Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1). Functional and structural characterization.
Pheromone binding proteins (PBPs) are small proteins (17 kDa on average) present at high concentrations ( approximately 10 mM) in the sensillum lymph of Lepidoptera antennae, where they play a key role in the perception of pheromones. By expression in Escherichia coli, we have obtained large quantities (2-3 mg.L-1) of pure, soluble, Mamestra brassicae PBP1 (MbraPBP1). These quantities are compatible with the requirements of X-ray and NMR studies. The recombinant protein has been characterized by native-polyacrylamide gel electrophoresis, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism, and NMR. Moreover, the recombinant MbraPBP1 has been shown to be able to bind the specific pheromone and a structural analogue, Z11-16:TFMK (cis-11-hexadecenyl trifluoromethyl ketone), in displacement experiments. Our results on MbraPBP1 confirm and extend previous findings on PBPs. MbraPBP1 and two PBPs from different species have been found to exist as dimers under nondenaturing conditions. The CD and structural prediction data confirm a markedly helical structure for insect PBPs rather than the beta-barrel fold found in vertebrates odorant binding proteins. We have tentatively identified the location of the helices and the short beta-strands with respect to the binding site. Currently we have obtained small diffracting crystals of the recombinant MbraPBP1 and determined their space group and molecular content.
Campanacci
V
V
AFMB, UPR 9039-CNRS, Marseille, France.
Longhi
S
S
Nagnan-Le Meillour
P
P
Cambillau
C
C
Tegoni
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003460:Crystallization; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017344:Genes, Insect; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007915:Lepidoptera / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010675:Pheromones / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D017931:DNA Primers; D019476:Insect Proteins; D010675:Pheromones; D011994:Recombinant Proteins