Copper sensing with a prion protein modified nanopipette

Abstract

Protein–metal interactions determine and regulate many biological functions. Nanopipettes functionalized with peptide moieties can be used as sensors for metal ions in solution.

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Solid-state nanopores are a class of nanomaterials that have been successfully applied to the study and detection of numerous biomolecular interactions.^1,2 A nanopore can be simply described as a nanometer-sized opening, separating two chambers filled with electrolyte solutions. When a voltage is applied across a nanopore, an ionic current is established, with a magnitude that depends on the size, geometry, and charge of the nanopore opening. Biomolecular analysis can be performed when analytes translocate through the nanopore, giving rise to temporary blockades of the ionic current which are characteristic of the size and charge of the analyte being studied. This configuration (called resistive pulse sensing) enables the analysis of single molecules, but lacks specificity, as biomolecules of similar size and charge cannot be discriminated. An alternative experimental configuration relies on the immobilization of a receptor on the nanopore surface. The interaction of the receptor with its target analyte (if present in the solution) causes a permanent current blockade. Our group pioneered the application of a particular class of nanopores, called nanopipettes, to the study of proteins,^3,4 toxins⁵ and metal ions.^6,7 In a nanopipette, the nanopore is situated at the tip of a very sharp quartz needle, which can be easily fabricated at the bench using a laser puller.

In this paper we describe the development of functionalized nanopipettes (Fig. 1a and b) to study peptide–metal interactions. As a model system, we take advantage of the unique metal-binding properties of the prion protein (PrP). This protein is responsible for a class of infectious neurodegenerative diseases termed transmissible spongiform encephalopathies (TSEs).^8,9 TSEs are spread via the misfolding of cellular PrP (PrP^C) into the infectious scrapie form, PrP^Sc, which in the process form PrP^Sc amyloid fibrils. It is now well established that PrP is a copper binding protein with an ability to take up several Cu²⁺ equivalents in its flexible N-terminal segment (Fig. 1c). Uptake of copper in this domain drives several physiological processes, including PrP^C trafficking and endocytosis.¹⁰

The PrP copper binding domain is composed of four tandem repeats of the highly-conserved eight amino-acid sequence PHGGGWGQ, termed the octarepeats.¹¹ The four octarepeats coordinate a single Cu²⁺ ion at low occupancy with a K_d of 0.12 nM, as determined by electron paramagnetic resonance (EPR). The histidine from each octarepeat forms a square-planar coordination environment around the Cu²⁺ ion. At higher Cu²⁺ concentration, however, each octarepeat coordinates its own Cu²⁺ ion with a K_d of 7–12 μM.^12,13 In addition, the octarepeats can weakly coordinate a single Zn²⁺ ion in a tetrahedral geometry with a K_d of ∼200 μM.¹⁴ Herein, we describe the immobilization of the prion peptide on quartz conical nanopipettes to probe its interaction with metal ions in solution, in order to develop an all-electrical Cu²⁺ sensor amenable to in vivo studies.

Fig. 1 Scanning electron micrographs of the conical nanopipettes used in this study. A) top view showing a 35 nm nanopipette pore and B) side view showing the conical shape. C) Three-dimensional rendering of the prion protein with copper ions included (cyan spheres).

The prion peptide was immobilized by physisorption onto the negatively charged nanopipette walls and the functionalization was followed in real time by chronoamperometry, as described in the Supporting Information.† During immobilization, addition of the prion peptide to the bath solution causes the ionic current to instantaneously drop by ∼35% of its initial value, due to the interaction of the positively charged lysine residues at the N-terminus of the peptide with the negatively charged carboxylic groups of the nanopipette walls (Fig. 2a). The electrostatic immobilization appeared to be stable and the peptide was removed only by treatment with diluted hydrochloric acid (data not shown). The 4-octarepeat is known to bind Cu²⁺ with micromolar affinity, as demonstrated with mass spectrometry¹⁵ and EPR.¹⁶ We then added 6 μM Cu²⁺ to the bath and measured in real-time the evolution of the ionic current (Fig. 2b).The current dropped by 15% in less than a minute, as Cu²⁺ ions were chelated by histidine moieties of the peptides. A bare nanopipette showed no response to Cu²⁺ up to mM concentrations. As expected, the binding is reversible by addition of EDTA (ethylenediaminetetraacetic acid) to the bath solution (Fig. S1†). Nanopipette sensors were regenerated up to 6 times with no loss in sensor performance (Fig. 3).

