Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

Takashi Yoshida, Norihisa Yasui, Yuko Kusakabe, Chiaki Ito, Miki Akamatsu, Atsuko Yamashita

Research output: Contribution to journalArticle

Abstract

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthostericbinding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested Lamino acids, but no D-amino acid, induced Tm shifts of T1r2a/T1r3LBD, indicating the broad L-amino acid specificities of the proteins probably with several different manners of recognition. The Tm shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF.

Original languageEnglish
Article numbere0218909
JournalPloS one
Volume14
Issue number10
DOIs
Publication statusPublished - Jan 1 2019

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fluorometry
Protein Binding
Fish
Fishes
Fluorometry
Ligands
Scanning
Amino Acids
receptors
amino acids
fish
Proteins
proteins
Assays
Temperature
assays
Stabilization
Hot Temperature
Satureja
Oryzias

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

Cite this

Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3. / Yoshida, Takashi; Yasui, Norihisa; Kusakabe, Yuko; Ito, Chiaki; Akamatsu, Miki; Yamashita, Atsuko.

In: PloS one, Vol. 14, No. 10, e0218909, 01.01.2019.

Research output: Contribution to journalArticle

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