Ratiometric fluorescent sensor proteins with subnanomolar affinity for Zn(II) based on copper chaperone domains

E.M.W.M. Dongen, van, L.M. Dekkers, K. Spijker, E.W. Meijer, L.W.J. Klomp, M. Merkx

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Abstract

The ability to image the concn. of transition metals in living cells in real time is important for further understanding of transition metal homeostasis and its involvement in diseases. The goal of this study was to develop a genetically encoded FRET-based sensor for copper(I) based on the copper-induced dimerization of two copper binding domains involved in human copper homeostasis, Atox1 and the fourth domain of ATP7B (WD4). A sensor has been constructed by linking these copper binding domains to donor and acceptor fluorescent protein domains. Energy transfer is obsd. in the presence of Cu(I), but the Cu(I)-bridged complex is easily disrupted by low mol. wt. thiols such as DTT and glutathione. To our surprise, energy transfer is also obsd. in the presence of very low concns. of Zn(II) (10-10 M), even in the presence of DTT. Zn(II) is able to form a stable complex by binding to the cysteines present in the conserved MXCXXC motif of the two copper binding domains. Co(II), Cd(II), and Pb(II) also induce an increase in FRET, but other, physiol. relevant metals are not able to mediate an interaction. The Zn(II) binding properties have been tuned by mutation of the copper-binding motif to the zinc-binding consensus sequence MDCXXC found in the zinc transporter ZntA. The present system allows the mol. mechanism of copper and zinc homeostasis to be studied under carefully controlled conditions in soln. It also provides an attractive platform for the further development of genetically encoded FRET-based sensors for Zn(II) and other transition metal ions.
Original languageEnglish
Pages (from-to)10754-10762
JournalJournal of the American Chemical Society
Volume128
Issue number33
DOIs
Publication statusPublished - 2006

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