Probe-dependence of competitive fluorescent ligand binding assays to odorant-binding proteins

Ligand binding experiments between small chemicals and proteins and the evaluation of dissociation constants of their complexes in competitive binding assays often rely on displacement of reporter probes by the tested ligand. The most widely adopted protocol uses a fluorescent ligand which changes its emission spectrum when bound to a protein. A decrease of fluorescence, caused by the addition of a second ligand to the complex is generally interpreted as displacement of the fluorescent probe by the ligand, and therefore as a measure of the affinity of the ligand for the protein. Working with an odorant-binding protein (OBP), we found drastic differences in the calculated affinities when using 1-aminoanthracene or N-phenyl-1-naphthylamine as the fluorescent reporter. This fact was quite unexpected, as OBPs are small compact proteins with a single binding pocket without allosteric sites. Such observation raises doubts on the reliability of the fluorescent binding assay, perhaps the most widely used approach to evaluate affinities of small organic compounds to OBPs and other binding proteins. We recommend that the results of fluorescent binding experiments with OBPs should be confirmed by using two different probes or alternative methods. The reliability of current protocols for ligand binding assays is rather limited, while we still wait for a label-free approach that could be simple, fast and free from the use of radioactive tracers.