PROJECT SUMMARY/ABSTRACT Genetically encoded sensors that can detect and quantify proteins and other ligands are useful tools for studies of target dynamics and biomedical applications. Despite this capability, however, the principal limitation of genetically-encoded sensor generations is a lack of flexible design strategies to produce a quantifiable readout upon target binding with minimizing perturbation of target function. One of the examples is a difficulty to generate a sensor for free (i.e., unconjugated) Nedd8. Nedd8 is a post-translational modifier that regulates the cullin-RING E3 ligases, a large class of ubiquitin ligases, via its conjugation, known as “neddylation”, to the cullin subunit. Neddylation enzymes are being actively explored as drug targets for treatment of heretofore undruggable solid- tumor cancers. The overall objective of this proposal is to establish a new design strategy to generate biosensors for quantification of free Nedd8 in vitro and in cells. My central hypothesis for free Nedd8 sensor can be designed that incorporates a free Nedd8 binder and two reporter domains. The rationale for this project is that such a sensor can be used to monitor and quantify free Nedd8 in vitro and in live cells. Importantly, the strategy we develop can be generalized as an approach to generate genetically encoded sensors for other targets. We have recently developed a selective free Nedd8 sensor (eDEN1-FRET) producing Forster Resonance Energy Transfer (FRET) signal for free Nedd8 quantification. Based on this preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Design, preparation, and characterization of genetically encoded sensors for free Nedd8 for in vitro and live-cell experiments;, 2) Systematic identification of structural factors affecting signal dynamic range for reproducible quantification of free Nedd8;, and 3) Measurement of free Nedd8 in reconstituted enzyme reactions, cell lysates, and live cells. This research proposal is innovative in that it offers flexible design strategies for sensors development without requiring target modification. The proposed research is significant because it is expected to provide free-Nedd8 sensors for studies of free Nedd8 dynamics in live cells and in vitro, with broader implications for biomedical applications. Importantly, students involved in this project will have the opportunity to develop new tools for biology. This creative experience will equip students with the confidence necessary to thrive in a scientific environment and facilitate their pursuit of careers in biomedical research.