Electrochemical Detection of Interaction between Copper(II) and

Subscriber access provided by WEBSTER UNIV

Article

Electrochemical detection of interaction between copper(II) and peptides related to pathological #-synuclein mutants Shaopei Li, and Kagan Kerman Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03612 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

ELECTROCHEMICAL DETECTION OF INTERACTION BETWEEN COPPER(II) AND PEPTIDES RELATED TO PATHOLOGICAL -SYNUCLEIN MUTANTS ShaoPei Li and Kagan Kerman* Department of Physical and Environmental Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto ON, M1C 1A4, Canada Abstract Herein we present a proof of concept study for electrochemical detection of the metal-binding site of -synuclein (-syn). Parkinson’s disease (PD) is associated with the aggregation and misfolding of -syn in dopaminergic neurons. Since copper homeostasis is deregulated in PD, it is of great significance to study the metal-binding site of wild-type (WT) -syn (48-53, VVHGVA), and its pathological mutants (H50Q, and G51D). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to monitor the formation of peptide-PEG mixed layer on gold surfaces. Differential pulse voltammetry (DPV) was used to detect and evaluate the interaction of copper(II) with the peptide layer. X-ray photoelectron spectroscopy (XPS) was used to characterize the formation and attachment of the peptide layer on gold surfaces. Isothermal titration calorimetry (ITC) was also utilized to evaluate the binding characteristics of the peptides with copper(II) ions. Our results indicated that the effect of a single amino acid mutation on the peptides drastically influenced their ability to interact with copper(II) ions. These results demonstrated that our electrochemical approach provided a rapid and cost-effective platform to study the strong interaction between α-syn and copper(II), which is implicated as one of the factors inducing structural changes in α-syn towards the progression of PD. Keywords: Bioelectrochemistry, -synuclein, copper, affinity, Parkinson’s disease.

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Parkinson's disease (PD) is a progressive neurodegenerative disease that affects the neuro-motor function. The symptomatic characteristic of PD includes dyskinesia, increase muscle rigidity, resting tremor and bradykinesia.1 Furthermore, PD patients can often develop other non-motor functions related symptoms such as psychosis, depression, and cognitive impairment.2,3 One of the leading theories of PD pathogenesis is the development of Lewy body inclusions at the synaptic terminal of mid-brain dopaminergic neurons.4 These inclusions have been shown to contain the high-level accumulation of alpha-synuclein (-syn).5,6 It was reported that the monomeric form of -syn contained two physiological states: cytosolic and membrane-bound. The monomers that are in the unbound state, are free-floating and unfolded, whereas the membranebound -syn proteins can rearrange into alpha-helical conformation. Both states of -syn have been shown to be able to undergo rearrangement and reconfiguration into β-sheet rich oligomers.7 The subsequent aggregation of β-sheet oligomers can lead to formation of fibrils resulting in cellular toxicity. There are many factors such as reactive oxygen species (ROS) and mutations that can all either alter or accelerate the aggregation of -syn in the pathological development of PD.8– 11

Since copper homeostasis is deregulated in PD, alteration in the levels of copper and increased

formation of -syn-copper complexes might play a key role in the production of ROS in the onset of PD.12 NMR spectroscopic analysis identified three regions of -syn with affinity towards copper(II) ions at residues: 3-11, 48-53 and 115-123, with residues 48-53 displaying the highest affinity.13 The formation of the copper(II) complex with α-syn and also, an N-terminal peptide, αsyn(1-19), was confirmed with electrospray-mass spectrometry (ESI-MS) and electrochemistry.14 Wang et al. 14 have also reported that the ROS generated reduced the viability of the neuroblastoma SY-HY5Y cells. The changes in the interfacial properties of α-syn preceding its aggregation were 2 ACS Paragon Plus Environment

Page 2 of 26

Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


determined using electrochemical techniques at hanging mercury drop electrodes by Palecek et al. 15,16

Recently, Lopes et al.17 have reported that electrochemical techniques can give reliable

information about the extent of α-syn fibrillation in vitro and offer an efficient tool for future in vivo monitoring of the protein conformational state.

Figure 1. The complete sequence of wild-type (WT) -syn with the copper-binding sites underlined in red. In recent findings, naturally occurring N-acetylated -syn has been shown to lower the copper(II) binding affinity at the C-terminal position (3-11), but position 48-53 retained its affinity towards copper(II).18 Thus, the metal binding behavior at residues 48-53 has become a topic of high interest. The gene for ⍺-syn has six known mutations that directly cause familial forms of PD. The pathological determinants of three of these mutants (A30P, E46K, and A53T) are wellcharacterized in diverse model systems with each mutant affecting the cellular toxicity in distinctive ways.19,20 The three more recently discovered familial mutants (H50Q, G51D, and A53E) are not extensively studied. 21–23 A genetic association was made between the familial form of PD and H50Q mutation, single amino acid substitution of histidine residue to glutamine (H50Q) was shown to accelerate the rate of fibril formation.21 The point mutation resulting in the replacement of glycine with aspartic acid (G51D) slowed down the rate of fibril formation with higher cellular toxicity.24 In this report, electrochemical techniques were explored in order to elucidate the effect of a single amino acid substitution on the interaction of peptides with copper(II). Understanding this interaction between both factors will shed light into the fundamental 3 ACS Paragon Plus Environment


