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Mohamad, M.* ; Goricanec, D.* ; Wagner, G.* ; Hagn, F.

NMR sample optimization and backbone assignment of a stabilized neurotensin receptor.

J. Struct. Biol. 215:107970 (2023)
Postprint DOI PMC
Open Access Green
G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Protein-coupled Receptor; Phospholipid-bilayer Nanodiscs; Alpha-subunit; Dynamics; Transduction; Rhodopsin; Binding
ISSN (print) / ISBN 1047-8477
e-ISSN 1047-8477
Zeitschrift Journal of Structural Biology
Quellenangaben Band: 215, Heft: 2, Seiten: , Artikelnummer: 107970 Supplement: ,
Verlag Elsevier
Verlagsort 525 B St, Ste 1900, San Diego, Ca 92101-4495 Usa
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Structural Biology (STB)
Förderungen
German Research Foundation DFG
National Instituted of Health (NIH)
Helmholtz Zentrum Munchen
Deutsche Forschungsgemeinschaft (DFG)