A dual-ligand-modulable fluorescent protein based on UnaG and calmodulin

UnaG is a green-emitting fluorescent protein that utilizes unconjugated bilirubin (BR) as its fluorophore. While BR has captured the attention of physiologists as an important antioxidant that scavenges reactive oxygen species in biological membranes, its excessive accumulation causes several clinical symptoms. Although the optimal regulation of BR concentration would result in clinical therapies for aging as well as reduce risks of clinical symptoms, UnaG hardly releases BR owing to its extremely high affinity for BR (K_d = 98 pM). Herein, we engineered the BR binding and fluorescence of UnaG to be Ca²⁺-sensitive via a genetic insertion of calmodulin (CaM). The resultant UnaG/CaM hybrid protein is a dual-ligand modulable fluorescent protein; binding of the fluorogenic ligand BR is negatively regulated by the other ligand, Ca²⁺ ion. The affinity for BR differed by three orders of magnitude between the Ca²⁺-free state (K_d = 9.70 nM) and Ca²⁺-saturated state (K_d = 9.65 μM). The chimeric protein can release nano- to micro-molar levels of BR with Ca²⁺ control, and is thus named BReleaCa (BR + releaser + Ca²⁺). Such a protein hybridization technique will be generally applicable to change the ligand binding properties of a variety of ligand-inducible functional proteins.