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

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

*この研究の対応する著者

研究成果: Article査読

11 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)872-879
ページ数8
ジャーナルBiochemical and Biophysical Research Communications
496
3
DOI
出版ステータスPublished - 2018 2 12

ASJC Scopus subject areas

  • 生物理学
  • 生化学
  • 分子生物学
  • 細胞生物学

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