Investigation of Cu2+ Binding to Human and Rat Amyloid Fragments

Alina Asandei , Irina Schiopu , Mauro Chinappi , Chang Ho Seo , Yoonkyung Park , and Tudor Luchian. ACS Applied Materials & Interfaces 2016 8 (20), 13...
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Investigation of Cu2+ Binding to Human and Rat Amyloid Fragments Aβ (1−16) with a Protein Nanopore Alina Asandei,†,∥ Irina Schiopu,‡,∥ Sorana Iftemi,‡ Loredana Mereuta,‡ and Tudor Luchian*,‡ †

Department of Interdisciplinary Research and ‡Department of Physics, Laboratory of Molecular Biophysics and Medical Physics, Alexandru Ioan Cuza University, Boulevard Carol I, No. 11, Iasi 700506, Romania S Supporting Information *

ABSTRACT: Recent evidence shows that metal coordination by amyloid beta peptides (Aβ) determines structural alterations of peptides, and His-13 from Aβ is crucial for Cu2+ binding. This study used the truncated, more soluble Aβ1−16 isoforms derived from human and rat amyloid peptides to explore their interaction with Cu2+ by employing the membrane-immobilized α-hemolysin (α-HL) protein as a nanoscopic probe in conjunction with single-molecule electrophysiology techniques. Unexpectedly, the experimental data suggest that unlike the case of the human Aβ1−16 peptide, Cu2+ complexation by its rat counterpart leads to an augmented association and dissociation kinetics of the peptide reversible interaction with the protein pore, as compared to the Cu2+-free peptide. Single-molecule electrophysiology data reveal that both human and rat Cu2+-complexed Aβ peptides induce a higher degree of current flow obstruction through the α-HL pore, as compared to the Cu2+-free peptides. It is suggested that morphology changes brought by Cu2+ binding to such amyloidic fragments depend crucially upon the presence of the His-13 residue on the primary sequence of such peptide fragments, and the α-HL protein-based approach provides unique opportunities and challenges to probing metal-induced folding of peptides.

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INTRODUCTION

modulatory effects induced by certain divalent metals on peptides and proteins folding processes.20−22 Owing to its tremendous social impact, a great deal of effort has been devoted thus far to understanding the interactions between various metal cations and amyloid peptides (Aβ), which are at the core of Alzheimer’s disease pathology (AD). AD is a neurodegenerative disease the onset of which is marked by the deposition of extracellular amyloid plaques and intracellular neurofibrillary tangles in the brain. The main components of the plaques are fibrils formed of 40−42 residue long Aβ peptides with a characteristic cross-β-sheet structure.23 The aggregation of Aβ monomers into pathogenic oligomer structures, which are thought to be even more neurotoxic than Aβ fibrils, is accepted to be considerably influenced among others by homeostasis of hydrogen ions as well as several metal ions, including Cu2+, Zn2+, and Fe2+.24−26 This paved the way to a new paradigm, suggesting that the perturbed cellular homeostasis of various metals may trigger the AD pathology. Extensive investigations aimed at understanding metal coordination by amyloid peptides suggested that Aβ−Cu2+ coordination assumes a square-planar arrangement and involves three intramolecular histidines (i.e., His-6, His-13, and His-14), whereas the fourth coordinate is either the amino group of the N terminus, an oxygen from Tyr-10, an oxygen from Glu-3, the

One of the most elegant approaches to sensing and identifying molecules at the unimolecular level, with promising applications in nanotechnology, relies on using protein nanopores.1−4 Inspired elegantly from the Coulter counting principle,5 and by nature in the use of proteins that sense chemical or physical stimuli, the robust α-hemolysin (α-HL) protein was demonstrated to generate real-time sensitive, specific, and reliable single-channel current signatures characteristic of various macromolecules and ions, mainly due to the fact that the pore block depends upon the topology, physical size, and noncovalent interactions of molecules in the pore.6−16 Typically, such experiments are carried out by applying a potential difference across an α-HL protein immobilized in a reconstituted planar lipid membrane, immersed in a buffer containing the analyte of interest. As individual analytes pass through the buffer-filled α-HL channel, driven by electrophoretic, electrosmotic, and diffusion effects, and partially obstruct its permeating pathway, transient blockages of the electric current recorded through the protein pore occur. The subsequent analysis of duration and amplitude of the blockage events, as well as their frequency of occurrence, can be used to monitor and investigate quantitatively various chemistries at the unimolecular level.17−19 Of particular relevance to this work, previous investigations demonstrated that protein nanopores are capable of revealing microscopic insights regarding © 2013 American Chemical Society

