Photoswitchable Protein-Protein Interactions: Engineering Light-Sensitive DARPins

Abstract

The recently developed technology of optogenetics utilizes photoactive proteins to enable control over intracellular processes. One of the well-studied families of photoactive proteins is the Light, Oxygen, Voltage (LOV) domain family, including the Avena sativa LOV2 protein that has been used to create a toolbox of photoactivatable signaling systems in vivo. Recently it was reported that by inserting LOV2 into a solvent-exposed loop of a protein of interest, it is possible to destabilize a protein upon light stimulation. Applying this concept to insert LOV2 domains in signaling proteins could allow for the creation of many photosowitchable versions of various signaling proteins. However, while this technique to create photoswitchable proteins is very promising, this method requires a solved crystal structure of each protein of interest for extensive calculations to correctly insert the LOV2 domains, which is not always feasible. Here, we propose a novel approach to introduce photoswitchability to DARPins, an entire class of highly conserved proteins that can bind and inhibit various proteins of interest with high affinity and high specificity. We insert LOV2 domains into two separate DARPins, ERK specific DARPin E40 and eGFP specific DARPin 3G124, and observe if similar insertion sites induce photoswitchability in both DARPins. We show the photoswitchability of one DARPin 3G124 construct, which was supported by both the yeast two-hybrid assay and mammalian binding assay, and demonstrate that the insertion site that induces the photoswitchability may be transferable to many other DARPins.