1-Aminoanthracene Transduction into Liposomes Driven by Odorant-Binding Protein Proximity

In this work, the anchorage of pig odorant binding protein (OBP-I) into liposomal membrane was promoted by the fusion of OBP-I with the anchor SP-DS3 peptide and with the (GQ)₂₀ spacer. The presence of the (GQ)₂₀ spacer in the construct confers flexibility to the protein and increases the distance between the OBP binding site and the liposomal surface. The engineered proteins, OBP::SP-DS3 and OBP::(GQ)₂₀::SP-DS3, were produced in Escherichia coli BL21(DE3) and characterized by circular dichroism spectroscopy and MALDI-TOF. The functionalization of liposomes with the OBP proteins was performed through ethanol injection, and similar liposomal anchorage (∼92-97%) was found for both OBP constructs. The effect of OBPs' proximity to the liposomes membrane on 1-aminoanthracene (1-AMA, model ligand) transduction was evaluated by measuring the amount of 1-AMA transduced into liposomes by fluorescence spectroscopy. While protein flexibility, given by the presence of the (GQ)₂₀ spacer, seems to influence the binding efficiency, ∼45% for OBP::(GQ)₂₀::SP-DS3 and ∼29% for OBP::SP-DS3, the distance between the proteins' binding site and the liposomal membrane determines their ability to transduce the 1-AMA into the liposomes (∼23% for OBP::SP-DS3 and ∼19% for OBP::(GQ)₂₀::SP-DS3). The anchorage capacity and proximity effect were confirmed by an experimental control where the wild-type (wt) OBP was added to the liposomes, resulting in low 1-AMA transduction (∼3.5%) and low binding to OBPwt (∼9%). These findings evidence the effect of anchorage, carrier protein's flexibility, and proximity as key features for the entrapment of molecules into the liposomal membrane. The developed OBP-based devices are thus promising anchorage systems for the capture and storage of odors with potential applications in textile and cosmetic industries.