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Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties - PubMed

  • ️Mon Jan 01 2018

Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties

Fariba M Assadi-Porter et al. Molecules. 2018.

Abstract

Taste signaling is a complex process that is linked to obesity and its associated metabolic syndromes. The sweet taste is mediated through a heterodimeric G protein coupled receptor (GPCR) in a species-specific manner and at multi-tissue specific levels. The sweet receptor recognizes a large number of ligands with structural and functional diversities to modulate different amplitudes of downstream signaling pathway(s). The human sweet-taste receptor has been extremely difficult to study by biophysical methods due to the difficulty in producing large homogeneous quantities of the taste-receptor protein and the lack of reliable in vitro assays to precisely measure productive ligand binding modes that lead to activation of the receptor protein. We report here a multimodal high throughput assay to monitor ligand binding, receptor stability and conformational changes to model the molecular ligand-receptor interactions. We applied saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) complemented by differential scanning calorimetry (DSC), circular dichroism (CD) spectroscopy, and intrinsic fluorescence spectroscopy (IF) to characterize binding interactions. Our method using complementary NMR and biophysical analysis is advantageous to study the mechanism of ligand binding and signaling processes in other GPCRs.

Keywords: G-coupled protein receptors (GPCRs); circular dichroism (CD) spectroscopy; differential scanning calorimetry (DSC); intrinsic fluorescence spectroscopy (IF); ligand binding; nuclear magnetic resonance spectroscopy (NMR); saturation transfer difference (STD)-NMR; sweet taste receptor.

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Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Figure 1
Figure 1

(A) Model of sweet receptor and its proposed interaction sites with sweet ligands. (B) SDS-PAGE of the expression and purification of human and mouse proteins. Lanes shown are: M. Novex Sharp Pertained Protein Standard (kDa); 1. −IPTG, total proteins; 2. +IPTG, total cell proteins containing His-TEV-ATD-hT1R2 (~56 kDa); 3. Pellet: His-ATD-hT1R2; 4. Supernatant: His-ATD-hT1R2; 5. Refolded His-ATD-hT1R2; 6. FPLC purified His-ATD-hT1R2; 7. +IPTG, total proteins, His-TEV-ATD-mT1R2 (~63 kDa); 8. Pellet of His-ATD-mT1R2+IPTG; 9. Supernatant of His-ATD-mT1R2; 10. Refolded His-ATD-mR2; 11. Purified His-ATD-mT1R2, by FPLC; 12. −IPTG; 13. +IPTG, His-Sumo-TEV-ATD-hT1R2; 14. Cut His-Sumo-TEV-ATD-hR2 (ATD-hT1R2, ~54 kDa and His-Sumo-TEV, ~12.5 kDa); 15. FPLC purified ATD-hT1R2. (C) Tev protease cleaved ATD protein peaks off the Superdex 200 prep grade FPLC column.

Figure 2
Figure 2

CD spectra of His tagged human and mouse T1R2 ATD ± ligands. Panel (A) human T1R2 ATD ± neotame, Panel (B) changes in ellipticity at 209 and 219 nm with increasing concentrations of neotame, black symbols are actual data and colored symbols are polynomial-fitted values; panel (C) human T1R2 ATD ± MSG; panel (D) mouse T1R2 ATD with addition of saturating concentrations of neotame, sucrose, or MSG.

Figure 2
Figure 2

CD spectra of His tagged human and mouse T1R2 ATD ± ligands. Panel (A) human T1R2 ATD ± neotame, Panel (B) changes in ellipticity at 209 and 219 nm with increasing concentrations of neotame, black symbols are actual data and colored symbols are polynomial-fitted values; panel (C) human T1R2 ATD ± MSG; panel (D) mouse T1R2 ATD with addition of saturating concentrations of neotame, sucrose, or MSG.

Figure 3
Figure 3

Intrinsic fluorescene emission spectra (excitation at 280 nm) of His tagged human and mouse T1R2 ATD. Panel (A) human T1R2 ATD ± neotame; (B) human T1R2 ATD ± sucralose; (C) human T1R2 ATD ± MSG; (D) changes in intrinsic fluorescence signal at 336 nm in the presence of human T1R2 ATD and increasing concentrations of either neotame, sucralose or MSG; (E) mouse T1R2 ATD ± sucrose; (F) mouse T1R2 ATD ± neotame; (G) mouse T1R2 ATD ± MSG; (H) changes in intrinsic fluorescence signal at 336 nm in the presence of mouse T1R2 ATD and increasing concentrations of either sucrose, neotame, or MSG.

Figure 3
Figure 3

Intrinsic fluorescene emission spectra (excitation at 280 nm) of His tagged human and mouse T1R2 ATD. Panel (A) human T1R2 ATD ± neotame; (B) human T1R2 ATD ± sucralose; (C) human T1R2 ATD ± MSG; (D) changes in intrinsic fluorescence signal at 336 nm in the presence of human T1R2 ATD and increasing concentrations of either neotame, sucralose or MSG; (E) mouse T1R2 ATD ± sucrose; (F) mouse T1R2 ATD ± neotame; (G) mouse T1R2 ATD ± MSG; (H) changes in intrinsic fluorescence signal at 336 nm in the presence of mouse T1R2 ATD and increasing concentrations of either sucrose, neotame, or MSG.

Figure 3
Figure 3

Intrinsic fluorescene emission spectra (excitation at 280 nm) of His tagged human and mouse T1R2 ATD. Panel (A) human T1R2 ATD ± neotame; (B) human T1R2 ATD ± sucralose; (C) human T1R2 ATD ± MSG; (D) changes in intrinsic fluorescence signal at 336 nm in the presence of human T1R2 ATD and increasing concentrations of either neotame, sucralose or MSG; (E) mouse T1R2 ATD ± sucrose; (F) mouse T1R2 ATD ± neotame; (G) mouse T1R2 ATD ± MSG; (H) changes in intrinsic fluorescence signal at 336 nm in the presence of mouse T1R2 ATD and increasing concentrations of either sucrose, neotame, or MSG.

Figure 3
Figure 3

Intrinsic fluorescene emission spectra (excitation at 280 nm) of His tagged human and mouse T1R2 ATD. Panel (A) human T1R2 ATD ± neotame; (B) human T1R2 ATD ± sucralose; (C) human T1R2 ATD ± MSG; (D) changes in intrinsic fluorescence signal at 336 nm in the presence of human T1R2 ATD and increasing concentrations of either neotame, sucralose or MSG; (E) mouse T1R2 ATD ± sucrose; (F) mouse T1R2 ATD ± neotame; (G) mouse T1R2 ATD ± MSG; (H) changes in intrinsic fluorescence signal at 336 nm in the presence of mouse T1R2 ATD and increasing concentrations of either sucrose, neotame, or MSG.

Figure 4
Figure 4

Saturation transfer difference spectra of His tagged human T1R2 ATD. NMR data were collected on a Varian VNMRS spectrometer operating at 800 MHz and equipped with a cryogenic probe. (A) Human T1R2 ATD ± neotame at 27 °C. Top panel shows one-dimensional (1D) 1H-NMR, and the bottom panel shows 1D STD NMR spectra of neotame at increasing concentrations (3–10 mM). The observed STD signals increase with the concentration of neotame, indicating that neotame binds to the receptor domain; (B) human T1R2 ATD ± MSG (negative control) measured at 7 and 27 °C. Top panel shows 1D 1H-NMR with MSG proton signals, and bottom panel shows STD spectra. Lack of STD signal in MSG spectra indicates no binding.

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