Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1)- Functional and structural characterization

Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) pr esent at high concentrations (approximate to 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of ph eromones. By expression in Escherichia coil, we have obtained large quantit ies (2-3 mg.L-1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). Thes e quantities are compatible with the requirements of X-ray and NMR studies. The recombinant protein has been characterized by native-polyacrylamide ge l electrophoresis, Western blotting, N-terminal sequencing, mass spectromet ry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a str uctural analogue, Z11-16:TFMK (cis-11-hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend pre vious findings on PBPs. MbraPBP1 and two PBPs from different species have b een found to exist as dimers under nondenaturing conditions. The CD and str uctural prediction data confirm a markedly helical structure for insect PBP s rather than the P-barrel fold found in vertebrates odorant binding protei ns. We have tentatively identified the location of the helices and the shor t P-strands with respect to the binding site. Currently we have obtained sm all diffracting crystals of the recombinant MbraPBP1 and determined their s pace group and molecular content.