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. 2018 Jun;1860(6):1372-1383.
doi: 10.1016/j.bbamem.2018.03.020. Epub 2018 Mar 26.

Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies

Fabian Bumbak  1 , Alastair C Keen  2 , Natalie J Gunn  3 , Paul R Gooley  4 , Ross A D Bathgate  5 , Daniel J Scott  6
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Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies

Fabian Bumbak et al. Biochim Biophys Acta Biomembr. 2018 Jun.
Free article

Abstract

Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS1). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS1 have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS1 however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS1 variants to identify an optimal variant for protein NMR studies. A variant termed enNTS1 exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS1 was subsequently labeled with 13CH3-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed 13CH3-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS1 represents a novel tool for investigating ligand induced conformational changes in NTS1 to gain insights into the molecular mechanisms underlying neurotensin signaling.

Keywords: GPCR NMR; GPCR signaling; Isotopic labeling; Neurotensin receptor; Stabilized GPCRs.

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