PHOTOLABELING OF ACETYLCHOLINE BINDING PROTEIN BY NICOTINOIDS & NEONICOTINOIDS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 乙酰胆碱结合蛋白(AChBP)是烟碱型ACh受体/离子通道复合体(NAChR)胞外结构域的模型/替代物。与nAChR的胞外结构域一样，AChBP有五个结合ACh或药物的口袋。AChBP由5个同源亚基(同源五聚体)组装而成，ACh结合部位位于亚基之间的界面区。 哺乳动物的nAChR选择性激动剂epbatidine(EPI)和昆虫的nAChR选择性激动剂吡虫啉(IMI，新烟碱类杀虫剂的代表)有一些共同的结构部分，但它们的物理化学性质明显不同。EPI有一个质子化的氮原子(阳离子)，但IMI有一个非质子化的电负性硝基(NO2)药效团。这些结构上的差异决定了哺乳动物和昆虫之间的选择性。作为证据，如果IMI失去硝基(Desnitro-IMI)，它现在是一种哺乳动物选择性化合物。 因此，即使两者埋在同一个空穴中，与EPI的阳离子作用的氨基酸残基(S)和与IMI的负性硝基氧(S)相互作用的氨基酸残基必然是不同的，即结合方向不同。为了探讨选择性相互作用的分子基础，我们设计并制备了两种类型的光亲和配体。AChBP结合阳离子EPI激动剂和电负性IMI杀虫剂。因此，本研究涉及LC/MS/MS分析的实验目标是鉴定这两种光亲和配体标记的特定氨基酸残基(S)。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acetylcholine (ACh) binding protein (AChBP) is the model/surrogate of the extra cellular domain for the nicotinic ACh receptor/ion channel complex (nAChR). As with the extra cellular domain of nAChR, the AChBP has five binding pockets for ACh or drug. The AChBP is assembled with five homologous subunits (homo-pentamer) and the ACh binding site exists on the interface region between subunits. Mammalian selective nAChR agonist epibatidine (EPI) and insect nAChR selective agonist imidacloprid (IMI, the representation neonicotinoid insecticide) share some common structural moieties, but they are distinctly different in their physicochemical properties. EPI has a protonated nitrogen atom (cation), but IMI has a non-protonatable and electronegative nitro (NO2) pharmacophore. These structural differences determine the selectivity between mammals and insects. As a proof, if IMI loses the nitro group (desnitro-IMI), it is now a mammalian selective compound. Therefore, amino acid residue(s) interacting with the cation of EPI and the electronegative nitro oxygen(s) of IMI must be quite different even if both compounds are buried in the identical hole, i.e., the binding orientations are different. To approach the molecular basis for the selective interaction, we designed and prepared two types of photoaffinity ligands. The Aplysia AChBP binds both the cationic EPI agonist and the electronegative IMI insecticide. Therefore, the experimental goals of this study involving the LC/MS/MS analyses are to identify specific amino acid residue(s) labeled by these two types of photoaffinity ligands.