Binding Site Structure of Insect Odorant Receptors.

描述（由申请人提供）：许多昆虫物种通过作为疾病传播媒介或通过造成农业损害对人类健康产生负面影响。化学感受器，气味受体（OR）和味觉受体，是昆虫发挥这些作用的能力的关键。昆虫化学感受受体是一种全新的受体类型，与任何已知的受体家族（包括哺乳动物化学感受受体）没有遗传或结构关系。因此，这些受体是开发用于昆虫控制的新化合物的有吸引力的靶标。要实现这一目标，需要对昆虫化学感受器的结构和功能有深入的了解。然而，这些受体最显著的特征之一是我们对其结构知之甚少。在这个项目中，我们将确定昆虫ORs的结构特征，这些结构特征在气味结合和识别中非常重要。我们已经建立了一个强大的功能测定昆虫气味受体使用非洲爪蟾卵母细胞表达系统和机器人电生理学。使用这种检测，我们提出了一个项目，将确定昆虫ORs识别气味的结构基础。在目标1中，我们将表达和筛选一个大面板的果蝇气味受体（DmORs）使用我们的检测。在目标2中，我们将通过用半胱氨酸反应性甲硫基磺酸盐试剂筛选受体组来鉴定DmOr的气味剂结合亚基上的感兴趣区域。该筛选将利用半胱氨酸残基在DmOR家族中的天然分布。在目标3中，我们将使用取代的半胱氨酸可及性方法（SCAM）来探索昆虫气味结合亚基的关键功能区域（在目标2中确定）。在目标4中，我们将使用定点突变和功能分析来揭示昆虫气味受体识别气味的结构基础。该项目将提供昆虫ORs在分子水平上识别气味配体的理解。该项目对人类健康很重要，因为昆虫OR是开发控制有害昆虫物种的化合物的新靶标。

DESCRIPTION (provided by applicant): Many insect species exert a negative impact on human health through serving as disease propagation vectors or through causing agricultural damage. Chemosensory receptors, odorant receptors (ORs) and taste receptors, are critical to the ability of insects to function in these roles. Insect chemosensory receptors are an entirely novel receptor class, having no genetic or structural relationship to any known receptor family (including mammalian chemosensory receptors). Thus, these receptors are appealing targets for the development of new compounds for insect control. Realizing this goal requires development of a detailed understanding of insect chemosensory receptor structure and function. However, one of the most notable characteristics of these receptors is how little we know about their structure. In this project, we will identify structural features of insect ORs that are important in odorant binding and recognition. We have established a robust functional assay for insect odorant receptors using the Xenopus oocyte expression system and robotic electrophysiology. Using this assay, we are proposing a project that will determine the structural basis for odorant recognition by insect ORs. In Aim 1, we will express and screen a large panel of Drosophila melanogaster odorant receptors (DmORs) using our assay. In Aim 2, we will identify regions of interest on the odorant binding subunits of DmOrs by screening the receptor panel with cysteine-reactive methanethiosulfonate reagents. This screen will take advantage of the natural distribution of cysteine residues across the DmOR family. In Aim 3, we will use the Substituted Cysteine Accessibility Method (SCAM) to explore critical functional regions (identified in Aim 2) of the odorant binding subunits of insect ORs. In Aim 4, we will use site-directed mutagenesis and functional analysis to reveal the structural basis for odorant recognition by insect odorant receptors. This project will provide an understanding of odorant ligand recognition by insect ORs at the molecular level. This project is important to human health because insect ORs are a novel target for the development of compounds to control deleterious insect species.