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Simple Summary: Odorant-binding proteins are soluble proteins abundantly secreted in the nasal mucus of vertebrates. Although their physiological functions are not known, these proteins are suspected to have a carrier role in solubilizing and carrying hydrophobic odorant molecules to the olfactory receptors. Here, we describe the expression of functional mouse mOBP5. The protein produced using bacteria was purified and characterized. We investigated its binding properties using a fluorescent competitive assay and microcalorimetry. Molecular docking experiments revealed hydrophobic residues in the binding cavity potentially involved in the stabilization of the odorant, thus explaining its binding properties. Odorant-binding proteins (OBPs) are abundant soluble proteins secreted in the nasal mucus of a variety of species that are believed to be involved in the transport of odorants toward olfactory receptors. In this study, we report the functional characterization of mouse OBP5 (mOBP5). mOBP5 was recombinantly expressed as a hexahistidine-tagged protein in bacteria and purified using metal affinity chromatography. The oligomeric state and secondary structure composition of mOBP5 were investigated using gel filtration and circular dichroism spectroscopy. Fluorescent experiments revealed that mOBP5 interacts with the fluorescent probe N-phenyl naphthylamine (NPN) with micromolar affinity. Competitive binding experiments with 40 odorants indicated that mOBP5 binds a restricted number of odorants with good affinity. Isothermal titration calorimetry (ITC) confirmed that mOBP5 binds these compounds with association constants in the low micromolar range. Finally, protein homology modeling and molecular docking analysis indicated the amino acid residues of mOBP5 that determine its binding properties. [ABSTRACT FROM AUTHOR]

Olfactory perception begins when odorous substances interact with specialized receptors located on the surface of dedicated sensory neurons. The recognition of smells depends on a complex mechanism involving a combination of interactions between an odorant and a set of odorant receptors (ORs), where molecules are recognized according to a combinatorial activation code of ORs. Although these interactions have been studied for decades, the rules governing this ligand recognition remain poorly understood, and the complete combinatorial code is only known for a handful of odorants. We have carefully analyzed experimental results regarding the interactions between ORs and molecules to provide a status report on the deorphanization of ORs, i.e. the identification of the first agonist for a given sequence. This meticulous analysis highlights the influence of experimental methodology (cell line or readout) on molecule-receptor association results and shows that 83% of the results are conserved regardless of experimental conditions. The distribution of another key parameter, EC50, indicates that most OR ligand activities are in the micromolar range and that impurities could lead to erroneous conclusions. Focusing on the human ORs, our study shows that 88% of the documented sequences still need to be deorphanized. Finally, we also estimate the size of the ORs' recognition range, or broadness, as the number of odorants activating a given OR. By analogously estimating molecular broadness and combining the two estimates we propose a basic framework that can serve as a comparison point for future machine learning algorithms predicting OR-molecule activity., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)