EAGER: Genetically Encoded Crosslinking Reagents to Map Protein Interaction Surfaces In Planta

该项目涉及开发一种新方法，用于识别允许植物细胞生长和茁壮成长的蛋白质。 该项目中使用的技术利用了一种新的方法来扩展植物细胞的“遗传密码”，使它们包含一种非天然氨基酸构建模块，可以将一种蛋白质与附近的另一种蛋白质化学连接起来。 这种联系将使研究人员能够确定植物的25，000种不同蛋白质中的每一种如何在植物种子发芽，生长和成熟为开花植物时相互作用。 这些知识是将基础研究应用于发现现实世界农业问题的关键，例如创造高产或耐胁迫的作物。该项目的另一个重要部分是揭开被称为“质谱”的关键复杂分析领域的神秘面纱，该领域用于识别哪些蛋白质与伴侣蛋白质相互作用。尽管质谱仪仪器是在机场、医院甚至火星上进行所有化学分析的基础，但许多科学家对这项技术并没有清晰的了解。 该项目将开发讲习班，向各级科学家教授质谱法，包括本科生和研究生，博士后研究人员和教师。 该项目的总体目标是开发一种新技术，通过利用基因工程将一种称为苯甲酰苯丙氨酸的基因编码的非天然氨基酸掺入植物蛋白质中，以鉴定蛋白质-蛋白质相互作用，苯甲酰苯丙氨酸可作为光亲和试剂。在长波长紫外光的存在下，这些蛋白质中的工程化氨基酸与位于三维空间附近的特定氨基酸形成共价交联，所述特定氨基酸位于所述蛋白质本身内或与其相互作用的其它蛋白质配偶体中。使用模式高等植物拟南芥，将产生表达tRNA及其tRNA合酶同源物的转基因植物，使得苯甲酰基苯丙氨酸可以掺入目的蛋白质内的任何位置。通过光活化交联和质谱，可以定义“相互作用组”。 该方法与目前使用的方法相比具有几个优点，因为它可以在植物中进行，它可以用于确定哪些特定氨基酸存在于两个蛋白质伴侣的相互作用表面，该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识产权进行评估来支持。优点和更广泛的影响审查标准。

This project involves the development of a new method for identifying proteins that allow plant cells to grow and thrive. The technology used in this project exploits a novel way to expand the 'genetic code' of plant cells so that they incorporate an unnatural amino acid building block that can chemically link one protein to another one that is nearby. Such linkages will allow researchers to identify how each of the plant's 25,000 different proteins interact with each other as the plant seeds germinate, grow and mature into a flowering plant. This knowledge is key for efforts to apply basic research to discovering real-world agricultural problems, such as creating crops with higher yields or stress tolerance. Another important part of this project is to demystify a key sophisticated analytical field known as 'mass spectrometry', which is used to identify which proteins interact with partner proteins. Despite the fact that mass spectrometer instruments are fundamental to all chemical analyses performed at airports, hospitals and even on Mars, many scientists do not have a clear understanding of this technology. This project will develop workshops to teach mass spectrometry to scientists at all levels, including undergraduate and graduate students, postdoctoral researchers, and faculty. The overall goal of this project is to develop a new technology for identifying protein-protein interactions by using genetic engineering to incorporate into plant proteins a genetically encoded unnatural amino acid, called bezoylphenylalanine, which acts as a photoaffinity reagent. In the presence of long wavelength UV light, the engineered amino acid in these proteins forms a covalent crosslink with specific amino acids that lie nearby in three-dimensional space, either within that protein itself, or in other protein partners that interact with it. Using the model higher plant, Arabidopsis thaliana, transgenic plants expressing a tRNA and its tRNA synthase cognate will be generated, such that benzoylphenylalanine can be incorporated into any position within a protein of interest. By photo-activated crosslinking and mass spectrometry, the 'interactome' can be defined. This method has several advantages over those currently in place, in that it can be performed in planta, it can be used to determine which specific amino acids are present at the interacting surface of the two protein partners, and it is not limited to the reactivity of any specific amino acid side chain.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.