6533b7dbfe1ef96bd1271223

RESEARCH PRODUCT

Expression hétérologue, repliement in vitro et caractérisation biophysique du domaine N-terminal de la sous-unité T1R3 du récepteur humain au goût sucré

subject

description

The sweet taste receptor is a heterodimer composed of two subunits called T1R2 and T1R3. Each subunit belongs to the class C of G protein-coupled receptors and is constituted by a large extracellular N-terminal domain (NTD) linked to the transmembrane domain by a cysteine-rich region. It has been shown that T1R2 and T1R3 NTDs are both able to bind natural sugars and sucralose with distinct affinities and undergo ligand-dependent conformational change (Nie et al., Curr Biol, 2005). However, the binding properties of T1R3 NTD and the relative contribution of the two subunits to the heterodimeric receptor function remained largely unknown. To characterize the binding properties of each subunit in greater depth a large quantity of proteins is required to use biochemical, biophysical and structural approaches. To accomplish this goal, we took advantage of bacterial expression strategy, which has been successfully used to produce functional mouse T1R2 and T1R3 NTDs (Nie et al., Curr Biol, 2005). Human T1R3 NTD was expressed in high level in Escherichia coli as insoluble aggregated protein (inclusion bodies). Transferring this protein into its native state by in vitro refolding requires screening to find buffer conditions and suitable additives. We established a factorial screen to detect folded functional T1R3 NTD based on intrinsic tryptophan fluorescence quenching by sucralose (known to bind mT1R3 NTD) and identified positive synergistic interactions between additives on refolding of T1R3 NTD. The soluble T1R3 NTD protein was then purified and characterized using electrophoresis, gel filtration, fluorescence and circular dichroism spectroscopy. T1R3 NTD is properly refolded and able to bind saccharide compounds with physiological relevant affinities. As expected, one free thiol could be measured in T1R3 NTD using Ellman’s assay suggesting that except one, all the cysteines are involved in disulfide linkages. This presence of this free cystein was also confirmed by mass spectrometry identification of the fluorescent peptide resulting from trypsin digestion. This free cysteine was used to covalently attach an environmentally sensitive fluorophore. This study also revealed that calcium and magnesium was able to bind T1R3 NTD. Due to the high quantities of functional NTD T1R3, the interactions with some sweeteners were characterized using microcalorimetry. Interestingly, we confirmed that T1R3 NTD is also able to bind numerous sweeteners with physiological affinities, suggesting that T1R3 NTD plays an important role in sweetener recognition.