1Architecture et Fonction des Macromolécules Biologiques, URA 9039, CNRS, IFR1, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
2INRA-Unité de Phytopharmacie et Médiateurs Chimiques- Bât. A, route de Saint-Cyr- 78026 Versailles Cedex, France.
Pheromone binding proteins (PBPs) are small proteins (17 kDa in average) present at high concentrations (ca 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. After subcloning the gene of the PBP1 of Mamestra brassicae, we have developed a method of expression in E. coli and purification, which allowed to obtain large quantities (2-3 mg/l) of pure, soluble, recombinant MbraPBP1 without the use of an unfolding-refolding procedure. The recombinant protein has been characterized by native-PAGE, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Our results on MbraPBP1 confirm and extend previous findings on PBPs of other species. MbraPBP1 has been found to be dimer under non-denaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs. We have tentatively identified the location of the helices and the short beta-strands, with respect to the binding site. Crystals of rPBP1 have been obtained. They belong to the P222 space group with the unit cell a=38.22, b=60.21, c=134.27. One data set to 2.7 Å resolution has been collected at the ESRF. Seleno-methionine substituted rPBP1 has been produced that should allow the phasing by MAD method and the obtention of the 3D structure of MbraPBP1, hus revealing the structural details of the previously unknown fold for this class of transport proteins. Our next target is to perform site-directed mutagenesis of putatively key residues.