Two Engineered OBPs with opposite temperature-dependent affinities towards 1-aminoanthracene.

Engineered odorant-binding proteins (OBPs) display tunable binding affinities triggered by temperature alterations. We designed and produced two engineered proteins based on OBP-I sequence: truncated OBP (tOBP) and OBP::GQ20::SP-DS3. The binding affinity of 1-aminoanthracene (1-AMA) to these proteins revealed that tOBP presents higher affinity at 25 °C (kd = 0.45 μM) than at 37 °C (kd = 1.72 μM). OBP::GQ20::SP-DS3 showed an opposite behavior, revealing higher affinity at 37 °C (kd = 0.58 μM) than at 25 °C (kd = 1.17 μM). We set-up a system containing both proteins to evaluate their temperature-dependent binding. Our data proved the 1-AMA differential and reversible affinity towards OBPs, triggered by temperature changes. The variations of the binding pocket size with temperature, confirmed by molecular modelling studies, were determinant for the differential binding of the engineered OBPs. Herein we described for the first time a competitive temperature-dependent mechanism for this class of proteins.