Loı̈c Briand

Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac protein

Aphrodisin is a soluble glycoprotein of hamster vaginal discharges, which stimulates male copulatory behavior. Natural aphrodisin was purified and its post‐translational modifications characterized by MALDI‐MS peptide mapping. To evaluate its ability to bind small volatile ligands, the aphrodisiac protein was expressed in the yeast Pichia pastoris as two major isoforms differing in their glycosylation degree, but close in conformation to the natural protein. Dimeric recombinant aphrodisins were equally able to efficiently bind odors (2‐isobutyl‐3‐methoxypyrazine and methyl thiobutyrate) and a pheromone (dimethyl disulfide), suggesting that they could act as pheromone carriers instead of, or in addition to, direct vomeronasal neuron receptor activators.

Relationships Between Molecular Structure and Perceived Odor Quality of Ligands for a Human Olfactory Receptor

Perception of thousands of odors by a few hundreds of olfactory receptors (ORs) results from a combinatorial coding, in which one OR recognizes multiple odorants and an odorant is recognized by a specific group of ORs. Moreover, odorants could act both as agonists or antagonists depending on the OR. This dual agonist-antagonist combinatorial coding is in good agreement with behavioral and psychophysical observations of mixture perception. We previously described the odorant repertoire of a human OR, OR1G1, identifying both agonists and antagonists. In this paper, we performed a 3D-quantitative structure-activity relationship (3D-QSAR) study of these ligands. We obtained a double-alignment model explaining previously reported experimental activities and permitting to predict novel agonists and antagonists for OR1G1. These model predictions were experimentally validated. Thereafter, we evaluated the statistical link between OR1G1 response to odorants, 3D-QSAR categorization of OR1G1 ligands, and their olfactory description. We demonstrated that OR1G1 recognizes a group of odorants that share both 3D structural and perceptual qualities. We hypothesized that OR1G1 contributes to the coding of waxy, fatty, and rose odors in humans.

Structural Basis of the Broad Specificity of a General Odorant-Binding Protein from Honeybee

General odorant-binding proteins (GOBPs) are believed to transport a wide range of volatile hydrophobic molecules across the aqueous sensillum lymph toward olfactory receptors in insects. GOBPs are involved in the first step of odorant recognition, which has a great impact in agriculture and in insect-mediated human disease control. We report here the first structural study of a GOBP, the honeybee ASP2, in complex with a small hydrophilic ligand. The overall fold of the NMR structure of ASP2 consists of the packing of six alpha-helices creating an internal cavity and closely resembles that of the related pheromone-binding proteins (PBPs). The predominantly hydrophobic internal cavity of ASP2 provides additional possible interactions (pi-stacking, electrostatic contact) for ligand binding. We also show that the internal cavity of ASP2 has the ability to bind ligands of different structures and properties, including a hydrophobic component of the floral scent [2-isobutyl-3-methoxypyrazine (IBMP)] and a small hydrophilic ligand. We further demonstrate that IBMP binds ASP2 with two stable alternative conformations inside the ASP2 binding pocket. The (15)N NMR relaxation study suggests that significant backbone mobility occurs at the ligand entry site at the millisecond rate, which likely plays a role in the recognition and the uptake-release mechanism of ligands by ASP2. We propose that the broad ligand specificity of GOBPs compared with PBPs is conferred by the cumulative effects of weak nonspecific protein-ligand interactions and of enhanced protein internal dynamics at the ligand entry site.

Impact of the lysine-188 and aspartic acid-189 inversion on activity of trypsin

The impact of the charge rearrangement on the specificity of trypsin was tested by an inversion of sequence K188D/D189K maintaining the integrity of the charges of the substrate binding pocket when switching their polarity. In native trypsin, aspartate 189 situated at the bottom of the primary substrate binding pocket interacts with arginine and lysine side chains of the substrate. The kinetic parameters of the wild‐type trypsin and K188D/D189K mutant were determined with synthetic tetrapeptide substrates. Compared with trypsin, the mutant K188D/D189K exhibits a 1.5‐ to 6‐fold increase in the K m for the substrates containing arginine and lysine, respectively. This mutant shows a ∼30‐fold decrease of its k cat and its second‐order rate constant k cat/K m decreases ∼40‐ and 150‐fold for substrates containing arginine and lysine, respectively. Hence, trypsin K188D/D189K displays a large increase in preference for arginine over lysine.

