printable |
| spellingShingle | Rony Nehmé Byron Carpenter Ankita Singhal Annette Strege Patricia C Edwards Courtney F White Haijuan Du Reinhard Grisshammer Christopher G Tate Mini-G proteins: Novel tools for studying GPCRs in their active conformation. PLoS ONE |
| title | Mini-G proteins: Novel tools for studying GPCRs in their active conformation. |
| title_full | Mini-G proteins: Novel tools for studying GPCRs in their active conformation. |
| title_fullStr | Mini-G proteins: Novel tools for studying GPCRs in their active conformation. |
| title_full_unstemmed | Mini-G proteins: Novel tools for studying GPCRs in their active conformation. |
| title_short | Mini-G proteins: Novel tools for studying GPCRs in their active conformation. |
| title_sort | mini g proteins novel tools for studying gpcrs in their active conformation |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0175642&type=printable |
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