Andrea K. Stoddard

Chapter 14 Determination of Zinc Using Carbonic Anhydrase-Based Fluorescence Biosensors

This chapter summarizes the use of carbonic anhydrase (CA)-based fluorescent indicators to determine free zinc in solution, in cells, and in subcellular organelles. Expression (both in situ and in vitro) and preparation of CA-based indicators are described, together with techniques of their use, and procedures to minimize contamination. Recipes for zinc buffers are supplied.

Fluorescence lifetime imaging of physiological free Cu(II) levels in live cells with a Cu(II)-selective carbonic anhydrase-based biosensor

Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable. We recently developed a variant of human apocarbonic anhydrase II for sensing metal ions that exhibits 25-fold better selectivity for Cu(II) over Zn(II) than the wild type protein, enabling us to accurately measure Cu(II) in the presence of ordinary cellular (picomolar) concentrations of free zinc. We inserted a fluorescent labeled Cu(II)-specific variant of human apocarbonic anhydrase into PC-12 cells and found that the levels are indeed extremely low (in the femtomolar range). We imaged the free Cu(II) levels in living cells by means of frequency-domain fluorescence lifetime microscopy. Implications of this finding are discussed.

In situ Measurement of Free Zinc in an Ischemia Model and Cell Culture Using a Ratiometric Fluorescence-Based Biosensor

Zinc ion is of growing interest in medicine and biology generally, and especially in the ischemic brain and other tissues. We have developed ratiometric fluorescence-based biosensors for the study of zinc in these systems; the biosensors use apocarbonic anhydrase variants as recognition elements that offer high sensitivity and selectivity. We report continuous in situ, in vivo measurement of nanomolar extracellular zinc in the brain of an animal model of ischemia using a ratiometric fiber optic biosensor. We also report the development of an expressible excitation ratiometric indicator of zinc ion suitable for use in cells that exhibits picomolar sensitivity. Finally, we also report the discovery that the Zn complex of the chelator TPEN seems to be comparably apoptogenic to the free chelator itself.

Long Wavelength Fluorescence Ratiometric Zinc Biosensor

A protein-based emission ratiometric fluorescence biosensor is described that exhibits sensitivity to free zinc ion in solution down to picomolar concentrations. Ratiometric measurements are widely used to assure accurate quantitation, and emission ratios are preferred for laser scanning microscopes such as confocal fluorescence microscopes. The relatively long emission wavelengths used are well suited to studies in tissues and other matrices which exhibit significant fluorescence background, and the apo-carbonic anhydrase moiety recognizes zinc ion with high and controllable specificity.

In vivo and intracellular sensing and imaging of free zinc ion

We describe new methods for the study of zinc in biological specimens. Intracellular free zinc was determined at levels down to picomolar using an excitation ratiometric fluorescence-based biosensing approach using a carbonic anhydrase variant as transducer. A new fiber optic sensor suitable for in vivo use is also described using laser excitation and an emission ratiometric approach; the zinc concentration range of sensor response can be selected to fit the application.

Cationic polymethacrylate-copolymer acts as an agonist for beta-amyloid and antagonist for amylin fibrillation

In human, amyloid-beta (Aβ) and islet amyloid polypeptide (hIAPP) aggregations are linked to Alzheimer’s disease and Type-2 Diabetes, respectively. There is significant interest in better understanding the aggregation process by using chemical tools. Here, we show the ability of a cationic polymethacrylate-copolymer (PMAQA) to quickly induce β-hairpin structure and promote fibrillation in Aβ40, and to constrain the conformational plasticity of hIAPP for several days and inhibit its aggregation at sub-micromolar concentrations. NMR experiments and atomistic molecular dynamics simulations reveal that PMAQA electrostatically interacts with Aβ40’s Glu22 and Asp23 followed by β-sheet induction while it binds strongly to the closest proximity of amyloid core domain (NFGAIL) of hIAPP and restrain its structural rearrangement. This study provides a valuable approach to develop polymer-based anti-amyloid inhibitors that may diminish the population of intermediates of Aβ40 or hIAPP.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimers amyloid-beta (Aβ40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aβ40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 μM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aβ40 aggregation and were also found to be suitable to trap Aβ40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aβ40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central β-sheet-forming residues of Aβ40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aβ40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aβ40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aβ40 intermediates for high-resolution structural and neurobiological studies.