His-tags as Zn(II) binding motifs in a protein-based fluorescent sensor.
Fluorescent indicators that allow real-time imaging of Zn(II) in living cells are invaluable tools for understanding Zn(II) homeostasis. Genetically encoded sensors based on fluorescence resonance energy transfer between fluorescent protein domains have important advantages over synthetic probes. We discovered that hexahistidine tags have a strong tendency to dimerize upon binding of Zn(II) in solution and we used this principle to develop a new protein-based sensor for Zn(II). Enhanced cyan and yellow fluorescent proteins were connected by long flexible peptide linkers and His-tags were incorporated at both termini of this fusion protein. The resulting sensor CLY9-2His allows the ratiometric fluorescent detection of Zn(II) in the nanomolar range. In addition, CLY9-2His is selective over the physiologically relevant metal ions Fe(II), Mn(II), Ca(II) and Mg(II). Our approach demonstrates the potential of using small peptides as metal-binding ligands in chelating fluorescent protein chimeras.
Evers
Toon H
TH
Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands.
Appelhof
Marieke A M
MA
Meijer
E W
EW
Merkx
Maarten
M
D015374:Biosensing Techniques / Q000379:methods*; D003001:Cloning, Molecular; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D005456:Fluorescent Dyes; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010455:Peptides; D011994:Recombinant Proteins; D006639:Histidine; D015032:Zinc