Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.)
Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.)
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DOI：
10.1046/j.1432-1327.2001.01927.x
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发表时间：
2001-02-01
期刊：
影响因子：
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通讯作者：
Pernollet, JC
中科院分区：
文献类型：
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作者：
Briand, L;Nespoulous, C;Pernollet, JC
在昆虫中，空气中的疏水性气味物质和信息素通过感器淋巴的运输通常被认为是由气味结合蛋白（OBP）完成的。我们报告的结构和功能特性的蜜蜂OBP称为ASP 2，异源表达的酵母毕赤酵母。ASP 2二硫键在经典胰蛋白酶解后通过离子喷雾质谱结合微测序进行归属。发现配对[Cys（I）-Cys（III），Cys（II）-Cys（V），Cys（IV）-Cys（VI）]与家蚕OBP的配对相同：表明这种模式通常发生在高度分化的昆虫OBP中。CD测量显示ASP 2与其他昆虫OBP一样主要由α螺旋组成，但与脂质运载蛋白样脊椎动物OBP不同。凝胶过滤分析表明，ASP 2是homodimeric在中性pH值，但在酸化或加入离液剂单体化。一般的挥发性气味结合试验，使我们能够检查吸收的一些气味和信息素的ASP 2。重组ASP 2结合所有测试的分子，除了β-紫罗兰酮，它不能与它相互作用。ASP 2对这些配体的亲和常数，在中性pH下通过等温滴定量热法测定，在微摩尔范围内，如脊椎动物OBP所观察到的。这些结果表明，气味占据每个二聚体的三个结合位点，可能是在每个单体的核心和另一个的位置和生物学作用是值得怀疑的。在酸性pH值下，没有观察到结合，与CD实验支持的单体化和局部构象变化相关。
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.