Kinetics and molecular properties of pheromone binding and release
Kinetics and molecular properties of pheromone binding and release
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DOI：
10.1073/pnas.0501447102
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发表时间：
2005-04-12
影响因子：
11.1
通讯作者：
Tsuruda, JM
中科院分区：
文献类型：
--
作者：
Leal, WS;Chen, AM;Tsuruda, JM
瞬时动力学研究表明，家蚕蛾(Bombyx Mori)的信息素结合蛋白(BmorPBP)对信息素(Bombykol)的摄取以0.068+/-0.01µM-1.s(-1)的“开启”速率进行。当感觉器淋巴中的PBP浓度较高时(10 MM)，信息素在体内的摄取半衰期约为1ms。与信息素释放相关的pH依赖性构象变化(BmorPBP(B)->；BmorPBP(A))为一级反应(k=74.1+/-0.32 S(-1)；t(1/2)，9.3ms)。在生理条件下，这两种反应的半衰期都在毫秒左右，这是昆虫以气味为导向导航所必需的。用一种新的结合分析方法分析了蚕豆酚与天然和突变的多溴联苯的分子相互作用。具有天然构象的重组蛋白(BmorPBP)在pH 7时表现出高亲和力(K-D=105 nM)，而在pH 5时表现出低亲和力(K-D=1,600 nM)。一个缺失C-末端片段的蛋白质(BmorPBP Delta P129-V142)在pH 7和pH 5时与蚕豆蛋白结合，其亲和力与天然蛋白质在pH 7时的亲和力相同，表明C-末端片段在低pH时是阻止结合所必需的。与三种突变蛋白(BmorPBPW37F、BmorPBPW127F和BmorPBPW37A)的结合研究表明，用Phe或Ala取代Trp-37或用Phe取代Trp-127不影响与家蚕酚的结合亲和力。荧光研究揭示了Trp-37和Trp-127排放对整体荧光的贡献。
Transient kinetic studies have shown that the uptake of the pheromone (bombykol) of the silkworm moth (Bombyx mori), by its pheromone-binding protein (PBP) BmorPBP, proceeds with an "on" rate of 0.068 +/- 0.01 mu M-1.s(-1). With the high concentration of PBP in the sensillar lymph (10 mM), the half-life for the uptake of pheromone in vivo is approximate to 1 ms. A pH-dependent conformational change (BmorPBP(B) -> BmorPBP(A)), associated with the release of pheromone, is a first-order reaction (k = 74.1 +/- 0.32 s(-1); t(1/2), 9.3 ms). Under physiological conditions, both reactions proceed with half-life times on the order of milliseconds, as is required for odorant-oriented navigation in insects. Molecular interactions of bombykol with both native and mutated PBPs were analyzed by a novel binding assay. A recombinant protein with the native conformation (BmorPBP) showed high binding affinity (K-D = 105 nM) at pH 7 but low affinity (K-D = 1,600 nM) at pH 5, when tested at both low and high KCl concentrations. A protein with a C-terminal segment deleted (BmorPBP Delta P129-V142) was found to bind bombykol at pH 7 and at pH 5 with the same affinity as the native protein at pH 7, indicating that the C-terminal segment is essential for preventing binding at low pH. Binding studies with three mutated proteins (BmorPBPW37F, BmorPBPW127F, and BmorPBPW37A) showed that replacing Trp-37 (with Phe or Ala) or Trp-127 (with Phe) did not affect the binding affinity to bombykol. Fluorescence studies shed light on the contributions of Trp-37 and Trp-127 emissions to the overall fluorescence.