title: Recombinant expression, in vitro refolding, and biophysical characterization of the N-terminal domain of T1R3 taste receptor.
abstract: The sweet taste receptor is a heterodimeric receptor composed of the T1R2 and T1R3 subunits, while T1R1 and T1R3 assemble to form the umami taste receptor. T1R receptors belong to the family of class C G-protein coupled receptors (GPCRs). In addition to a transmembrane heptahelical domain, class C GPCRs have a large extracellular N-terminal domain (NTD), which is the primary ligand-binding site. The T1R2 and T1R1 subunits have been shown to be responsible for ligand binding, via their NTDs. However, little is known about the contribution of T1R3-NTD to receptor functions. To enable biophysical characterization, we overexpressed the human NTD of T1R3 (hT1R3-NTD) using Escherichia coli in the form of inclusion bodies. Using a fractional factorial screen coupled to a functional assay, conditions were determined for the refolding of hT1R3-NTD. Far-UV circular dichroism spectroscopic studies revealed that hT1R3-NTD was well refolded. Using size-exclusion chromatography, we found that the refolded protein behaves as a dimer. Ligand binding quantified by tryptophan fluorescence quenching and microcalorimetry showed that hT1R3-NTD is functional and capable of binding sucralose with an affinity in the millimolar range. This study also provides a strategy to produce functional hT1R3-NTD by heterologous expression in E. coli; this is a prerequisite for structural determination and functional analysis of ligand-binding regions of other class C GPCRs.
authors.affiliation: Centre des Sciences du Goût et de l'Alimentation, UMR6265 CNRS, UMR1324 INRA, Université de Bourgogne, F-21000 Dijon, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Maîtrepierre
authors.forename: Maud
authors.initials: M
authors.lastname: Sigoillot
authors.forename: Laurence
authors.initials: L
authors.lastname: Le Pessot
authors.forename: Loïc
authors.initials: L
authors.lastname: Briand
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D058849:Protein Refolding; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013395:Sucrose / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C431926:taste receptors, type 1; D013395:Sucrose; C026285:trichlorosucrose