Probing GPCR-Gα interactions: a functional study by EM and SPR
<p>The G protein-coupled receptor (GPCR), neurotensin receptor type 1 (NTS1), is pharmacologically important and activated by the tridecapeptide hormone, neurotensin (NT), initiating a cascade of interactions through G proteins to effect cellular responses. The mechanisms by which these occur...
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| Format: | Thèse |
| Langue: | English |
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2014
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| _version_ | 1826311158610526208 |
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| author | Adamson, RJ |
| author2 | Watts, A |
| author_facet | Watts, A Adamson, RJ |
| author_sort | Adamson, RJ |
| collection | OXFORD |
| description | <p>The G protein-coupled receptor (GPCR), neurotensin receptor type 1 (NTS1), is pharmacologically important and activated by the tridecapeptide hormone, neurotensin (NT), initiating a cascade of interactions through G proteins to effect cellular responses. The mechanisms by which these occur have only recently begun to be examined structurally, and standard assays involving downstream effectors or radioactive GTPγS G protein activation to describe GPCR-G protein interactions do not assay the interactions directly. Two methods have been used here to study the interaction of NTS1 with the signalling partners, Gαs and Gαi1. A novel DNA-nanotechnological approach for preparing samples for electron microscopy (EM) has been used to study NTS1 and Gαi1, both separately and complexed, on a functionalised 2D DNA lattice, providing the first direct evidence of this interaction. Single particle reconstruction methods were used to determine a structure of NTS1 at a resolution of ~15 Å, a structure of Gαi1 at ~ 15 Å, and the interaction of Gαi1 with NTS1 has been observed using EM. </p> <p>A further nanotechnological approach, using the increasingly popular method of reconstitution of membrane proteins in nanodiscs, has been used to study NTS1-Gαs and NTS1-Gαi1 interactions, using EM and surface plasmon resonance (SPR). The ligand (NT) affinity of detergent-solubilised NTS1 and NTS1 reconstituted into nanodiscs was &Tilde;1 nM, and for the first time, the affinity of binding of Gαi1 and Gαs to NTS1 was directly measured and determined as 15 nM and 31 nM, respectively. These results will facilitate cryo-EM studies on GPCRs with interacting partners, using a tethering system that both activates the GPCR and maintains it in a concentrated form within a restricted, two-dimensional plane; and also will aid a wealth of mutational and lipid-dependent studies whereby the effect on coupling and GPCR-protein interactions of specific residues and lipid types can be directly measured. This thesis primarily demonstrates the development and effective application of a biophysical methodology for measuring the coupling kinetics of GPCRs to G proteins; as well as demonstrating that this coupling can be visually evidenced under EM using gold-labelled G proteins.</p> |
| first_indexed | 2024-03-07T08:04:13Z |
| format | Thesis |
| id | oxford-uuid:d24b4be6-65d6-4b3c-b049-5c6d1e737f1e |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T08:04:13Z |
| publishDate | 2014 |
| record_format | dspace |
| spelling | oxford-uuid:d24b4be6-65d6-4b3c-b049-5c6d1e737f1e2023-10-13T09:53:03ZProbing GPCR-Gα interactions: a functional study by EM and SPRThesishttp://purl.org/coar/resource_type/c_db06uuid:d24b4be6-65d6-4b3c-b049-5c6d1e737f1eMicroscopyBiochemistryBiophysicsMolecular biophysics (biochemistry)Membrane proteinsBiosensorsProtein chemistryNano-biotechnologyEnglishHyrax Deposit2014Adamson, RJWatts, A<p>The G protein-coupled receptor (GPCR), neurotensin receptor type 1 (NTS1), is pharmacologically important and activated by the tridecapeptide hormone, neurotensin (NT), initiating a cascade of interactions through G proteins to effect cellular responses. The mechanisms by which these occur have only recently begun to be examined structurally, and standard assays involving downstream effectors or radioactive GTPγS G protein activation to describe GPCR-G protein interactions do not assay the interactions directly. Two methods have been used here to study the interaction of NTS1 with the signalling partners, Gαs and Gαi1. A novel DNA-nanotechnological approach for preparing samples for electron microscopy (EM) has been used to study NTS1 and Gαi1, both separately and complexed, on a functionalised 2D DNA lattice, providing the first direct evidence of this interaction. Single particle reconstruction methods were used to determine a structure of NTS1 at a resolution of ~15 Å, a structure of Gαi1 at ~ 15 Å, and the interaction of Gαi1 with NTS1 has been observed using EM. </p> <p>A further nanotechnological approach, using the increasingly popular method of reconstitution of membrane proteins in nanodiscs, has been used to study NTS1-Gαs and NTS1-Gαi1 interactions, using EM and surface plasmon resonance (SPR). The ligand (NT) affinity of detergent-solubilised NTS1 and NTS1 reconstituted into nanodiscs was &Tilde;1 nM, and for the first time, the affinity of binding of Gαi1 and Gαs to NTS1 was directly measured and determined as 15 nM and 31 nM, respectively. These results will facilitate cryo-EM studies on GPCRs with interacting partners, using a tethering system that both activates the GPCR and maintains it in a concentrated form within a restricted, two-dimensional plane; and also will aid a wealth of mutational and lipid-dependent studies whereby the effect on coupling and GPCR-protein interactions of specific residues and lipid types can be directly measured. This thesis primarily demonstrates the development and effective application of a biophysical methodology for measuring the coupling kinetics of GPCRs to G proteins; as well as demonstrating that this coupling can be visually evidenced under EM using gold-labelled G proteins.</p> |
| spellingShingle | Microscopy Biochemistry Biophysics Molecular biophysics (biochemistry) Membrane proteins Biosensors Protein chemistry Nano-biotechnology Adamson, RJ Probing GPCR-Gα interactions: a functional study by EM and SPR |
| title | Probing GPCR-Gα interactions: a functional study by EM and SPR |
| title_full | Probing GPCR-Gα interactions: a functional study by EM and SPR |
| title_fullStr | Probing GPCR-Gα interactions: a functional study by EM and SPR |
| title_full_unstemmed | Probing GPCR-Gα interactions: a functional study by EM and SPR |
| title_short | Probing GPCR-Gα interactions: a functional study by EM and SPR |
| title_sort | probing gpcr gα interactions a functional study by em and spr |
| topic | Microscopy Biochemistry Biophysics Molecular biophysics (biochemistry) Membrane proteins Biosensors Protein chemistry Nano-biotechnology |
| work_keys_str_mv | AT adamsonrj probinggpcrgainteractionsafunctionalstudybyemandspr |