Characterization of cT1R1-NTD using far-UV circular dichroism spectroscopy.

<p>Protein concentration in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1mM DTT and 0.1 mM DDM was approximately 0.2 mg/ml. Light path: 0.01 cm.</p

Strategy used for expression of the cat T1R1-NTD in bacteria.

<p>(A) The N-terminal domain (NTD) of cT1R1 was expressed independently from the transmembrane heptahelical domain (HD), minus a short putative signal peptide (S), and a cysteine-rich region (CRR). (B) The pET28-cT1R1-NTD plasmid encodes a fusion protein that contains an N-terminal His-tag that can be cleaved with thrombin, followed by cT1R1-NTD (Leu21-Ser495) and a C-terminal His-tag. (C) Full-length cT1R1 is presented according to its primary amino acid sequence deduced from DNA sequence. The numerical positions of amino acid residues of cT1R1 are indicated.</p

cT1R1-NTD binds L-amino acids and IMP.

<p>Normalized maximal fluorescence intensity of cT1R1-NTD before and after addition of ligands (100 μM final concentration). L-Cys does not affect cT1R1-NTD fluorescence. Fluorescence of cT1R1-NTD alone was defined as 100% in absence of ligand. Excitation and emission wavelength were 295 nm and 340 nm, respectively. cT1R1-NTD concentration was 0.5 μM. Data values are the mean ± SEMs of more than nine independent replicates of at least three independently refolded protein samples. *, Significantly different from cT1R1-NTD before addition of ligands (one-way ANOVA followed by Dunnett’s, p ≤ 0.05; for L-Arg p ≤ 0.08).</p

Dissociation constants (<i>K</i>_d) values of ligands for cT1R1-NTD.

<p>Dissociation constants (<i>K</i>_d) values of ligands for cT1R1-NTD.</p

SDS-PAGE analysis of purified cT1R1-NTD inclusion bodies.

<p>cT1R1-NTD is indicated with an arrow while the star indicates a band corresponding to a N-terminal fragment of cT1R1-NTD. The proteins were separated by 12% SDS-PAGE and stained with Coomassie blue. The molecular mass markers are in lane M.</p

SEC-MALS analysis of cT1R1-NTD.

<p>The column was equilibrated and cT1R1-NTD eluted with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 mM DDM. The chromatograms show the readings of the light scattering (LS), the differential refractive index (dRI) and UV detectors in red, blue and green, respectively. The scale for the LS detector is shown in the right-hand axis. The thick black line indicates the calculated molecular mass of the eluting protein throughout the chromatogram (scale on the left-hand axis). cT1R1-NTD has a fitted molecular mass of 52.5 kDa; its theoretical monomer molecular mass value is 55.4 kDa.</p