Design of an ultrabright biosensor for dynamic imaging of kinase activity in cells
- Xiaoquan Li
- Sophia K. Tan
- Chan-I Chung
- A. Katherine Hatstat
- Qian Zhao
- Jingyu Luo
- William F. DeGrado
- Xiaokun Shu
2026-05-04
Protein kinases regulate almost every major signaling pathway. Visualizing spatiotemporal dynamics of kinase activity is thus essential to understand cell signaling. Here, we report a de novo–designed activity reporter of kinase, dubbed NOVARK, which contains a single polypeptide chain with multiple modular motifs that act as specific kinase substrates and reporters. NOVARK undergoes phosphorylation-induced higher-order assembly, which are detectable as ultrabright green fluorescent protein (GFP) droplets with a large dynamic range. We designed versions of NOVARK that rapidly and reversibly report intracellular activity of protein kinase A, C, and extracellular signal-regulated kinase (ERK) following stimulation/inhibition by upstream G protein-coupled receptor (GPCR) agonists. Our work provides a generalizable platform that enables the design of ultrabright biosensors for illuminating dynamic architecture of kinase signaling.