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

Yoh Shitashima, Togo Shimozawa, Toru Asahi, Atsushi Miyawaki*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)


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 (Kd = 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 (Kd = 9.70 nM) and Ca2+-saturated state (Kd = 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.

Original languageEnglish
Pages (from-to)872-879
Number of pages8
JournalBiochemical and Biophysical Research Communications
Issue number3
Publication statusPublished - 2018 Feb 12


  • Bilirubin
  • Calmodulin
  • Fluorescent protein
  • UnaG

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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