mechanisms triggering ROS production in the -syn pathology of PD. Our preliminary electrochemical results indicated that a single mutation in the -syn metal-binding peptide sequence not only altered the aggregation patterns, but also influenced their interaction with copper(II) ions. In addition, our isothermal titration calorimetry (ITC) studies supported the electrochemical ones demonstrating that electrochemical approach can provide a rapid and costeffective platform to study α-syn-copper(II) interactions. With further studies of this platform, we envisage that our electrochemical approach can also be applied to screen large libraries of small neuroprotective molecules that are designed to decrease ROS production by targeting the metalbinding site 48-53 region of α-syn.

Methods Hexapeptides related to the metal-binding site 48-53 of wild-type (WT) -syn and its pathological mutants (H50Q and G51D) were purchased from CanPeptide (Québec, Canada) and were used as received. Full-sequence WT -synuclein, the truncated forms of -synuclein with sequence 1-60 (-syn1-60) and sequence 61-140 (-syn61-140) were purchased from rPeptide (Georgia, USA) and a buffer exchange procedure was performed using ZebaTM desalting columns from Thermo-Fischer Scientific (Massachusetts, USA). All other chemicals and reagents were purchased from SigmaAldrich (Oakville, ON), and were used as received unless otherwise stated.


Page 4 of 26

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Schematic illustration depicting the modification of gold electrode surfaces using HSPEG and HS-PEG-Peptides. Due to the interaction between the peptides and copper(II) ions, the electrochemical reduction current signal of copper(II) was observed only in the presence of peptides on the electrode surface. The hexamer peptides represent the metal-binding region 48-53 of the wild-type (WT) -syn and its mutants (H50Q and G51D).

Electrode modification with peptides Gold electrodes were purchased from CH Instruments (99.99% gold, Austin, TX). The electrodes were polished to obtain a mirror-like finish using 1.0 mm alumina slurry, followed by 0.3 mm and 0.05 mm alumina slurries on a micro cloth pad. In between polishing procedures, the electrodes were washed with Milli-Q water (18 MΩ cm, Millipore, Canada) to remove remaining alumina slurry. The polished electrodes were finally sonicated in Milli-Q water for the final removal of trace alumina powder. Electrochemical etching was then performed by cycling the electrode potential between 0 and 1.5 V in 0.1 M NaOH followed by in 0.1 M H2SO4 until reproducible cyclic voltammograms were obtained. For the physisorption of the thiolated-peptides on surfaces, 1 mM HS-PEG-peptides and 1 mM HS-PEG were mixed in different ratios and directly pipetted 5 ACS Paragon Plus Environment


Page 6 of 26

(20 µL) on the working electrode surface. Full sequence and truncated forms of -syn were immobilized by covalent attachment of the proteins on gold electrode surfaces. The modification of the gold surface was initiated by dipping the polished and cleaned electrode in 1 mM 6mercaptohexanoic acid and SH-PEG3 ethanolic solution for 12 h, followed by rinsing the electrode in

95%

(v/v)

ethanol.

Then,

the

electrodes

were

immersed

in

0.1

M

2-(N-

morpholino)ethanesulfonic acid (MES) (pH 5.5) with 15 mM N-hydroxysuccinimide (NHS), and 75 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 1 h. The resulting electrode was dipped into 200 µM protein solution in 10 mM PBS (pH 7.5) for 8 h and rinsed with the same buffer. All the modified electrodes were incubated at room temperature for 12 h and washed with 10 mM PBS (pH 7.4). The modified electrodes were then exposed to 200 L of copper(II) sulfate solutions at desired concentrations in 50 mM ammonium acetate (pH 7.4) with agitation overnight. The electrodes were then washed with copper(II)-free ammonium acetate and transferred into a cell containing blank 50 mM ammonium acetate (pH 7.4) with 50 mM NaOCl as the supporting electrolyte. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were performed using a conventional three-electrode system, with a gold working electrode, a platinum wire as the auxiliary electrode and an Ag/AgCl reference electrode (CHInstruments, Austin, TX) in connection with an Autolab PGSTAT12 potentiostat (Metrohm Canada, Mississauga, ON). All measurements were carried out at room temperature. CV was performed between -0.5 V and 0 V with a scan rate of 0.1 V.s-1. Differential pulse voltammetry was performed from +0.5 V to -0.3 V with a pulse amplitude of 25 mV, a step potential of 4 mV at a scan rate of 0.01 V.s-1. EIS measurements were conducted at a constant applied potential of -0.200 V over the frequency range from 0.1 Hz to 10 kHz, using in 1 mM [Ru(NH3)6]3+/2+ as the redox probe with 0.1 M NaOCl as the supporting electrolyte.


Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Molecular structures of the metal-binding site 48-53 of WT -syn, its pathological mutant (H50Q and G51D) peptides and the spacer molecule (HS-PEG).

XPS analysis of the modified electrodes X-ray photoelectric spectra were collected on a Thermo Scientific K-Alpha spectrometer with a monochromatic A1 Ka source (1486.6 eV). The accumulated angle was 90° with a 20 eV pass energy with the analyzer at a 10-8 mbar vacuum chamber.

Isothermal titration calorimetry (ITC): ITC analyses were performed using MicroCal ITC 200 (Malvern Panalytical, Northampton, MA). Stock solutions of peptides and copper(II) were prepared in 10 mM PBS (pH 7.4). An aliquot (100 7 ACS Paragon Plus Environment


L) of 1 mM copper(II) was injected into the sample cell containing 250 M peptides. The reaction occurred at 1000 rpm stirring rate with a 2 L per injection for a total of 19 injections at 25°C, and an interval of 180 s between each injection. The blank signal was obtained by titrating 1 mM copper(II) into PBS. The thermal signals obtained were used and subtracted as “background signal” from subsequent analyses that were performed by titrating 1 mM copper(II) with peptides. The area under the curve of peaks resulted from each injection were integrated and plotted against the molar ratio () of peptides to copper(II) ions. Origin software with Levenberg-Marquardt algorithm was employed to analyze the data. Single-set binding model from the algorithm was used to fit the data obtained under the assumption that the peptides have identical binding sites. The following parameters correlated to peptide-copper(II) ion interactions, association constant (Ka), stoichiometric ratio (n), enthalpy (H), and entropy (S) were obtained and described in the isotherm parameters. c value which is a unitless parameter that indicates the accuracy of the model fitting to obtain Ka values, was also calculated using 𝑐 = 𝑛 ∙ 𝐾𝑎 ∙ 𝐶.

Results and Discussion Surface characterizations The immobilization of the peptide layer on gold electrode surface was first monitored using cyclic voltammetry. In Figure 4a, clear cathodic peak shifts and slight anodic peak shifts were observed compared to bare gold electrodes on the cyclic voltammograms. This was an indication that peptides were immobilized on the gold electrode surface, obstructing the electron exchange process between the electrode surface and the [Ru(NH3)6]3+/2+. The subsequent EIS measurements were performed to analyze the charge-transfer resistance (Rct) characteristics of gold surfaces prior 8 ACS Paragon Plus Environment

Page 8 of 26

Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and post-modification. As shown in Fig. 4b, the Nyquist plots indicated that the Rct obtained from the bare electrode surface was approximately 37.549  7.01 . After the immobilization of the peptides, Rct values for WT-, H50Q- and G51D-modified electrodes increased to 27798.17  2499.1 , 21853.56  1260.9 , and 23324.90  2056.6  respectively. This significant increase in Rct was attributed to the immobilization of peptides on the gold electrode surface. In an attempt to decrease the non-specific adsorption of copper(II) on gold surfaces, a mixed-layer of peptides with PEG was prepared by co-incubation of the thiolated-PEG (containing three units of ethylene glycol) with peptides. As shown in Supplementary Fig. 1, gold electrodes were modified with different ratios of the thiolated and PEGylated peptides (HS-PEG-peptide) and HS-PEG, then exposed to 0.1 M copper(II) ions. Co-immobilization of HS-PEG with of HS-PEGpeptide mixture in the ratio of 1:4 peptide:PEG (0.2 mM HS-PEG-peptide: 0.8 mM HS-PEG in a volume of 100 µL in PBS) demonstrated a strong cathodic peak current response with relatively low standard deviation across 3 trials (n=3) (Supplementary Fig. 1). However, with increasing PEG concentration immobilized on the electrode surface, the reproducibility of the results across the repeated trials became increasingly challenging. When 100% PEG was immobilized on the electrode surface (Supplementary Fig. 2), the cathodic peak current corresponding to the reduction of copper(II) significantly diminished. This was attributed to the suppression of the non-specific adsorption of copper(II) ions on the HS-PEG-modified electrodes. The modification of gold electrode surface with only HS-peptides could create a highly compact layer and hinder the ability of copper(II) ions to interact with the metal-binding site. With the addition of HS-PEG acting as a spacer molecule, copper(II) ions could diffuse to the immobilized peptide layer and interact with histidine residues, which was observed as a strong cathodic peak current in the voltammograms. In order to demonstrate the stability of the mixed layer, multi-cycle CVs were performed in the



