(513b) Engineering a Blue Light Inducible Spycatcher System (BLISS) As a Tool for the in Vitro Photo-Pattering of Proteins and Optically Controlled Intracellular Protein Activity

The SpyTag-SpyCatcher protein conjugation system has recently exploded in popularity due to its ability to form irreversible, isopeptide bonds with fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. We can further expand the utility of this system by introducing the ability to spatially and temporally control the conjugation event. Taking inspiration from photoreceptor proteins in nature, we have rationally designed a method to integrate light dependency into the protein conjugation reaction by exploiting the light-oxygen-voltage 2 domain of Avena sativa (AsLOV2), which undergoes a dramatic conformational change in response to blue light. We have thus genetically fused the SpyTag into the AsLOV2 domain to create a Blue Light Inducible SpyCatcher System (BLISS). In this design, the SpyTag is blocked from reacting with the SpyCatcher in the dark. Upon irradiation with blue light, the Jα-helix of the AsLOV2 undocks to expose the SpyTag. We screened several likely insertion points in the Jα-helix, and found a variant with desirable light switching behavior where the reaction is 80% completed in the light, while only 10% of the AsLOV2-SpyTag protein is reacted in the dark after one hour. This reaction is tightly controlled by light exposure, as it can be quenched in the dark within minutes and quickly switched on and off through repeated cycling between light and dark states. We demonstrated the spatial aspect of this control mechanism through photopatterning proteins onto surfaces. We anticipate the precise control our strategy provides will be a strong tool for making protein microarrays, crafting biomaterial composition, as well as controlling protein activity in select cells for various optogenetic, synthetic biology applications.