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Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor

Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the...

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Autores principales: Pacull, Emelyne M., Sendker, Franziska, Bernhard, Frank, Scheidt, Holger A., Schmidt, Peter, Huster, Daniel, Krug, Ulrike
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723455/
https://www.ncbi.nlm.nih.gov/pubmed/33324203
http://dx.doi.org/10.3389/fphar.2020.562113
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author Pacull, Emelyne M.
Sendker, Franziska
Bernhard, Frank
Scheidt, Holger A.
Schmidt, Peter
Huster, Daniel
Krug, Ulrike
author_facet Pacull, Emelyne M.
Sendker, Franziska
Bernhard, Frank
Scheidt, Holger A.
Schmidt, Peter
Huster, Daniel
Krug, Ulrike
author_sort Pacull, Emelyne M.
collection PubMed
description Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded in vitro and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for (13)C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane α-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged (1)H-(13)C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly (13)C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding of the receptor dynamics under equilibrium conditions.
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spelling pubmed-77234552020-12-14 Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor Pacull, Emelyne M. Sendker, Franziska Bernhard, Frank Scheidt, Holger A. Schmidt, Peter Huster, Daniel Krug, Ulrike Front Pharmacol Pharmacology Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded in vitro and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for (13)C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane α-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged (1)H-(13)C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly (13)C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding of the receptor dynamics under equilibrium conditions. Frontiers Media S.A. 2020-10-29 /pmc/articles/PMC7723455/ /pubmed/33324203 http://dx.doi.org/10.3389/fphar.2020.562113 Text en Copyright © 2020 Pacull, Sendker, Bernhard, Scheidt, Schmidt, Huster and Krug http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Pharmacology
Pacull, Emelyne M.
Sendker, Franziska
Bernhard, Frank
Scheidt, Holger A.
Schmidt, Peter
Huster, Daniel
Krug, Ulrike
Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor
title Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor
title_full Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor
title_fullStr Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor
title_full_unstemmed Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor
title_short Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor
title_sort integration of cell-free expression and solid-state nmr to investigate the dynamic properties of different sites of the growth hormone secretagogue receptor
topic Pharmacology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723455/
https://www.ncbi.nlm.nih.gov/pubmed/33324203
http://dx.doi.org/10.3389/fphar.2020.562113
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