Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.)

被引:81
作者
Briand, L
Nespoulous, C
Huet, JC
Takahashi, M
Pernollet, JC
机构
[1] INRA, Unite Rech 477, F-78352 Jouy En Josas, France
[2] Univ Paris 11, CNRS, UMR 216, F-91405 Orsay, France
[3] Univ Paris 11, Inst Curie, Orsay, France
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2001年 / 268卷 / 03期
关键词
dimerization; insect; odorant binding; protein; olfaction; pheromone;
D O I
10.1046/j.1432-1327.2001.01927.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In insects, the transport of airborne, hydrophobic odorant:; and pheromones through the sensillum lymph is generally thought to be accomplished by odorant-binding proteins (OBPs). We report the structural and functional properties of a honeybee OBP called ASP2, heterologously expressed by the yeast Pichia pastoris. ASP2 disulfide bonds wen: assigned after classic trypsinolysis followed by ion-spray mass spectrometry combined with microsequencing. The pairing [Cys(I)-Cys(III), Cys(II)-Cys(V), Cys(IV)-Cys(VI)] was found to be identical to that of Bombyx mori OBP: suggesting that this pattern occurs commonly throughout the highly divergent insect OBPs. CD measurements revealed that ASP2 is mainly constituted of alpha helices, like other insect OBPs, but different from lipocalin-like vertebrate OBPs. Gel filtration analysis showed that ASP2 is homodimeric at neutral pH, but monomerizes upon acidification or addition of a chaotropic agent. A general volatile-odorant binding assay allowed us to examine the uptake of some odorants and pheromones by ASP2. Recombinant ASP2 bound all tested molecules, except beta -ionone, which could not interact with it at all. The affinity constants of ASP2 for these ligands, determined at neutral pH by isothermal titration calorimetry, are in the micromolar range, as observed for vertebrate OBP. These results suggest that odorants occupy three binding sites per dimer, probably one in the core of each monomer and another whose location and biological role are questionable. At acidic pH, no binding was observed, in correlation with monomerization and a local conformational change supported by CD experiments.
引用
收藏
页码:752 / 760
页数:9
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