PROJECT SUMMARY Fluorescent methods have revolutionized our ability to study biological processes in cellular environments. Despite an ever-expanding fluorescent toolkit, current methods are often limited in their ability to report on dynamic events (e.g. protein-protein interactions, ligand binding, or the flux of metal ions in cells) that underpin a majority of biological processes. The proposed research seeks to address this challenge by combining computational protein design methods with technology allowing the cellular incorporation of fluorescent non-canonical amino acids (fNCAAs) in proteins to generate novel classes of protein-based fluorescent sensors with enhanced properties. Our efforts will focus on developing new protein-based tools in which environmentally sensitive fluorophores— those whose fluorescent properties are modified in response to changes in the surrounding environment—serve as sensors of dynamic cellular processes. The ability to use such dyes to study dynamic processes in cellular environments is often limited by the fact that fluorophores are generally attached to the surfaces of target biomolecules. Alternatively, genetically encoded NCAAs are incorporated directly in the peptide backbone and are therefore uniquely suited to respond to subtle changes within protein scaffolds. This suggests that fNCAAs could serve as a platform for the creation of a novel class of protein-based sensors that dynamically respond to a host of stimuli. However, achievement of this goal would require the ability to identify optimal sites of fNCAA incorporation such that a well-defined change in fluorescence is expected without disrupting natural protein function. We recently structurally characterized proteins containing an fNCAA with a 7-hydroxycoumarin (7-HC) side chain that are responsive to protein-protein and protein-small molecule interactions. These data provide insight into how changes in the environment surrounding the 7-HC fluorophore translate into changes in its spectrum, thereby paving the way for the rational design of fluorescent biosensors of protein-small molecule interactions. To explore this possibility, our recently obtained structural data will serve as inputs to computational protein design methods that will be used to engineer new fluorescent sensors of small molecule metabolites. In a second aim, we will develop highly selective metal ion sensors based on NCAAs containing either 7-HC or 8- hydroxyquinoline (8-HQ) as a side chain. Again, computational protein design methods will be used to sculpt the protein environments surrounding these NCAAs in order to generate new fluorescent proteins that are sensitive to a wide range of biologically relevant metal concentrations and can be selectively produced in cells in a spatiotemporally controlled fashion. Finally, because many existing fNCAAs exhibit fluorescent properties that are not readily amenable to cell-based assays we will expand the toolkit of existing fluorescent...