Protein Sensors

Protein-based biosensors provide sophisticated structural scaffolds that can, in principle, offer more selective recognition sites for Zn2+ compared to truncated peptide analogues. These macromolecular Zn2+-responsive probes can be grouped into one of two classes: sensor systems that combine synthetic flu-orophores with natural Zn2+-binding proteins, and sensor systems that couple fluorescent proteins to an engineered Zn2+-recognition site.

The most well-developed protein-based probes for Zn2+ rely on carbonic anhydrase as a Zn2+-binding scaffold and inhibition of its metal-bound (holo) form by fluorescent aryl sulfonamides as a signaling mechanism [164]. In the absence of Zn2+, the aryl sulfonamide has negligible affinity for metal-free (apo) CA. The free fluorophore is surrounded by polar aqueous solvent and exhibits a low fluorescence intensity, short excited-state lifetime, and small anisotropy. Upon addition of Zn2+, the aryl sulfonamide binds to the Zn2+ center of holo CA. Encasement of the aryl sulfonamide fluorophore in the hydrophobic environment of the protein triggers an increase in fluorescence intensity, lifetime, and anisotropy. Several sulfonamide reporters have been employed for CA-based sensing of Zn2+, with excitation and emission profiles spanning the ultraviolet to visible range [57,165-172]. These probes, which include 8 through 12, can detect down to picomolar amounts of free Zn2+ in aqueous solution (Figure 4). This strategy has been extended to detect Zn2+ by FRET using CA mutants

Figure 4. Zinc reporters based on carbonic anhydrase and fluorescent sulfonamides.

hydrophobic CA pocket

Figure 4. Zinc reporters based on carbonic anhydrase and fluorescent sulfonamides.

SO3H

SO3H

Figure 5. Carbonic anhydrase probes for zinc detection by fluorescence resonance energy transfer.

Figure 5. Carbonic anhydrase probes for zinc detection by fluorescence resonance energy transfer.

labeled with a fluorescent donor or acceptor in combination with an appropriate free aryl sulfonamide partner. Systems 14 and 15 (Figure 5) are illustrative of this approach [57,172]. Some of the CA biosensors have been applied to image release of labile Zn2+ from live hippocampal slices [111].

Maltose binding protein (MBP) has been exploited as a macromolecular template for Zn2+ sensing [173]. This periplasmic bacterial protein consists of a single polypeptide chain with two domains connected by a hinge region. Lig-and binding to MBP triggers a conformational change of the protein from an open form to a closed form through a combined bending-twisting motion around the hinge. MBP labeled with N-([2-(iodoacetoxy)ethyl]-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD) has been converted to Zn2+ sensor 16 with the aid of the iterative computational design program DEZYMER (Figure 6).

1329F MBP

NO O

"N 16

SO3- SO3

SO3- SO3

SO3- SO3

SO3- SO3

ECFP

18: Receptor = zif268 motif 19: Receptor = hMT-2

R1""

Zn 20

Figure 6. Protein-based zinc fluorophores.

The I329F mutant of MBP changes the specificity of ligand binding to this protein from maltose to Zn2+, providing a His2Glu2 site for Zn2+ with moderate affinity (Kd = 0.35 |M).

The natural Zn2+-buffering protein metallothionein has been modified for fluorescence detection of Zn2+. Recombinant human metallothionein 2 (hMT-2) has a free N-terminal amino group, and introduction of a S32C mutation into this protein provides a second site with orthogonal chemical reactivity for fluorophore labeling. Notably, this additional thiol perturbs neither the overall structure of the protein nor the formation of metal-thiolate clusters. Alexa 488 and Alexa 546 were attached specifically to the mutated hMT-2 at the N terminus and Cys32 positions, respectively, to afford protein probe 17 (Figure 6) [174]. FRET between the Alexa 488 donor and Alexa 546 acceptor allows monitoring of metal binding and conformational changes in the N-terminal ^-domain of the protein.

Intrinsic fluorescent proteins like green fluorescent protein (GFP) and its enhanced yellow (EYFP) and cyan (ECFP) derivatives have been genetically modified with metal-binding sites for analytical detection of Zn2+. These types of probes are promising for nonhuman in vivo optical imaging applications because they can be introduced noninvasively to cells and tissue by trans-fection and/or can be targeted to specific tissues, organelles, or other cellular compartments by genetic methods [157]. The design of zinc sensor protein 1 (ZS-1, 18, Figure 6) is based on the popular chameleon-type FRET probes for Ca2+. ZS-1 contains ECFP and EYFP units connected through a 39-amino-acid linker corresponding to a zinc-finger motif from a mouse transcription factor, zif268 [175]. Upon excitation at 432nm, the ratio of emission intensities at 535 and 480nm increases from 3.5 to 6.7 in the presence of Zn2+. A similar fusion construct using human type 2a metallothionein (hMT-2a, 19, Figure 6) as a hinged Zn2+-recognition site sandwiched between ECFP and EYFP has been employed to study the interaction between MT and NO in live mammalian cells [176]. Zn2+ chelation to the MT hinge in 19 brings the fluorescent protein pair closer together to increase FRET. An improved ECFP/EYFP FRET system has been obtained by engineering a bidentate or tridentate Zn2+ site at the interface between heterodimers of ECFP and EYFP [177]. Upon excitation at 433 nm, the ratio of emission intensities at 530 and 475 nm increases by 8-to 10-fold in the presence of millimolar concentrations of Zn2+. Finally, a GFP modified with a tridentate tripyrrole ligand (20, Figure 6) exhibits fluorescence increases upon Zn2+ binding, whereas Cu2+ chelation results in fluorescence quenching [178].

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