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PEDS Advance Access published online on May 23, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn029
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

His-tags as Zn(II) binding motifs in a protein-based fluorescent sensor

Toon H. Evers, Marieke A.M. Appelhof, E.W. Meijer and Maarten Merkx1

Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands

1 To whom correspondence should be addressed. E-mail: m.merkx{at}tue.nl

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.

Keywords: fluorescent probes/FRET/GFP/hexahistidine tag/Zn(II) sensor

Received January 4, 2008; revised April 3, 2008; accepted April 29, 2008.


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