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

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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.