Optimization and (CH3)-C-13 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies
Fabian Bumbak, Alastair C Keen, Natalie J Gunn, Paul R Gooley, Ross AD Bathgate, Daniel J Scott
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES | ELSEVIER SCIENCE BV | Published : 2018
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 experimen..View full abstract
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We thank Sharon Layfield, Tania Ferraro and Alice Whitehead for cell-based assays (all from The Florey Institute of Neuroscience and Mental Health); Fei Yan (from the Department of Biochemistry & Molecular Biology and Bio21 Institute) for assistance with protein expression and purification; Yee-Fong Mok (from the Department of Biochemistry & Molecular Biology and Bio21 Institute) for assistance with CD; Shuai Nie, Cheng-Sing Ang and Nicholas Williamson for assistance with mass spectrometry (all Bio21 Institute); Tim Ryan and Nigel Kirby for assistance with SAXS (both from the Australian Synchrotron); Lisa Mckenzie Williams (The Florey Institute of Neuroscience and Mental Health) for saturation and competition binding assays; and Ashish Sethi for assistance with NUS (from the Department of Biochemistry & Molecular Biology and Bio21 Institute). This work was supported by NHMRC project grants 1081801 (D.J.S) and 1081844 (R.A.D.B, P.R.G, D.J.S); ARC equipment grant LE120100022. D.J.S. is an NHMRC Boosting Dementia Research Leadership Fellow. R.A.D.B. is an NHMRC Senior Research Fellow. Studies at The Florey Institute of Neuroscience and Mental Health were supported by the Victorian Government's Operational Infrastructure Support Program.