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Production of a Human Histamine Receptor for NMR Spectroscopy in Aqueous Solutions
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 spectr...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146376/ https://www.ncbi.nlm.nih.gov/pubmed/33923140 http://dx.doi.org/10.3390/biom11050632 |
Sumario: | 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 H(1) receptor (H(1)R) 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 H(1)R over initial efforts in defined media. The expressed receptor could be purified to homogeneity and was found to respond to the addition of known H(1)R ligands. Two-dimensional transverse relaxation-optimized spectroscopy (TROSY) NMR spectra of stable-isotope labeled H(1)R show well-dispersed and resolved signals consistent with a properly folded protein, and (19)F-NMR data register a response of the protein to differences in efficacies of bound ligands. |
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