Variation of linker length in ratiometric fluorescent sensor proteins allows rational tuning of Zn(II) affinity in the picomolar to femtomolar range

Ratiometric fluorescent sensor proteins with a very high and tunable affinity for Zn(II) were created by connecting two fluorescently labeled metal binding domains, CFP-Atox1 and WD4-YFP, using a series of flexible peptide linkers. A simple random-coil model describing the conformational distribution of the linker allowed a quant. understanding of the effect of the linker length on both the change in emission ratio and the Zn(II) affinity

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

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

Genetically encoded fluorescent probes for Intracellular Zn2+ imaging

In this chapter we provide an overview of the various genetically encoded fluorescent Zn2+ sensors that have been developed over the past 5 to 10 years. We focus on sensors based on Förster resonance energy transfer (FRET), as these have so far proven to be the most useful for detecting Zn2+ in biological samples. Our goal is to provide a balanced discussion of the pros and cons of the various sensors and their application in intracellular imaging. Following the description of the various sensors, several recent applications of these sensors are discussed. We end the chapter by identifying remaining challenges in this field and discussing future perspectives

Genetically encoded fluorescent probes for Intracellular Zn2+ imaging

In this chapter we provide an overview of the various genetically encoded fluorescent Zn2+ sensors that have been developed over the past 5 to 10 years. We focus on sensors based on Förster resonance energy transfer (FRET), as these have so far proven to be the most useful for detecting Zn2+ in biological samples. Our goal is to provide a balanced discussion of the pros and cons of the various sensors and their application in intracellular imaging. Following the description of the various sensors, several recent applications of these sensors are discussed. We end the chapter by identifying remaining challenges in this field and discussing future perspectives