Fig. 2 a) Interaction of the prion peptide with the nanopipette surface causes a decrease in the normalized ionic current. 20 μg of the prion peptide was added to the bath. b) Addition of 2 μM Cu²⁺ causes a further 15% drop in the ionic current, indicative of the interaction of copper with the prion peptide. No change in current is seen when copper is added to an unmodified nanopipette.

Fig. 3 Reversibility of the Cu²⁺ interaction with the peptide-modified nanopipette. Copper binding was reversed by addition of 1 mM EDTA, and solution was exchanged with fresh buffer. Cu²⁺ concentration 5 μM.

The current response is concentration dependent (Fig. 4A). We used the functionalized nanopipette to study response of the prion peptide to metal ions. The nanopipette responds linearly to increasing Cu²⁺ concentrations (Fig. 4A, inset). The modulation of normalized current vs. Cu²⁺ concentration is analogous to a Langmuir adsorption isotherm. Assuming that copper binding is an equilibrium process with independent binding sites, and the variation of the normalized current is proportional to the number of Cu²⁺ bound to the nanopipette, the thermodynamic affinity constant K for Cu²⁺ binding can be estimated using the following equation:

where I_n is the signal normalized to the one measured in pure buffer, I_max is the signal when all the binding sites are occupied, and c is the concentration of Cu²⁺. The plot of 1/I_nvs. 1/c is linear (Fig. 4a, inset) and the affinity constant K for Cu²⁺ binding can be calculated from the slope. We obtained a value of 1 μM⁻¹, which is in excellent agreement with values reported in the literature.¹⁷ This result demonstrates that the immobilization of the prion peptide on the nanopipette walls does not affect its binding properties. We then evaluated the specificity of the binding compared to other divalent metals. As expected, neither Ca²⁺ nor Mg²⁺ showed any interaction with the prion peptide (data not shown). By contrast, Zn²⁺ and Mn²⁺ did interact. We found an affinity constant K of 500 μM⁻¹ and 600 μM⁻¹ for zinc and manganese, respectively (Fig. S2†). These values are consistent with values reported in the literature,^14,18,19 confirming that Zn²⁺ and Mn²⁺ bind to the prion peptide but with a much lower affinity than Cu²⁺.

Fig. 4 Dose-response and metal screening. a) Dose-response of the peptide-modified nanopipette to increasing concentrations of Cu²⁺. Inset shows linear fit to the Langmuir isotherm (R² = 0.99). b) Dose-response to other divalent metals (Mn²⁺, Zn²⁺) compared to Cu²⁺.

Jeremy Lee's group pioneered the application of nanopores to study the interaction of the prion peptide with metals²⁰ and antibodies.²¹ Lee's group employed a biological nanopore, α-haemolysin, to study metal-induced conformational changes in the prion peptide. They employed resistive-pulse sensing to monitor the change of the translocation rate of prion peptides upon interaction with metal ions. Their approach has the advantage of qualitatively studying peptide–metal interactions at the single-molecule level, but it cannot be used for analytical screening. Our approach, which relies on the immobilization of the prion peptide on the nanopipette walls, allows the quantitative monitoring of peptide–metal interactions and extrapolation of thermodynamic affinity constants. Furthermore, nanopipettes can be easily integrated with nanomanipulators to comprise a scanning ion conductance microscope. This allows positioning of the nanopore sensor over living cells with nanometer resolution.^22–24 We propose that, with further improvements, these nanopipette sensors could be used to monitor in real time the extra- and intra-cellular concentrations of metal ions in and around living cells.

In conclusion, electrostatic immobilization of the prion peptide on a conical quartz nanopipette allows the monitoring of peptide-metal interactions. The sensor takes advantage of the unique properties of the PrP octarepeat domain and can be regenerated several times by EDTA treatment without any significant loss of performance, thus demonstrating the stability of the peptide interaction with nanopipette walls. Our findings pave the way for the development of peptide-encoded nanopipette sensors. As shown by Scott Banta's group, peptides selected from phage display can be used as recognition elements in biosensing experiments,²⁵ which expands the applications of nanopipette sensors in biomolecular analysis.

Acknowledgements

We thank Prof. Holger Schmidt and the Keck Center for Nanoscale Optofluidics at UC Santa Cruz for electron microscopy of the nanopores. This work was supported in part by grants from the National Aeronautics and Space Administration Cooperative Agreements NCC9-165 and NNX08BA47A, the National Institutes of Health grants: P01-35HG000205, and GM065790.

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Footnotes

† Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c2ra21730a

‡ These authors contributed equally to the work

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