Responses of the Pheromone-Binding Protein of the Silk Moth Bombyx mori on a Graphene Biosensor Match Binding Constants in Solution

An electronic biosensor for odors was assembled by immobilizing the silk moth Bombyx mori pheromone binding protein (BmorPBP1) on a reduced graphene oxide surface of a field-effect transistor. At physiological pH, the sensor detects the B. mori pheromones, bombykol and bombykal, with good affinity and specificity. Among the other odorants tested, only eugenol elicited a strong signal, while terpenoids and other odorants (linalool, geraniol, isoamyl acetate, and 2-isobutyl-3-methoxypyrazine) produced only very weak responses. Parallel binding assays were performed with the same protein and the same ligands, using the common fluorescence approach adopted for similar proteins. The results are in good agreement with the sensor’s responses: bombykol and bombykal, together with eugenol, proved to be strong ligands, while the other compounds showed only poor affinity. When tested at pH 4, the protein failed to bind bombykol both in solution and when immobilized on the sensor. This result further indicates that the BmorPBP1 retains its full activity when immobilized on a surface, including the conformational change observed in acidic conditions. The good agreement between fluorescence assays and sensor responses suggests that ligand-binding assays in solution can be used to screen mutants of a binding protein when selecting the best form to be immobilized on a biosensor.

Pheromone binding protein shapes olfactory temporal resolution

Male moths are capable of orienting towards conspecific females using sex pheromones. Since pheromones are distributed as discontinuous plumes owing to air turbulence, tracking intermittent stimuli with high temporal resolution is suggested to be important for efficient localisation. Here, using a pheromone binding protein (BmPBP1) knockout silkmoth, we revealed that the loss of functional pheromone binding protein altered antennal response kinetics resulting in reduced temporal resolution to intermittent pheromone stimuli on the antennae. Behavioural analysis revealed that BmPBP1-knockout males exhibited significantly less straight walking, which occurs when detecting pheromone stimuli, especially to high frequency stimuli. Accordingly, BmPBP1-knockout males took a significantly longer time to locate pheromone sources and females than did wild-type males. Together, BmPBP1 plays a critical role in determining temporal antennal response kinetics and that an appropriate range of temporal sensory and behavioural resolutions is essential for tracking pheromone plumes for efficient pheromone source localisation in the silkmoth.