absence of copper(II) ions. As shown in Supplementary Fig. 3, no significant change in the CV responses was observed after 15 consecutive cycles. This indicated that the layer was stable and did not detach from the surface after repetitive CV scans. Therefore, in all future analysis, all the electrodes were co-immobilized with peptide and PEG in the ratio of 1:4. To further characterize the surface coverage efficiency, using the optimal peptide to PEG co-immobilization ratio of 1:4, a desorption study was performed. Under basic conditions, sulfur can be electrochemically desorbed form gold in a reductive manner: AuRS + e- ⇄ Au + RS-.25 Thus, the surface coverage of the peptide immobilized, as well as peptide and PEG co-immobilized process can be studied, and calculated using Γ = 𝑄/𝑛𝐹𝐴, where Q is the reductive desorption charge, n is 1 mol of e-, F is the Faraday constant (96.485 C/mol), and A is the electrode surface area (0.24 cm2). As Table 1 shows, the surface coverage of gold electrodes modified with WT, H50Q, and G51D along with HS-PEG was significantly higher than the ones determined in the absence of HS-PEG. The binding efficiency of HS-PEG-peptides appeared to be higher compared to the previously reported system using cysteine as a surface linker molecule. 26,27

Figure 4. The cyclic voltammograms (a) and Nyquist plots (b) displaying the immobilization of the HS-PEG-peptide (200 M) layer on gold surfaces; bare gold surface (blue), WT-(yellow), 10 ACS Paragon Plus Environment

Page 10 of 26

Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


G51D-(orange), and H50Q-(grey) modified gold electrode. Rct values were obtained by fitting the results with a Randles equivalent circuit for EIS (inset). (The thermodynamic parameters of Ru(NH3)6Cl3 are described in Supplementary Table 1).

Table 1. The mean surface coverage (n=3) determined by the reduction and desorption of sulfur on gold surface in 0.5 M KOH. Mutation WT H50Q G51D

Surface coverage without HS-PEG blocking (pmol/cm2) 9430  320 9290  400 9400  590

Surface coverage with HS-PEG blocking (pmol/cm2) 12700  370 12400  830 11800  940

Finally, X-ray photoelectric spectroscopy (XPS) was performed to obtain further molecular information of the modified surfaces. In Fig. 5 a-c (i), the S 2p signal for WT, H50Q, and G51D was not significantly visible on the bare gold surface prior to the modification with peptide-PEG mixed-layer. After the modification (ii), the S 2p spectrum showed a 163.5 eV signal correlating to the Au-S structure. This indicated that the HS-PEG-peptide molecules were immobilized on the gold surfaces. In the N 1s spectrum, low level of nitrogen signal was observed prior to modification (Fig. 5. d-f (i)). After the immobilization process (Fig. 5. d-f (ii)), an increase was observed in the signal of nitrogen on at N 1s spectrum at 399.58. This is a characteristic signal for the -NH2 moiety, a common amino acid N-terminal substituent. The O 1s spectrum, compared to the non-modified surface (Fig. 5. g-I (i)) the modified surface showed an increase of signal at 532.08 eV position for Fig. 5. g-i (ii). This indicated that a new oxygen species was present. In addition, there was a 1.6 eV separation between the 533.68 eV and 532.08 eV peaks. This was attributed to the presence of -C=O and the -OH groups. Moreover, significant changes were observed in the C 1s spectrum, as well. As shown on Fig. 5 j-l (ii), after the modification, the peak in 285.15 eV displayed a



significant increase. This was the result of -C-O groups present in the modified surface, which was a characteristic molecular structure of PEG. Finally, the Au 4f7/2 spectrum (Fig. 5. g-i) of both modified (ii) and non-modified (i) surfaces showed an identical signal at 83.68 eV. This indicated that the gold surface was not changed during the modification process indicating the overall coimmobilization of HS-PEG-peptide and HS-PEG on the gold surface, was successful.


Page 12 of 26

Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Photoelectron analyses of HS-PEG-peptide and HS-PEG modified surfaces before (i), and after (ii) modification. a-c S 2p spectra, d-f N 1s spectra, g-i O 1s spectra, j-l C 1s spectra, and m-o Au f7/2 spectra. Experimental details of XPS survey spectra are described in the Supplementary Information section. Interaction of copper(II) with α-syn peptides To assess the interaction of immobilized peptides with copper(II), the peptide-modified electrodes were exposed to copper(II) ions followed by a stringent rinse and DPV measurements in blank 50 mM ammonium acetate with 50 mM NaOCl solution (pH 7.4). As shown in Fig. 6a, after the exposure of copper(II), the WT-modified electrode displayed an anodic and a cathodic peak at 0.21 V, and 0.17 V (vs/ Ag/AgCl), respectively. Similarly, DPV voltammograms showed a clear peak at 0.21 V (vs. Ag/AgCl) corresponding to the copper(II) ion reduction signal (Fig. 6b) in agreement with previous studies.26 To demonstrate that copper(II) ions were bound to the peptide (Fig. 7), the dependence of peak scan rate on current signal was analyzed. In all the peptide13 ACS Paragon Plus Environment


modified electrodes, with increasing scan rate the anodic peak current increased linearly and the reduction current peaks decreased linearly. This result was attributed to an adsorption-controlled redox process indicating the strong interaction of copper(II) ions with the surface-bound peptides.

