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

Autoři: Takashi Yoshida aff001;  Norihisa Yasui aff001;  Yuko Kusakabe aff002;  Chiaki Ito aff001;  Miki Akamatsu aff003;  Atsuko Yamashita aff001
Působiště autorů: Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Okayama, Japan aff001;  Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan aff002;  Graduate School of Agriculture, Kyoto University, Kyoto, Kyoto, Japan aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0218909


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.

Klíčová slova:

Amino acid analysis – Binding analysis – Fluorescence resonance energy transfer – Glutamate – Melting – Sensory receptors – Taste – G protein coupled receptors


1. Yarmolinsky DA, Zuker CS, Ryba NJ. Common sense about taste: from mammals to insects. Cell. 2009;139(2):234–44. doi: 10.1016/j.cell.2009.10.001 19837029

2. Liman ER, Zhang YV, Montell C. Peripheral coding of taste. Neuron. 2014;81(5):984–1000. doi: 10.1016/j.neuron.2014.02.022 24607224

3. Berridge KC. Measuring hedonic impact in animals and infants: microstructure of affective taste reactivity patterns. Neurosci Biobehav Rev. 2000;24(2):173–98. 10714382

4. Shi P, Zhang J. Contrasting modes of evolution between vertebrate sweet/umami receptor genes and bitter receptor genes. Mol Biol Evol. 2006;23(2):292–300. Epub 2005/10/07. doi: 10.1093/molbev/msj028 16207936

5. Hoon MA, Adler E, Lindemeier J, Battey JF, Ryba NJ, Zuker CS. Putative mammalian taste receptors: a class of taste-specific GPCRs with distinct topographic selectivity. Cell. 1999;96(4):541–51. doi: 10.1016/s0092-8674(00)80658-3 10052456

6. Pin JP, Galvez T, Prezeau L. Evolution, structure, and activation mechanism of family 3/C G-protein-coupled receptors. Pharmacol Ther. 2003;98(3):325–54. Epub 2003/06/05. doi: 10.1016/s0163-7258(03)00038-x 12782243

7. Nelson G, Hoon MA, Chandrashekar J, Zhang Y, Ryba NJ, Zuker CS. Mammalian sweet taste receptors. Cell. 2001;106(3):381–90. Epub 2001/08/18. doi: 10.1016/s0092-8674(01)00451-2 11509186

8. Li X, Staszewski L, Xu H, Durick K, Zoller M, Adler E. Human receptors for sweet and umami taste. Proc Natl Acad Sci U S A. 2002;99(7):4692–6. Epub 2002/03/28. doi: 10.1073/pnas.072090199 11917125

9. Nelson G, Chandrashekar J, Hoon MA, Feng L, Zhao G, Ryba NJ, et al. An amino-acid taste receptor. Nature. 2002;416(6877):199–202. Epub 2002/03/15. doi: 10.1038/nature726 11894099

10. Xu H, Staszewski L, Tang H, Adler E, Zoller M, Li X. Different functional roles of T1R subunits in the heteromeric taste receptors. Proc Natl Acad Sci U S A. 2004;101(39):14258–63. Epub 2004/09/09. doi: 10.1073/pnas.0404384101 15353592

11. Nuemket N, Yasui N, Kusakabe Y, Nomura Y, Atsumi N, Akiyama S, et al. Structural basis for perception of diverse chemical substances by T1r taste receptors. Nat Commun. 2017;8:15530. doi: 10.1038/ncomms15530 28534491

12. Oike H, Nagai T, Furuyama A, Okada S, Aihara Y, Ishimaru Y, et al. Characterization of ligands for fish taste receptors. J Neurosci. 2007;27(21):5584–92. Epub 2007/05/25. 27/21/5584 doi: 10.1523/JNEUROSCI.0651-07.2007 17522303

13. Toda Y, Nakagita T, Hayakawa T, Okada S, Narukawa M, Imai H, et al. Two distinct determinants of ligand specificity in T1R1/T1R3 (the umami taste receptor). J Biol Chem. 2013;288(52):36863–77. doi: 10.1074/jbc.M113.494443 24214976

14. Kunishima N, Shimada Y, Tsuji Y, Sato T, Yamamoto M, Kumasaka T, et al. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature. 2000;407(6807):971–7. Epub 2000/11/09. doi: 10.1038/35039564 11069170

15. Geng Y, Bush M, Mosyak L, Wang F, Fan QR. Structural mechanism of ligand activation in human GABA(B) receptor. Nature. 2013;504(7479):254–9. Epub 2013/12/07. doi: 10.1038/nature12725 24305054

16. Ashikawa Y, Ihara M, Matsuura N, Fukunaga Y, Kusakabe Y, Yamashita A. GFP-based evaluation system of recombinant expression through the secretory pathway in insect cells and its application to the extracellular domains of class C GPCRs. Protein Sci. 2011;20(10):1720–34. Epub 2011/08/02. doi: 10.1002/pro.707 21805523

