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

Workflow of the study.

<p>Strategy to improve knowledge of olfactory perception and biological roles of odorant molecules. First an OR-OR association network identifies novel odorant-OR interactions for odorant candidates. Second, pathways linked to proteins are integrated in the OR-OR network allowing deciphering odor-disease connections. The last step involves scoring and ranking of odorant candidates for biological targets within the pharmacological space.</p

List of ORs predicted to interact with molecules carrying the “anis” note.

<p>List of ORs predicted to interact with molecules carrying the “anis” note.</p

Efficient Production and Characterization of the Sweet-Tasting Brazzein Secreted by the Yeast Pichia pastoris

Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and ¹H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms

Concentration-response curves of odorants for the human cannabinoid receptor CB1.

<p>Predicted compounds, tributyl acetyl citrate, 2-nonanone and 2-phenylethyl hexanoate acted as inverse agonists. GloSensor assays were carried out in the absence (•) or in the presence (○) of pertussis toxin-treated cells. Data points and EC₅₀ values are means ± s.e.m. from three experiments.</p