Figure 6. (a) Cyclic voltammograms of WT peptide-modified electrodes in 50 mM ammonium acetate (pH 7.4) with 50 mM NaOCl before (green), and after exposure to 0.1 M copper(II) (blue). (b) Differential pulse voltammograms of WT peptide-modified electrodes before (green), and after exposure to 0.1 M copper(II) (blue) in 50 mM ammonium acetate (pH 7.4) with 50 mM NaOCl. Other conditions were as described in the Methods section.

Figure 7. Plots for anodic (blue) and cathodic (orange) peak current vs. scan rate obtained from the cyclic voltammograms of gold electrodes modified with HS-PEG-peptides of wild-type


Page 14 of 26

Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(WT), G51D, and H50Q sequences after exposure to 0.1 M copper(II). Other conditions were as described in the Methods section. The copper(II) binding is of significant interest due to different point mutations on the 4853 region that can drastically alter the aggregation process of -syn, as well as the production of ROS in subsequent pathological pathways. The variation of copper(II) binding with -syn related peptides are shown in Fig. 8. With increasing concentration of copper(II), an increase in cathodic peak current was observed for all peptides. An observed limit of detection for copper(II) ions was determined as 50 µM (S/N=3). The strongest interaction between copper(II) and peptides was observed from G51D peptides followed by WT and H50Q ones. Our results were in agreement with previous findings that H50Q mutation displayed weak affinity towards copper(II).28,29 Initial attachment of copper(II) ions to the peptide was hypothesized to take place at the histidine residue in the metal-binding site (VVHGVA) located at 48-53 residues of -syn. Then, subsequent intermolecular arrangement would engulf the bound copper(II) ions.30 In H50Q mutants, the histidine residue is substituted with a glutamine. The charged imidazole group is substituted by a polar uncharged glutamine side-chain. This substitution was hypothesized to reduce the initial binding between copper(II) and -syn, thus resulting in a low metal-binding efficiency of H50Q mutant species. In contrast, for G51D mutants, glycine residue is substituted with aspartic acid residue. With a neutral side chain exchanged to a negatively charged organic acid group, this allowed strong intermolecular stabilization post-binding between copper(II) ions and histidine residue with a rapid aggregation rate. In addition, due to the single amino acid mutation, the hydrodynamic behavior of the mutant peptides varied significantly. Mutants containing hydrophobic amino acids prefers to undergo aggregation rapidly, and thus, have lower affinity or



access to free copper(II) ions, hence the aggregation and metal binding rates differed between the analyzed peptides in this study.

Figure 8. Mean cathodic peak current from differential pulse voltammograms (n=3) of copper(II) on peptide-modified (WT, H50Q, G51D) gold electrodes (vs. Ag/AgCl reference electrode) after exposure to various concentrations of copper(II) ions in 50 mM ammonium acetate with 50 mM NaOCl (pH 7.4). Other conditions were as described in the Methods section.

To support our electrochemical results, isothermal titration calorimetry (ITC) was used to assess the thermodynamic properties of the interaction between -syn peptides and copper(II) ions. For ITC measurements, non-thiolated and non-PEGylated WT, G51D and H50Q peptides were utilized in order to avoid the non-specific interaction of copper(II) with thiol and PEG groups (Supplementary Fig. 4). As shown in Fig. 9, the cal ∙ mol-1 of injectant between three peptides were plotted against the molar ratio of copper(II) ions. By fitting the integrated heat signal from ITC 16 ACS Paragon Plus Environment

Page 16 of 26

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


experiments with a single-binding site model, the n stochiometric ratio, enthalpy (H), entropy (S), and the association constant (Ka) were obtained. As shown in Table 2, G51D demonstrated the highest association constant (Ka) towards copper(II) ions, followed by WT and H50Q. This correlates with the trend observed in electrochemical analysis. The acidic side chain of the aspartic acid of the single amino acid substitution on G51D mutants facilitated the binding of copper(II) ions after it has interacted with the histidine residue. Thus, a higher association constant was observed. In contrast, due to the histidine substitution by glutamine on H50Q mutants, the initial interaction between copper(II) ions and the peptide was hypothesized to be decreased. Thus, a lower association constant was observed between H50Q and copper(II) ions.31

Figure 9. ITC binding isotherms of 250 M wild-type (WT, triangle), H50Q (square), and G51D (circle) peptides upon titration with 1 mM copper(II) in 10 mM PBS at 25°C. Other conditions were as described in the Methods section.

