Engineering a blue light inducible SpyCatcher system (BLISS) as a tool for protein photopatterning and optogenetics

The SpyTag-SpyCatcher protein conjugation system has recently exploded in popularity due to its fast kinetics and high yield under biologically favorable conditions in both in vitro and intracellular settings. We imagine 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 designed a method to integrate light dependency into the protein conjugation reaction. The light-oxygen-voltage 2 domain of Avena sativa (AsLOV2) 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 (Figure 1), the SpyTag is blocked from reacting with the SpyCatcher in the dark, but 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 after one hour of irradiation, the reaction is 80% complete, while only 10% of the AsLOV2-SpyTag protein reacted in the dark. This reaction can be quenched within minutes by returning the reaction to the dark. We demonstrated the spatial aspect of this light control mechanism through photopatterning proteins onto Ni-NTA coated slides. As our system is made solely from protein components, which can be genetically encoded, we can extend the same spatiotemporal control of proteins inside cells. We anticipate BLISS will be a strong tool for fabricating protein microassays, crafting biomaterial composition, as well as optically controlling enzyme activity and protein localization in cells. Please click Additional Files below to see the full abstract

ENGINEERING THE ASLOV2 DOMAIN FOR OPTICALLY CONTROLLED PROTEIN CONJUGATION

Wilfred Chen, Ph. D.The ability to specifically conjugate proteins together has been a useful tool in cellular monitoring and controlling expression. The SpyTag:SpyCatcher systems has been developed as a robust, irreversible peptide tagging system used in vitro and in vivo. The Sortase A transpeptidation system, derived from Gram-positive bacteria, is a reversible, enzyme mediated conjugation system with applications in purification and tagging. We can imagine that if we can control the conjugation event in time and space, the system could become more versatile with a metaphorical “on/off” switch. Learning from photoreceptors found in nature, constructs were generated to control protein conjugation events. Blue light dependence was introduced via the light-oxygen-voltage 2 domain from Avena sativa (AsLOV2). When irradiated with blue light the domain undergoes a dramatic conformational change that renders the C-terminus “undocked”. Blue light dependent conjugation was tested by genetically fusing the AsLOV2 domain to a reactive partner of either the SpyTag:SpyCatcher system or Sortase A system. Fusion of SpyTag to LOV showed the best results for the irreversible system. After 5 hours, the conjugation reaction was 3-fold more complete with blue light irradiation. The Sortase A system has been generated and expressed but has not been tested. If deemed feasible, application of these types of systems include but are not limited to intracellular protein localization and the dynamic decoration of hydrogels.Chemical Engineerin