Effect of pea and bovine trypsin inhibitors on wild-type and modified trypsins

In order to modify the catalytic properties of trypsin, lysine‐188 (S1) of the substrate binding pocket was substituted by an aromatic amino acid residue (Phe, Tyr, Trp) or by a histidyl residue. Two other mutants were obtained by displacement or elimination of the negative charge of aspartic acid‐189 (K188D/D189K and G187W/K188F/D189Y, respectively). The high affinity inhibitors, like PSTI II and BPTI, behaved as specific substrates of the trypsin and its mutants. Their inhibiting effect toward modified trypsins was studied. The bovine inhibitor had a higher affinity for all tested enzymes than pea inhibitor. The inhibition constants differed according to the mutations on the protease.

Dynamics of Odorant Binding to Thin Aqueous Films of Rat-OBP3

Uptake, retention and release of 5 selected odorants (benzaldehyde, 2-methylpyrazine, 2-isobutyl-3-methoxypyrazine, 2-isobutylthiazole, and 2,4,5-trimethylthiazole) by recombinant rat odor-binding protein 3 (rat-OBP3) were measured in a model system under nonequilibrium conditions. Gaseous odorants were introduced into a 100 mm section of a polar deactivated capillary in which aqueous rat-OBP3 films were formed to mimic the olfactory epithelium (OE), and the change in the gas-phase concentration of the outflow gas was monitored in real time using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The 5 odorants were chosen because they exhibited a broad range of dissociation constants with rat-OBP3 and because they were amenable to detection by on-line APCI-MS. All 5 odorants were quantitatively bound by rat-OBP3, which resulted in an effective concentration of the odorants in the aqueous layer (about 50 000-fold). Odorant release from the rat-OBP3-odorant complex into the gas phase showed that odorant release was governed by the dissociation constant of the complex and the flow rate of odorant-free air. When 2 odorants were introduced into the system, odorant uptake and release were influenced by the method of introduction and their relative affinities for the protein. Because rat-OBP3 exhibits typical odorant-binding characteristics, the results not only provide fundamental information on the kinetics of odorant mass transfer induced by the presence of OBPs in the olfactory mucus layer but also support the possibility that vertebrate OBPs may facilitate the accumulation of odorants in the OE.

Engineering of trypsin and its impact on β-casein processing

Tryptic processing of β-casein yields several important nutraceutic and nutritious peptides. However, a final product peptide (1-105) stops the processing, inhibiting the enzyme. In attempt to modulate catalytic properties of this protease, K188 was replaced with aromatic amino acid residues. This aimed amplification of local hydrophobic and electrostatic interactions at the substrate binding site. The catalytic properties of obtained mutants (K188F, K188Y, and K188W) were measured at pH 7, 8, 9, and 10 with synthetic substrates and β-casein. Kinetic analysis revealed that all the mutants conserve the capacity to split peptide bonds involving arginyl and lysyl residues. However, depending on mutation, the optimum pH of activity changes. As shown only by proteolysis of a natural substrate, produced mutants cleaved near 30 new peptide bonds compared to wild-type trypsin, 8 of them involving asparagine and glutamine amino acids. Some of the new cleavage sites can be related to the nature of the amino acid residue introduced in position 188. Consequently, only the joint use of several methods (synthetic substrate, protein substrate, influence of pH) can help to define better the differences of catalytic properties of wild-type and mutant proteases. Modifications introduced by the mutations are at the origin of the alteration of the specificity of the studied enzymes which are cleaving β-casein in many places, hydrolysing well the fragment 1-105. Since many tryptic inhibitors contain amidated Glu and Asp, and form amyloid structures, the new mutants could be used in hydrolysing resistant proteic structures.

Evidence for antagonism between odorants at olfactory receptor binding in humans

Abstract The odorant repertoire of two human olfactory receptors (ORs) belonging to two major phylogenetic classes representing ORs from aquatic (class I) and terrestrial animals (class II) were elucidated. For this purpose, a new biomimetic screening assay based on calcium imaging on HEK293 cells expressing an OR and the promiscuous G protein G α16 was developed. Class I OR52D1 is functional, exhibiting a narrow repertoire related to that of its orthologous murine OR, demonstrating that this class I OR is not an evolutionary relic. In contrast, class II OR1G1 was broadly tuned towards odorants of 9 to 10 carbons chain length, with diverse functional groups. The existence of antagonism between odorants at level of OR binding was demonstrated. OR1G1 antagonists were observed to be OR specific and structurally related to its agonists with a shorter size.