Table 2. Thermodynamic parameters of -syn peptides in the presence of copper(II) ions, n (stoichiometric ratio), Ka, H, and S values were all obtained by integrating the heat peaks from 17 ACS Paragon Plus Environment


Page 18 of 26

ITC measurements and fitted using a single-site model. c-value is an unitless value depicting the accuracy of fit (equation described in materials and method section). All values reported here show the mean of triplicate measurements with  standard deviation. Species

n

Ka (M-1)

H (cal mol-1)

S

c-value

WT

0.523  0.0326

6.50  0.64  105

-11133  856.7

(cal/mol/deg) -9.65  1.8

87.9  15.6

H50Q

0.457  0.045

4.24  0.63  105

-2368  398.5

17.33  1.4

49.4  11.6

G51D

0.482  0.056

8.05  0.99  105

-157273  2375.5

-10.93  4.4

95.0  19.3

To further analyze the modified electrode surfaces after incubation with copper(II) ions, XPS analyses were performed. As shown in Fig. 10 a-c (ii), the Cu 2p3/2 spectra indicated that copper species were present on the surfaces. An intense peak was found at 932 eV after the exposure to copper(II) ions correlated to the signal of surface-bound copper(II) species. These results were in parallel with the previously reported ones, in which copper(II) ions were successfully attached via coordination to a peptide monolayer on a gold electrode surface.32

Figure 10. Cu 2p3/2 and N 1s X-ray photoelectron spectra of WT(a,d), H50Q(b,e), and G51D (c,f) modified gold surfaces before (i), and after (ii) exposure to copper(II) ions. Other conditions were as described in the Methods section. To evaluate our hypothesis that isolating the copper(II) ion binding site of -syn as a short peptide is a feasible approach, electrochemical studies using full and truncated sequences of -syn


Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were performed. As described in literature, a definitive evidence using NMR spectroscopy indicated that the primary copper(II) ion binding site was located in the region 48-53.33 Thus, two truncated forms of -syn were utilized in our study; -syn1-60 that contains the identified copper(II) binding site, and -syn61-140, where the copper(II) binding site is absent. Using DPV, the interaction of copper(II) with these -syn proteins was detected, and the cathodic current peak height values were interpreted to generate the mean ∆Ip,c values (n=3). As shown in Fig. 11, WT full-sequence -syn displayed the highest ∆Ip,c, value indicating the strong interaction with copper(II) ions. The truncated -syn1-60 also displayed a high current response indicating copper(II) binding, whereas -syn61-140 displayed a negligible current response. Interestingly, the gold elecrodes modified with -syn48-53 showed ∆Ip,c value that was significantly similar to WT full-sequence -syn. Previous reports identified copper binding with -syn as an important factor to induce and accelerate its conformational change.28 Therefore, WT full-sequence -syn displayed highest current response after interaction with copper(II). Conversely, -syn61-140, due to the absence of copper(II) ion binding site, the interaction of the truncated -syn61-140 with copper(II) ions, was significantly decreased, which was observed by the ∆Ip,c value similar to the control assays. In the control assays, no protein modifications were performed and the EDC/NHS activated gold surfaces were quenched with 100 mM ethanolamine before exposure to copper(II) ions. Finally, the isolated short peptide -syn48-53 displayed a high ∆Ip,c value similar to the -syn WT one. This supported our hypothesis that, even in a short peptide sequence, the isolated copper(II) binding site -syn48-53 was still able to retain its strong interaction with copper(II).



Figure 11. The average peak height of differential pulse voltammograms (n=3) on gold electrodes modified with 200 µM WT full-sequence -syn (blue), -syn1-60 (yellow), -syn61-140 (pink), short peptide WT -syn48-53 (green), and control (no prior protein modifications, purple) after incubation with 200 µM copper(II) solution. DPV measurements were performed in 50 mM ammonium acetate buffer (pH 7.0) with 50 mM NaOCl as described in the Methods section.

Conclusions In this proof-of-concept study, we presented an electrochemical approach to shed light on the interaction of copper(II) ions specifically with the metal-binding region of -syn and its mutant forms, which are important as one of the pathological pathways of PD. Our data indicated that the mixed-layer of HS-PEG-peptides and HS-PEG immobilized on gold surfaces were stable and able to interact with copper(II) ions. To the best of our knowledge, we were able to generate a trend of affinity between short peptides and copper(II) ions for the first time, as follows: G51D>WT>H50Q 20 ACS Paragon Plus Environment

Page 20 of 26

Page 21 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


using electrochemical techniques. We have also confirmed this trend as Ka values using ITC. The electrochemical approach provides a platform for cost-effective and rapid screening of therapeutic agents that might be synthesized to target the metal-binding site of -syn. Furthermore, the electrochemical system was challenged to observe if the presence and the absence of copper(II) ion binding site can influence the behavior of both truncated and full-sequence of -syn with copper(II). In conclusion, we envisage that our electrochemical approach can be extended into the study of other metal-binding peptides and proteins such as amyloid-β and tau related to Alzheimer’s disease.

