Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori

Disulfide bond formation is the only known posttranslational modification o f insect pheromone binding proteins (PBPs), In the PBPs from moths (Lepidop tera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkag e or redox status, We used a multiple approach of enzymatic digestion, chem ical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, follo wed by digestion with endoproteinase Lys-C to determine the disulfide conne ctivity in the PBP from Bombyx mori (BmPBP), Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spe ctrometry (LC/ESI-MS) and protein sequencing supported the assignment of di sulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The di sulfide linkages were identical in the protein obtained by periplasmic expr ession in Escherichia coli and in the native BmPBP. (C) 1999 Federation of European Biochemical Societies.