Dissecting the ABA signalling pathway with a biological chemistry approach

脱落酸（ABA）是一种重要的植物激素，通过植物合成ABA启动各种防御机制，控制着从植物发育到生物和非生物胁迫反应的广泛过程。在这个过程中起关键作用的一个家族是ABA受体，它是所谓的pyrabactin resistance （PYR）和PYR like （PYL）蛋白的成员。这个多样化的PYR/PYL蛋白家族在拟南芥中至少有14个成员，它们都在ABA结合和随后的信号传导中起作用。虽然先前对PYL1蛋白的结构研究已经揭示了一个保守的ABA结合袋和一个负责信号转导的2C蛋白磷酸酶（PP2C）成员的结合位点，但PYL1蛋白在发育中的多种作用尚不清楚。目的：我们已经确定了一系列对植物生长发育具有不同生物学效应的化合物，这些化合物被认为是PYL1配体。这些化合物可以作为化学探针，进一步阐明不同家族成员在应激反应和信号转导中的作用。此外，这些化合物具有控制发芽和发育的潜力，这是我们的工业合作伙伴Tozer Seeds， Lt.非常感兴趣的领域。该项目的目的是利用生物物理和结构方法的结合来研究配体结合的分子基础，以设计和合成具有改进生物活性的化合物。方法：蛋白质靶点将在大肠杆菌中以成熟的合成基因构建方法表达。生物物理方法的范围从高通量热移法（TSA）到高精度等温滴定量热法（ITC）。共结晶实验将从已知的PYL1条件开始，然后扩展到其他家庭成员。结构和生物物理数据将用于指导新化合物的合成。该项目代表了与英国最大的独立蔬菜种子公司Tozer Seeds Ltd的合作，在那里将进行为期3个月的生物测试。

Abscisic acid (ABA) is a key plant hormone that controls a wide range of processes from plant development to biotic and abiotic stress responses as plants synthesize ABA to initiate various defense mechanisms. One family of key players in this process are the ABA receptors that are members of the so-called pyrabactin resistance (PYR) and PYR-like (PYL) proteins. This diverse family of PYR/PYL proteins consists of at least 14 members in Arabidopsis that all function in ABA binding and consequent signalling. While previous structural studies of the PYL1 protein have revealed a well-conserved ABA binding pocket and a binding site for members of the 2C protein phosphatases (PP2C) responsible for signal transduction, the diverse roles of the PYL1 proteins in development are poorly understood.Aim: We have identified a series of chemical compounds with distinct biological effects on plant growth and development that are thought to be PYL1 ligands. These compounds can be used as chemical probes to further elucidate the roles of different family members in stress response and signal transduction. Furthermore, the compounds possess the potential to control germination and development, an area of great interest to our industrial partner, Tozer Seeds, Lt. The aim of this project is to investigate the molecular basis of ligand binding using a combination of biophysical and structural methods to design and synthesize chemical compounds with improved biological activities.Methods: Protein targets will be expressed in E. coli - based well-established methods using synthetic gene constructs. Biophysical methods range from high-throughput thermal shift assays (TSA) to highly accurate isothermal titration calorimetry (ITC). Co-crystallisation experiment will begin with the known condition for PYL1 and will then be extended to other family members. Structural and biophysical data will be used to guide the synthesis of new compounds. This project represents a collaboration with Tozer Seeds Ltd, UK's largest independent vegetable seed company,where biological tests will be performed during a 3-month placement.