Associated content Supporting information: optimization of the ratio used between HS-PEG-peptides and the surface blocking HS-PEG molecules, CVs for the peptide layer stability, ITC thermograms, XPS survey spectra, and CVs for the sulfur desorption studies used to determine the surface coverage of peptides on gold surfaces.

Acknowledgments This work was financially supported by the Canada Research Chair Tier-2 award for “Bioelectrochemistry of Proteins” (Project no. 950-231116), Ontario Ministry of Research and Innovation (Project no. 35272), Discovery Grant (Project no: 3655) from Natural Sciences and Engineering Research Council of Canada (NSERC), and Canada Foundation for Innovation (Project no. 35272). We thank Mr. Bhargav R. Patel and Ms. Piryanka Sasidharan for their kind support with the ITC analyses. We also thank Surface Interface Ontario at University of Toronto, and Dr. Rana Sodhi for his support on XPS analyses.



Author information Kagan Kerman (Corresponding author) * Email: [email protected]; Phone: (416)-287-7250 Shaopei Li * Email: [email protected]; Phone: (416)-287-7250 References

(1)

Polymeropoulos, M. H.; Lavedan, C.; Leroy, E.; Ide, S. E.; Dehejia, A.; Dutra, A.; Pike, B.; Root, H.; Rubenstein, J.; Boyer, R.; Stenroos, E. S.; Chandrasekharappa, S.; Athanassiadou, A.; Papaetropoulos, T.; Johnson, W. G.; Lazzarini, A. M.; Duvoisin, R. C.; Di Iorio, G.; Golbe, L. I.; Nussbaum, R. L. Science 1997, 276, 2045–2047.

(2)

Reijnders, J. S. A. M.; Ehrt, U.; Weber, W. E. J.; Aarsland, D.; Leentjens, A. F. G. Mov. Disord. 2008, 2, 183–189.

(3)

Giladi, N.; Treves, T. A.; Paleacu, D.; Shabtai, H.; Orlov, Y.; Kandinov, B.; Simon, E. S.; Korczyn, A. D. J. Neural Transm. 2000, 107, 59–71.

(4)

Spillantini, M. G.; Schmidt, M. L.; Lee, V. M. Y.; Trojanowski, J. Q.; Jakes, R.; Goedert, M. Nature 1997, 388, 839–840.

(5)

Gibb, W. R. G.; Lees, A. J. J. Neurol. Neurosurg. Psychiatry 1988, 51, 745–752.

(6)

Recasens, A.; Dehay, B.; Bové, J.; Carballo-Carbajal, I.; Dovero, S.; Pérez-Villalba, A.; Fernagut, P. O.; Blesa, J.; Parent, A.; Perier, C.; Farinas, I.; Obeso, J. A.; Bezard, E.; Vila, M. Ann. Neurol. 2014, 75, 351–362.

(7)

Baba, M.; Nakajo, S.; Tu, P. H.; Tomita, T.; Nakaya, K.; Lee, V. M.; Trojanowski, J. Q.; Iwatsubo, T. Am. J. Pathol. 1998, 152, 879–884.

(8)

Rego, A. C.; Oliveira, C. R. Neurochem. Res. 2003, 28, 1563–1574.

(9)

Ghosh, D.; Sahay, S.; Ranjan, P.; Salot, S.; Mohite, G. M.; Singh, P. K.; Dwivedi, S.; 22 ACS Paragon Plus Environment

Page 22 of 26

Page 23 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Carvalho, E.; Banerjee, R.; Kumar, A.; Maji, S. K. Biochemistry 2014, 53, 6419–6421. (10)

Fares, M. B.; Ait-Bouziad, N.; Dikiy, I.; Mbefo, M. K.; Jovičić, A.; Kiely, A.; Holton, J. L.; Lee, S. J.; Gitler, A. D.; Eliezer, D.; Lashuel, H. A. Hum. Mol. Genet. 2014, 23, 4491– 4509.

(11)

Lomakin, A.; Chung, D. S.; Benedek, G. B.; Kirschner, D. A.; Teplow, D. B. Proc. Natl. Acad. Sci. 1996, 93, 1125–1129.

(12)

Villar-Piqué, A.; Lopes da Fonseca, T.; Sant’Anna, R.; Szegö, É. M.; Fonseca-Ornelas, L.; Pinho, R.; Carija, A.; Gerhardt, E.; Masaracchia, C.; Abad Gonzalez, E.; Rossetti, G.; Carloni, P.; Fernandez, C. O.; Foquel, D.; Milosevic, I.; Zweckstetter, M.; Ventura, S.; Outeiro, T. F. Proc. Natl. Acad. Sci. 2016, 113, E6506–E6515.

