Metal-ion-dependent GFP emission in vivo by combining a circularly permutated green fluorescent protein with an engineered metal-ion-binding coiled-coil.
Coordination of metal ions significantly contributes to protein structures and functions. Here we constructed a fusion protein, consisting of a de novo designed, metal-ion-binding, trimeric coiled-coil and a circularly permutated green fluorescent protein (cpGFP), where the fluorescent emission from cpGFP was induced by metal ion coordination to the coiled-coil. A circularly permutated GFP, (191)cpGFP(190), was constructed by connecting the original N- and C-termini of GFP(UV) by a GGSGG linker and cleaving it between Asp(190) and Gly(191). The metal-ion-binding coiled-coil, IZ-HH, was designed to have three alpha-helical structures, with 12 His residues in the hydrophobic core of the coiled-coil structure. IZ-HH exhibited an unfolded structure, whereas it formed the trimeric coiled-coil structure in the presence of divalent metal ions, such as Cu(2+), Ni(2+), or Zn(2+). The fusion protein (191)cpGFP(190)-IZ-HH was constructed, in which (191)cpGFP(190) was inserted between the second and third alpha-helices of IZ-HH. Escherichia coli cells, expressing (191)cpGFP(190)-IZ-HH, exhibited strong fluorescence when the Cu(2+) and Zn(2+) ions were present in the medium, indicating that they passed through the cell membrane and induced the proper folding of the (191)cpGFP(190) domain. This strategy, in which protein function is regulated by a metal-ion-responsive coiled-coil, should be applicable to the design of various metal-ion-responsive, nonnatural proteins that work both in vitro and in vivo.
Mizuno
Toshihisa
T
Graduate School of Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan. toshitcm@nitech.ac.jp
Murao
Kaori
K
Tanabe
Yoichi
Y
Oda
Masayuki
M
Tanaka
Toshiki
T
D000595:Amino Acid Sequence; D002413:Cations, Divalent; D002942:Circular Dichroism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D019216:Metals, Heavy / Q000737:chemistry*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D002413:Cations, Divalent; D019216:Metals, Heavy; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins