Project Summary The cellular environment is crowded with a concentration of macromolecules between 200-400 grams per liter. This crowding affects protein interactions, binding affinities, and diffusion. These conditions are not replicated in the dilute solutions used for in vitro studies. To have a full understanding of protein function, it is necessary to study proteins in complex environments that mimic the in vivo environment. However, the high concentration of macromolecules makes it difficult to perform structural studies in these systems. Owing to this, it is necessary to develop new methods to study protein structure in complex model systems. Here, we propose to further establish the protein footprinting method fast photochemical oxidation of proteins (FPOP) for studying complex model systems. FPOP utilizes hydroxyl radicals to oxidatively modify solvent accessible amino acids in proteins. The in vitro method can identify protein-ligand and protein-protein interaction sites as well as regions of protein conformation changes. My group has further expanded FPOP for studies in cells (IC-FPOP) and in vivo (IV-FPOP) in C. elegans, an animal model for human disease. We have demonstrated that IC- and IV-FPOP can oxidatively modify hundreds to thousands of proteins in these complex systems. The next step in method development is to establish their efficacy for identifying protein interactions in these model systems by studying specific applications. For the next 5 years, we plan to apply IC- and IV-FPOP to study protein folding and aggregation. The identification of protein interactions involved in misfolding and aggregation will help design new therapeutics. We also plan to extend the method into another three-dimensional model system, ex vivo tissue. This will provide structural information in a model system that more closely resembles the in vivo environment than monolayer cell culture.