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

Citation
V. Campanacci et al., Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1)- Functional and structural characterization, EUR J BIOCH, 264(3), 1999, pp. 707-716
Citations number
50
Categorie Soggetti
Biochemistry & Biophysics
Journal title
EUROPEAN JOURNAL OF BIOCHEMISTRY
ISSN journal
00142956 → ACNP
Volume
264
Issue
3
Year of publication
1999
Pages
707 - 716
Database
ISI
SICI code
0014-2956(199909)264:3<707:RPBP1F>2.0.ZU;2-9
Abstract
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.