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Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling
G protein‐coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, rang...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821118/ https://www.ncbi.nlm.nih.gov/pubmed/32881260 http://dx.doi.org/10.1002/cbic.202000541 |
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author | Goba, Inguna Goricanec, David Schum, Dominik Hillenbrand, Matthias Plückthun, Andreas Hagn, Franz |
author_facet | Goba, Inguna Goricanec, David Schum, Dominik Hillenbrand, Matthias Plückthun, Andreas Hagn, Franz |
author_sort | Goba, Inguna |
collection | PubMed |
description | G protein‐coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein‐stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope‐labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist‐induced activity of the receptor. Furthermore, this assay can be used as a readout when re‐introducing agonist‐dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively (19)F‐labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis. |
format | Online Article Text |
id | pubmed-7821118 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-78211182021-01-26 Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling Goba, Inguna Goricanec, David Schum, Dominik Hillenbrand, Matthias Plückthun, Andreas Hagn, Franz Chembiochem Communications G protein‐coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein‐stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope‐labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist‐induced activity of the receptor. Furthermore, this assay can be used as a readout when re‐introducing agonist‐dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively (19)F‐labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis. John Wiley and Sons Inc. 2020-11-06 2021-01-05 /pmc/articles/PMC7821118/ /pubmed/32881260 http://dx.doi.org/10.1002/cbic.202000541 Text en © 2020 The Authors. Published by Wiley-VCH GmbH This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. |
spellingShingle | Communications Goba, Inguna Goricanec, David Schum, Dominik Hillenbrand, Matthias Plückthun, Andreas Hagn, Franz Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling |
title | Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling |
title_full | Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling |
title_fullStr | Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling |
title_full_unstemmed | Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling |
title_short | Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site‐Directed Methyl Labeling |
title_sort | probing the conformation states of neurotensin receptor 1 variants by nmr site‐directed methyl labeling |
topic | Communications |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821118/ https://www.ncbi.nlm.nih.gov/pubmed/32881260 http://dx.doi.org/10.1002/cbic.202000541 |
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