# Red-Light-Only Control of Protein–Protein Interactions Using a Cyanobacteriochrome (UNICYCL)

## TL;DR

A new red-light-controlled system for protein interactions is introduced, offering a simpler and smaller alternative to existing tools.

## Contribution

A novel red-light-only optogenetic system using a cyanobacteriochrome is developed for controlling protein–protein interactions.

## Key findings

- The system forms a 1:1 complex in the dark with a binding affinity of 1–5 μM.
- Red light causes dissociation of the complex by reducing binding affinity >25-fold.
- The system is suitable for controlling interactions in vitro and in living cells.

## Abstract

Most optogenetic tools are controlled by blue light.
Red-light-responsive
tools enable multiwavelength applications and allow greater biological
tissue penetration with reduced toxicity. Current red-light tools
are primarily based on phytochromes, large dimeric proteins with a
structurally complex mode of interaction with their binding partners.
Here we introduce a small red-light-only responsive system composed
of a BNp-Red-1.2 (6 kDa) that binds to a cyanobacteriochrome (CBCR)
GAF domain NpF2164g6 (17 kDa) with a Kd ≈ 1–5
μM to form a 1:1 complex in the dark. Red light causes dissociation
of the complex by causing a > 25-fold decrease in binding affinity.
The CBCR GAF domain reverts to the dark state with a half-life of
∼ 1 min and the complex reforms. Structural analysis using
NMR measurements combined with molecular docking and dynamics simulations
shows that the binder interacts with the GAF domain and senses isomerization
of the bilin chromophore at a site that overlaps the critical tongue
domain of phytochromes. This system provides a small, simple red-light-only
optogenetic tool that can operate to control protein–protein
interactions in vitro and in living cells.