Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives

Abstract

Carbonic anhydrases are archetypical zinc metalloenzymes and as such, they have been developed as the recognition element of a family of fluorescent indicators (sensors) to detect metal ions, particularly Zn(2+) and Cu(2+). Subtle modification of the structure of human carbonic anhydrase II isozyme (CAII) alters the selectivity, sensitivity, and response time for these sensors. Sensors using CAII variants coupled with zinc-dependent fluorescent ligands demonstrate picomolar sensitivity, unmatched selectivity, ratiometric fluorescence signal, and near diffusion-controlled response times. Recently, these sensors have been applied to measuring the readily exchangeable concentrations of zinc in the cytosol and nucleus of mammalian tissue culture cells and concentrations of free Cu(2+) in seawater.

Figure 1

Biosensor paradigm: A sensor is termed a “biosensor” if the analyte is recognized by a molecule that is biological in origin.

Figure 2

Ribbon diagram of human carbonic anhydrase II: Metal binding site highlighting: the zinc ion as a sphere; the direct ligand histidines, H94, H96, H119; the second shell ligands Q92, E117, T199; and the hydrophobic residues F93, F95, W97.

Figure 3

Schematic diagram of the active site of WT CAII: Substitutions in both direct (H94) and indirect (E117, T199, Q92) ligands decrease the zinc affinity of CAII by 5- to 10⁵ –fold, and dramatically reduce equilibration time.

Figure 4

Divalent metal ion affinities of WT CAII: The metal ion affinity of CAII follows the inherent metal ion-ligand affinity trend termed the Irving-William series (Mn²⁺<Co²⁺<Ni²⁺<Cu²⁺>Zn²⁺).

Figure 5

Metal ion selectivity of CAII variants with mutations in the direct ligands: The affinities (pK_D = − log K_D) for copper, zinc, and nickel are compared of the wild type and three variants, each with a mutation in one of the direct ligands. The Asn and Gln variants coordinate metals via an uncharged carbonyl oxygen while the Asp variant coordinates metals with a carboxylate oxygen.

Figure 6

FRET excitation ratiometric zinc sensing scheme: In the presence of zinc, dapoxyl sulfonamide binds to holo-CAII and transfers energy to the acceptor, AlexaFluor 594. The ratio of the acceptor fluorescence emission with excitation of the donor to that with excitation of the acceptor -is measured. The FRET intensity ratio increases with zinc concentration as the fraction of zinc-bound CAII increases, producing a binding curve with picomolar affinity (inset).

Figure 7

False color ratio image of free zinc ion in PC-12 cells. The cells were stained with TAT-tagged H36C CAII labeled with Alexa Fluor 594 and Dapoxyl sulfonamide. The ratio image was constructed from two fluorescence micrographs taken with excitation at 380 nm, emission 617 nm; and excitation 540 nm, emission 617 nm. The calibration is measured using the same filters in the microscope with samples containing known free zinc ion concentrations maintained with NTA buffers.

Figure 8

Principle of CA-based fluorescence sensing of Cu²⁺. Oregon Green maleimide is conjugated to cysteine inserted in place of a leucine (L198C) close to the active site. In the absence of Cu²⁺ (left) the Oregon Green is unquenched, but when Cu²⁺ binds in the active site the Oregon Green is partially quenched and exhibits reduced fluorescence intensity and lifetime.

Figure 9

Cu(II) sensor calibration. The figure depicts measured phase angles (open circles) and modulation ratios (squares) at 200 MHz (which are functions of the fluorescence lifetime) and fluorescence intensities as a function of free Cu(II) concentration in NTA buffers for L198C-apoCA II conjugated with the fluorophore Alexa Fluor 660. Reproduced with permission from Zeng, et al.

Scheme 1

Proposed mechanism of zinc binding to CAII.

Scheme 2

Proposed mechanism for catalysis of zinc exchange by DPA.