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

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 orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested l-amino 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.

Chloride ions evoke taste sensations by binding to the extracellular ligand-binding domain of sweet/umami taste receptors

“…To investigate the Cl − actions on T1r functions, we first examined the properties of the Cl − binding to medaka T1r2a/T1r3LBD using various biophysical techniques. For this purpose, the purified T1r2a/T1r3LBD was subjected to differential scanning fluorimetry (DSF), which we previously used for the amino acid-binding analysis ( Yoshida et al, 2019 ). In order to prepare a Cl − -free condition, Cl − in the sample was substituted with gluconate, as it is unlikely accommodated in the Cl − site due to its much larger size.…”

“…The EC 50 for Cl − -induced FRET signal change was determined as ~1 mM ( Table 2 ). Note that both DSF and FRET estimations have some degree of error: the former produced slightly lower values than the latter, particularly in the case of weak affinities in the mM concentration range ( Yoshida et al, 2019 ). As such, although the EC 50 value determined by FRET was slightly higher than the apparent K d value of Cl − determined by DSF, the two are most likely relevant.…”

“…DSF was performed as previously described ( Yoshida et al, 2019 ). The purified medaka T1R2a/3LBD heterodimer protein was prepared ( Nango et al, 2016 ) and dialyzed with buffer A (20 mM HEPES–NaOH, 300 mM sodium gluconate, pH 7.5) to remove Cl − .…”

“…The apparent melting transition temperature ( T m ) was determined using the maximum of the derivatives of the melt curve (dFluorescence/d T ) by Protein Thermal Shift Software version 1.3 (Applied Biosystems). The apparent dissociation constant ( K d-app ) for Cl − derived from the T m values at different NaCl concentrations was estimated using a thermodynamic model proposed by Schellman, 1975 as described ( Yoshida et al, 2019 ). The sample sizes for the analyses by DSF, as well as FRET and isothermal titration calorimetry described below, were set to obtain reliable values based on the experiences in the previous studies ( Nango et al, 2016 ; Nuemket et al, 2017 ; Yoshida et al, 2019 ).…”

eSPC: an online data-analysis platform for molecular biophysics

“…Additionally to the isothermal analysis, in FoldAffinity we allow the fitting of a thermodynamic-based model to estimate the binding affinities directly from the observed melting temperatures (Yoshida et al, 2019;Schellman, 1975; Supplementary Fig. S1).…”