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Media type:
E-Article
Title:
Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions
Contributor:
Mulry, Emma;
Ray, Arka Prabha;
Eddy, Matthew T.
Published:
MDPI AG, 2021
Published in:
Biomolecules, 11 (2021) 5, Seite 632
Language:
English
DOI:
10.3390/biom11050632
ISSN:
2218-273X
Origination:
Footnote:
Description:
G protein-coupled receptors (GPCRs) bind a broad array of extracellular molecules and transmit intracellular signals that initiate physiological responses. The signal transduction functions of GPCRs are inherently related to their structural plasticity, which can be experimentally observed by spectroscopic techniques. Nuclear magnetic resonance (NMR) spectroscopy in particular is an especially advantageous method to study the dynamic behavior of GPCRs. The success of NMR studies critically relies on the production of functional GPCRs containing stable-isotope labeled probes, which remains a challenging endeavor for most human GPCRs. We report a protocol for the production of the human histamine H1 receptor (H1R) in the methylotrophic yeast Pichia pastoris for NMR experiments. Systematic evaluation of multiple expression parameters resulted in a ten-fold increase in the yield of expressed H1R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H1R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H1R show well-dispersed and resolved signals consistent with a properly folded protein, and 19F-NMR data register a response of the protein to differences in efficacies of bound ligands.