17. Nango E, Akiyama S, Maki-Yonekura S, Ashikawa Y, Kusakabe Y, Krayukhina E, et al. Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains. Sci Rep. 2016;6:25745. doi: 10.1038/srep25745 27160511

18. Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc. 2007;2(9):2212–21. doi: 10.1038/nprot.2007.321 17853878

19. Poklar N, Lah J, Salobir M, Macek P, Vesnaver G. pH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence. Biochemistry. 1997;36(47):14345–52. doi: 10.1021/bi971719v 9398152

20. Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen. 2001;6(6):429–40. doi: 10.1177/108705710100600609 11788061

21. Yamashita A, Nango E, Ashikawa Y. A large-scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand-binding domains of taste receptor. Protein Sci. 2017;26(11):2291–301. doi: 10.1002/pro.3271 28833672

22. Schellman JA. Macromolecular Binding. Biopolymers. 1975;14:999–1018.

23. Hansch C, Fujita T. ρ−σ−π Analysis. A Method for the Correlation of Biological Activity and Chemical Structure. J Am Chem Soc. 1964;86:1616–26.

24. Asao M, Shimizu R, Nakao K, Fujita T. QREG 2.05. Society of Comuputer Chemistry, Japan1997.

25. Koehl A, Hu H, Feng D, Sun B, Zhang Y, Robertson MJ, et al. Structural insights into the activation of metabotropic glutamate receptors. Nature. 2019;566(7742):79–84. doi: 10.1038/s41586-019-0881-4 30675062

26. Sleigh SH, Seavers PR, Wilkinson AJ, Ladbury JE, Tame JR. Crystallographic and calorimetric analysis of peptide binding to OppA protein. J Mol Biol. 1999;291(2):393–415. doi: 10.1006/jmbi.1999.2929 10438628

27. Gilli P, Ferretti V, Gilli G, Borea PA. Enthalpy-Entropy Compensation in Drug-Receptor Binding. J Phys Chem-Us. 1994;98(5):1515–8. doi: 10.1021/j100056a024

28. Assadi-Porter FM, Radek J, Rao H, Tonelli M. Multimodal Ligand Binding Studies of Human and Mouse G-Coupled Taste Receptors to Correlate Their Species-Specific Sweetness Tasting Properties. Molecules. 2018;23(10). doi: 10.3390/molecules23102531 30282936

29. Luan CH, Light SH, Dunne SF, Anderson WF. Ligand screening using fluorescence thermal shift analysis (FTS). Methods Mol Biol. 2014;1140:263–89. doi: 10.1007/978-1-4939-0354-2_20 24590724

30. Shrake A, Ross PD. Ligand-induced biphasic protein denaturation. J Biol Chem. 1990;265(9):5055–9. 2318882

31. Bjork I, Pol E. Biphasic transition curve on denaturation of chicken cystatin by guanidinium chloride. Evidence for an independently unfolding structural region. FEBS Lett. 1992;299(1):66–8. doi: 10.1016/0014-5793(92)80102-m 1544477

32. Olofsson L, Felekyan S, Doumazane E, Scholler P, Fabre L, Zwier JM, et al. Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy. Nat Commun. 2014;5:5206. doi: 10.1038/ncomms6206 25323157

33. Manglik A, Kobilka B. The role of protein dynamics in GPCR function: insights from the beta2AR and rhodopsin. Curr Opin Cell Biol. 2014;27:136–43. Epub 2014/02/19. doi: 10.1016/j.ceb.2014.01.008 24534489

34. Nie Y, Vigues S, Hobbs JR, Conn GL, Munger SD. Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli. Curr Biol. 2005;15(21):1948–52. Epub 2005/11/08. doi: 10.1016/j.cub.2005.09.037 16271873

35. Maitrepierre E, Sigoillot M, Le Pessot L, Briand L. Recombinant expression, in vitro refolding, and biophysical characterization of the N-terminal domain of T1R3 taste receptor. Protein Expr Purif. 2012;83(1):75–83. doi: 10.1016/j.pep.2012.03.006 22450161

36. Jiang P, Cui M, Zhao B, Snyder LA, Benard LM, Osman R, et al. Identification of the cyclamate interaction site within the transmembrane domain of the human sweet taste receptor subunit T1R3. J Biol Chem. 2005;280(40):34296–305. doi: 10.1074/jbc.M505255200 16076846

37. Jiang P, Ji Q, Liu Z, Snyder LA, Benard LM, Margolskee RF, et al. The cysteine-rich region of T1R3 determines responses to intensely sweet proteins. J Biol Chem. 2004;279(43):45068–75. doi: 10.1074/jbc.M406779200 15299024

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