6533b828fe1ef96bd1288bbc

RESEARCH PRODUCT

Characterization of ligands for the human T1R2 sweet taste receptor (obtention "Manfred Rothe Poster Award")

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description

poster ayant obtenu le prix "Manfred Rothe Poster Award"; The human sweet-taste receptor is a heterodimer composed of two class C G-protein coupled receptors (GPCR), T1R2 and T1R3. Both subunits possess a large N-terminal domain (NTD) linked to a heptahelical transmembrane domain (TM) by a cysteine rich domain (CRD) [1]. Cellular assays, molecular docking and mutagenesis studies have revealed that small sweet ligands interact primarily with the NTD of T1R2 (T1R2-NTD) [2, 3]. To further elucidate the contribution of T1R2-NTD to small sweet ligand binding, we overexpressed T1R2-NTD in Escherichia coli as inclusion bodies [4, 5]. Human T1R2-NTD was refolded and the correct formation of secondary structures was verified using circular dichroism. T1R2-NTD was then characterized for its ability to interact with a number of known sweet tasting ligands using intrinsic tryptophan fluorescence. We found the measured affinities with the wild-type T1R2-NTD are in agreement with cellular based assays conducted on the full-length receptor and sensory analysis in humans. Using site-directed mutagenesis, we confirmed the binding site of sucralose. This strategy will be used to study the binding mechanism of other sweeteners including sweet-tasting proteins, such as brazzein and monellin. 1.Nelson, G., et al., Mammalian sweet taste receptors. Cell, 2001. 106(3): p. 381-90.2.Masuda, K., et al., Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds. PLoS One, 2012. 7(4): p. e35380.3.Xu, H., et al., Different functional roles of T1R subunits in the heteromeric taste receptors. Proc Natl Acad Sci U S A, 2004. 101(39): p. 14258-63.4.Maitrepierre, E., et al., Recombinant expression, in vitro refolding, and biophysical characterization of the N-terminal domain of T1R3 taste receptor. Protein Expr Purif, 2012. 83(1): p. 75-83.5.Nie, Y., et al., Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli. Curr Biol, 2005. 15(21): p. 1948-52.