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Recombinant pheromone binding protein 1 from Mamestra brassicae (MbraPBP1). Functional and structural
V Campanacci1, S Longhi, P Nagnan-Le Meillour
1AFMB, UPR 9039-CNRS, Marseille, France.
European Journal of Biochemistry
|September 22, 1999
Summary
We successfully produced recombinant Mamestra brassicae PBP1 (MbraPBP1) for structural studies. This pheromone binding protein binds specific pheromones and exhibits a helical structure, differing from vertebrate odorant binding proteins.
Area of Science:
- Biochemistry
- Structural Biology
- Insect Physiology
Background:
- Pheromone binding proteins (PBPs) are crucial for insect olfaction, particularly in Lepidoptera.
- PBPs are abundant in the sensillum lymph of antennae, facilitating pheromone perception.
Purpose of the Study:
- To produce recombinant Mamestra brassicae PBP1 (MbraPBP1) in large quantities for structural and functional analysis.
- To characterize the biophysical and biochemical properties of MbraPBP1.
Main Methods:
- Recombinant protein expression in Escherichia coli.
- Characterization using native-PAGE, Western blotting, N-terminal sequencing, mass spectrometry, gel filtration, circular dichroism (CD), and NMR.
- Pheromone binding assays and crystallographic studies.
Main Results:
- High yields of pure, soluble MbraPBP1 were obtained, suitable for structural studies.
- MbraPBP1 demonstrated binding affinity for its specific pheromone and a structural analogue.
- CD and structural predictions indicated a predominantly helical structure for MbraPBP1, distinct from vertebrate odorant binding proteins.
- MbraPBP1 exists as a dimer under nondenaturing conditions.
Conclusions:
- Recombinant MbraPBP1 production is feasible for detailed structural investigations.
- MbraPBP1 possesses a helical fold characteristic of insect PBPs, with identified potential binding site regions.
- The dimeric nature and helical structure provide insights into PBP function in pheromone perception.