(13)

Fernández, C. O.; Hoyer, W.; Zweckstetter, M.; Jares-Erijman, E. A.; Subramaniam, V.; Griesinger, C.; Jovin, T. M. EMBO J. 2004, 23, 2039–2046.

(14)

Wang, C.; Liu, L.; Zhang, L.; Peng, Y.; Zhou, F. Biochemistry 2010, 49, 8134-8142.

(15)

Paleček, E.; Ostatná, V.; Masařík, M.; Bertoncini, C. W.; Jovin, T. M. Analyst 2008.133, 76-84.

(16)

Paleček, E.; Tkáč, J.; Bartošík, M.; Bertók, T.; Ostatná, V.; Paleček, J. Chem. Rev. 2015, 115, 2045-2108.

(17)

Lopes, P.; Dyrnesli, H.; Lorenzen, N.; Otzen, D.; Ferapontova, E. E. Analyst 2014, 139, 749–756.

(18)

Binolfi, A.; Quintanar, L.; Bertoncini, C. W.; Griesinger, C.; Fernández, C. O. Coord. Chem. Rev. 2012, 256, 2188–2201.

(19)

Outeiro, T. F.; Lindquist, S. Science 2003, 302, 1772-1775.

(20)

Zarranz, J. J.; Alegre, J.; Gómez-Esteban, J. C.; Lezcano, E.; Ros, R.; Ampuero, I.; Vidal,



L.; Hoenicka, J.; Rodriguez, O.; Atarés, B.; Llorens, V.; Gomez Tortosa, E.; del Ser, T.; Munoz, D. G.; de Yebenes, J. G. Ann. Neurol. 2004, 55, 164-173. (21)

Khalaf, O.; Fauvet, B.; Oueslati, A.; Dikiy, I.; Mahul-Mellier, A. L.; Ruggeri, F. S.; Mbefo, M. K.; Vercruysse, F.; Dietler, G.; Lee, S. J.; Eliezer, D.; Lashuel, H. A. J. Biol. Chem. 2014, 289, 21856–21876.

(22)

Rutherford, N. J.; Moore, B. D.; Golde, T. E.; Giasson, B. I. J. Neurochem. 2015, 131, 859–867.

(23)

Ghosh, D.; Mondal, M.; Mohite, G. M.; Singh, P. K.; Ranjan, P.; Anoop, A.; Ghosh, S.; Jha, N. N.; Kumar, A.; Maji, S. K. Biochemistry 2013, 52, 6925–6927.

(24)

Lesage, S.; Anheim, M.; Letournel, F.; Bousset, L.; Honoré, A.; Rozas, N.; Pieri, L.; Madiona, K.; Dürr, A.; Melki, R.; Verny, C.; Brice, A.; French Parkinson's Disease Genetics Study Group Ann. Neurol. 2013, 73, 459–471.

(25)

Lara Carrillo, J. A.; Fierro Medina, R.; Manríquez Rocha, J.; Bustos Bustos, E.; Insuasty Cepeda, D. S.; García Castañeda, J. E.; Rivera Monroy, Z. J. Molecules 2017, 22, 1970.

(26)

Yang, W.; Gooding, J. J.; Hibbert, D. B. J. Electroanal. Chem. 2001, 516, 10–16.

(27)

Zhang, J.; Chi, Q.; Nielsen, J. U.; Friis, E. P.; Andersen, J. E. T.; Ulstrup, J. Langmuir 2000, 16, 7229–7237.

(28)

Ranjan, P.; Ghosh, D.; Yarramala, D. S.; Das, S.; Maji, S. K.; Kumar, A. Biochim. Biophys. Acta - Gen. Subj. 2017, 1861, 365–374.

(29)

Rasia, R. M.; Bertoncini, C. W.; Marsh, D.; Hoyer, W.; Cherny, D.; Zweckstetter, M.; Griesinger, C.; Jovin, T. M.; Fernandez, C. O. Proc. Natl. Acad. Sci. 2005, 102, 4294– 4299.

(30)

Rose, F.; Hodak, M.; Bernholc, J. Sci. Rep. 2011, 1, 11.


Page 24 of 26

Page 25 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(31)

Zhang, Y.; Akilesh, S.; Wilcox, D. E. Inorg. Chem. 2000, 39, 3057–3064.

(32)

Bi, X.; Yang, K. L. Langmuir 2007, 23, 11067–11073.

(33)

Sung, Y. H.; Rospigliosi, C.; Eliezer, D. BBA-Proteins Proteom 2006, 1764, 5–12.



Metal-binding sites of wild-type α-syn (48-53, VVHGVA) and its pathological mutants (H50Q, and G51D) were studied using electrochemical techniques. 192x73mm (96 x 96 DPI)

ACS Paragon Plus Environment

Page 26 of 26

Electrochemical Detection of Interaction between Copper(II) and

Recommend Documents