PHOTOLABELING OF ACETYLCHOLINE BINDING PROTEIN BY NICOTINOIDS & NEONICOTINOIDS

烟碱类化合物对乙酰胆碱结合蛋白进行光标记

• 批准号: 8363771
• 负责人: JOHN E CASIDA
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363771
• 关键词: Acetylcholine; Agonist; Amino Acids; Aplysia; Binding; Binding Proteins; Binding Sites; Biology; Chemicals; DNA Sequence Rearrangement; Environment; Face; Funding; Gated Ion Channel; Grant; Insecticides; Investigation; Label; Ligand Binding; Ligand Binding Domain; Ligands; Maps; Mass Spectrum Analysis; Methionine; Molecular Conformation; Mutagenesis; National Center for Research Resources; Nicotinic Receptors; Pharmaceutical Preparations; Photoaffinity Labels; Play; Positioning Attribute; Principal Investigator; Property; Proteins; Research; Research Infrastructure; Resolution; Resources; Role; Scanning; Site; Source; Structure; Therapeutic Agents; Tyrosine; United States National Institutes of Health; X-Ray Crystallography; comparative; cost; design; extracellular; mutant; neurotransmission; nitrene; receptor; response; structural biology; toxicant

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The nicotinic acetylcholine (ACh) receptor (nAChR) is the prototypical agonist-gated ion channel responsible for rapid excitatory neurotransmission. The nAChR agonists include many toxicants, potential therapeutic agents, and the important neonicotinoid insecticides. Initial attempts to understand receptor-agonist interactions involved site-directed or chimeric mutagenesis, estimating the role of specific region(s) or amino acid(s) on the pharmacological response (electrophysiological or ligand binding). The structural biology approach of high-resolution X-ray crystallography reveals geometries of functional amino acids in the drug-bound state and conformational rearrangement of the protein upon ligand interaction. Alternatively, incorporation of an unnatural amino acid or photoaffinity labeling provides a direct and physiologically-relevant definition of the recognition properties of the ligand binding environment. Binding site interactions of nicotinoid and neonicotinoid agonists have been characterized by comparative structural and chemical biology approaches using mollusk ACh binding protein (AChBP), which is a suitable structural surrogate of the extracellular ligand-binding domain of the nAChR. 5-Azido-6-chloropyridin-3-yl nicotinic photoaffinity probes have played an important role in studying agonist-nAChR interactions. In principle, these probes bind to the specific site and then the reactive nitrene intermediate, generated by photoirradiation, reacts covalently with the target protein. On photoaffinity labeling of Aplysia californica AChBP, the azidochloropyridinyl photoprobes derivatized at only one position at the interface between loop C Y195 on the principal or (+)-face subunit and loop E M116 on the partnering or (-)-face subunit, establishing a bound ligand position and conformation identical to those observed in crystal structures. Interestingly, a nicotinic photoaffinity ligand of this type exclusively labeled the ¿4 subunit of the chick ¿4¿2 nAChR subtype, in which loop C Y225 on the ¿4 subunit and loop E F137 on the ¿2 subunit are spatially equivalent to Y195 and M116, respectively, of AChBP. These observations may be rationalized by the difference in the reactivity between amino acids and the photoactivated probe molecule. Therefore, the present investigation designs a methionine- and tyrosine-scanning approach principally on the loop E domain involving 17 AChBP mutants regarding photoreactivity of the 5-azido-6-chloropyridin-3-yl nicotinic photoprobes, ultimately mapping the specific site undergoing photoderivatization by mass spectrometry analyses and precise position and conformation of the bound ligand.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 烟碱乙酰胆碱（ACH）受体（NACHR）是负责快速兴奋性神经传递的原型激动剂门控通道。 NACHR激动剂包括许多毒性，潜在的治疗剂和重要的新烟碱杀虫剂。最初的尝试了解受体激动剂相互作用涉及位置定向或嵌合诱变，估计特异性区域或氨基酸在药物反应（电生理学或配体结合）中的作用。高分辨率X射线晶体学的结构生物学方法揭示了在药物结合状态和配体相互作用时蛋白质重排的功能氨基酸的几何形状。另外，掺入不自然的氨基酸或光性标签可为配体结合环境的识别特性提供直接且与物理相关的定义。使用软体动物ACH结合蛋白（ACHBP）的比较结构和化学生物学方法，烟碱类和新烟碱激动剂的结合位点相互作用的特征是，这是NACHR的细胞外配体结合结构域的合适结构替代物。 5-氮杂-6-氯吡啶素-3-烟碱光性探针在研究激动剂-NACHR相互作用中起着重要作用。原则上，这些问题与特定位点结合，然后是通过光辐射产生的反应性硝酸盐中间体与靶蛋白共价反应。在Aplysia californica ACHBP的光附近标记上，偶氮氯吡啶基光合过程仅在主要或（+）face c Y195之间的一个位置衍生的位置或（+） - 脸部亚基和环路E m116在伴侣或（ - ）face ununit上，建立一个构造的构造和综合构成的构造，以实现这些构造，以实现这些构造，以实现这些构成。有趣的是，这种类型的烟碱光亲和力配体专门标记了小鸡的4个亚基€4€2 NACHR子类型，其中oop oop c y225上的循环c y225在€4亚基和loop e f137上通常相当于2个亚基，通常等于y195和y195和m116和m116和m116和m116和sar b。这些观察结果可能是通过氨基酸与光活化探针分子之间反应性差异合理化的。因此，本研究设计主要是在涉及17个ACHBP突变体的循环域的方法和酪氨酸扫描方法，该方法涉及5-氮杂-6-氯吡啶-3-基基的烟碱光螺旋体的光电反应性，最终绘制了通过质量谱分析和精确构图的特定范围，并最终映射了特定的光电培训和精确的位置。