Tools that can rapidly and conditionally perturb protein function are critical for the quantitative characterization of complex biological behavior. While numerous small molecule-based approaches have proven useful in advancing our understanding of protein signaling, the precise spatiotemporal regulation of protein activity remains a significant challenge. To address current limitations, this project aims to develop a general strategy to control the levels of any protein-of-interest with high spatiotemporal precision. Key to the success of this methodology is the design of a photoswitchable degradation (PSD) sequence that becomes metabolically unstable when irradiated with a specific wavelength of light. In particular, the photoswitchable degradation system will comprise a mutant of a light-sensitive phototropin domain that contains an embedded tetrapeptide degron. In the absence of light, the photoswitchable degradation domain will adopt a conformation that cages the degron and prevents its recognition by the endogenous cellular quality control pathways. Upon irradiation with light, the photoswitch mutant will undergo conformational changes that expose the degron and promote its degradation. This light- dependent instability will be conferred to any protein-of-interest genetically fused to the PSD domain.
The proposed project, which is co-funded by the program Instrument Development for Biological Research, will result in a novel, unique tool to (1) control the activity of any protein-of-interest and (2) elucidate the mechanistic details of protein-protein interactions. During the tenure of the fellowship, the fellow will obtain extensive training in the fields of biochemistry, cell biology, and structural biology. In addition, the fellow will mentor underrepresented minority undergraduates and K-12 students through the Stanford Summer Research Program and the Stanford Science in Service Program, respectively.
