Ligand-binding properties and structural characterization of a novel rat odorant-binding protein variant

After characterization of a novel odorant-binding protein (OBP) variant iso lated from the rat nasal mucus, the corresponding cDNA was cloned by RT-PCR . Recombinant OBP-1F, the sequence of which is close to that of previously reported rat OBP-1, has been secreted by the yeast Pichia pastoris at a con centration of 80 mg.L-1 in a form identical to the natural protein as shown by MS, N-terminal sequencing and CD. We observed that, in contrast with po rcine OBP-1, purified recombinant OBP-1F is a homodimer exhibiting two disu lfide bonds (C44-C48 and C63-C155), a pairing close to that of hamster aphr odisin. OBP-1F interacts with fluorescent probe 1-aminoanthracene (1-AMA) w ith a dissociation constant of 0.6 +/- 0.3 mu m. Fluorescence experiments r evealed that 1-AMA was displaced efficiently by molecules including usual s olvents such as EtOH and dimethylsulfoxide. Owing to the large OBP-1F amoun ts expressed, we set up a novel biomimetic assay (volatile-odorant binding assay) to study the uptake of airborne odorants without radiolabelling and attempted to understand the odorant capture by OBP in the nasal mucus under natural conditions. The assay permitted observations on the binding of air borne odorants of different chemical structures and odors (2-isobutyl-3-met hoxypyrazine, linalool, isoamyl acetate, 1-octanal, 1-octanol, dimethyl dis ulfide and methyl thiobutyrate). Uptake of airborne odorants in nearly phys iological conditions strengthens the role of OBP as volatile hydrophobic od orant carriers in the mucus of the olfactory epithelium through the aqueous barrier towards the chemo-sensory cells.