Tuning a light-regulated allosteric switch for enhanced temporal control of protein activity

Abstract

Optogenetics enables researchers to control protein localization, interactions, and activity using photosensitive domains. The key desired properties for optogenetic tools include broad applicability, tight light-regulated control with high dynamic range, and tunability. Previously, we described an engineered light-sensitive switch, LightR, composed of two VVD domains connected by a flexible linker, enabling light-dependent allosteric control of protein activity through site-specific insertion. Here, we introduce enhanced LightR variants with improved dynamic range and faster activation kinetics. Through targeted modifications to the VVD domains and linker region, we optimized a LightR-regulated Src kinase (LightR-Src) activity and generated two LightR-Src variants: one supporting sustained Src activation comparable to constitutively active Src, and another enabling rapid, reversible control, ideal for modeling transient signaling events suitable to mimic Src signaling in living cells. These modifications expand the versatility of LightR-based tools, facilitating their use in diverse optogenetic applications requiring high dynamic range of regulation and fast control of targeted proteins.