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

Yoh Shitashima, Togo Shimozawa, Toru Asahi, Atsushi Miyawaki

    Research output: Contribution to journalArticle

    3 Citations (Scopus)

    Abstract

    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
    Volume496
    Issue number3
    DOIs
    Publication statusPublished - 2018 Feb 12

    Fingerprint

    Calmodulin
    Bilirubin
    Ligands
    Proteins
    Biological membranes
    Fluorophores
    Green Fluorescent Proteins
    Reactive Oxygen Species
    Antioxidants
    Aging of materials
    Fluorescence
    Ions
    Membranes

    Keywords

    • Bilirubin
    • Calmodulin
    • Fluorescent protein
    • UnaG

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    A dual-ligand-modulable fluorescent protein based on UnaG and calmodulin. / Shitashima, Yoh; Shimozawa, Togo; Asahi, Toru; Miyawaki, Atsushi.

    In: Biochemical and Biophysical Research Communications, Vol. 496, No. 3, 12.02.2018, p. 872-879.

    Research output: Contribution to journalArticle

    Shitashima, Yoh ; Shimozawa, Togo ; Asahi, Toru ; Miyawaki, Atsushi. / A dual-ligand-modulable fluorescent protein based on UnaG and calmodulin. In: Biochemical and Biophysical Research Communications. 2018 ; Vol. 496, No. 3. pp. 872-879.
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