Received: October 10, 2013 Published: November 25, 2013 15634

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Figure 1. Typical single-channel current traces reflecting the unimolecular interaction between the human Aβ1−16 peptide with an α-HL pore, in the absence (panel a) and presence of trans-added Cu2+ at various concentrations (panels b−d), at an applied potential of −100 mV. Upward current spikes reflect the transient association of a single peptide with the protein pore. The amplitude peak denoted “*” reflects the average current through the unblocked, peptide-free, α-HL pore. The extent of the current magnitude blockage induced by the peptide on the current flow through the protein (ΔIblock) is quantified by the difference between the current measured in the absence of interaction (i.e., average values marked with “*”) and the residual current through the protein hosting a single peptide (i.e., average values marked with “#” or “$”; see also text). involving the human Aβ1−16 fragment) and between 100 and 400 μM (for experiments involving the rat Aβ1−16 fragment). Unless stated otherwise, all reagents used herein were purchased from SigmaAldrich, Germany. Measurements were carried out at room temperature (∼23 °C), and the bilayer chamber was housed in a Faraday cage (Warner Instruments, U.S.A), mechanically isolated with a vibrationfree platform (BenchMate 2210, Warner Instruments, U.S.A.). Ionic current fluctuations, reflecting Cu2+-free or Cu2+-complexed peptide interaction with a single α-HL protein, were recorded in the voltageclamp mode, with either an Axopatch 200B (Molecular Devices, U.S.A.) or an EPC 8 (Heka, Germany) patch-clamp amplifier. Amplified electric signals were low-pass filtered at a corner frequency ( fc) of 10 kHz for experiments involving the human Aβ1−16 fragment or 20 kHz for those involving the rat Aβ1−16 fragment. Data acquisition was performed using an NI PCI 6221 or an NI 6251 acquisition board (National Instruments, U.S.A.) at a sampling frequency of 50 kHz within the LabVIEW 8.20 (National Instruments, U.S.A.) environment.6,22

carboxylate group of Asp-1, or the amide CO of Ala-2.27 Importantly, His-13 was proposed to play a critical role in coordinating the Cu2+ ion, since rat amyloid which lacks a histidine in position 13, does not trigger AD-like pathology, and Cu2+-induced aggregation of rat amyloid is virtually absent.28 However, investigating metal binding by the full-length Aβ is hampered by several drawbacks, especially aggregation of the peptides. Disparate data exist regarding quantitative estimations of the dissociation constants for the binding affinity of metal ions to Aβ, with reported values ranging from attomolar to 11 μM for Cu2+ and 2−300 μM for Zn2+.29−31 Herein, we used the truncated, more soluble Aβ1−16 peptide isoforms from human and rats, and demonstrated the potential of a sensing element based on a single α-HL protein incorporated in a planar lipid membrane, to examine at the single-molecule level the interactions between Cu2+ ions and two Aβ peptide fragments.

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MATERIALS AND METHODS

RESULTS When added on the trans side of a planar lipid membrane containing a single inserted α-HL protein, and subjected to a negative (trans) potential, the human Aβ1−16 peptide is driven electrophoretically into the lumen of the protein pore and produces reversible blockages of the protein-mediated ionic current (Figure 1a). The lumen occlusion by the peptide is not complete, mainly due to slight geometrical differences between the effective size of the peptide and the inner diameter of the protein pore, and a residual current is present (Figure 1e). Previous data established firmly that the weak noncovalent interactions that occur between individual peptides and the αHL pore result in a reversible association between the peptide and the pore, which is best described kinetically by a bimolecular interaction model.6,14 During the course of

Single-molecule electrophysiology measurements were performed on planar lipid membranes made from L-α-phosphatidylcholine obtained as described previously.32,33 Both cis (grounded) and trans chambers of the bilayer setup contained 2 M KCl, buffered in 10 or 50 mM HEPES at pH 7.3. Single-channel insertion of an α-HL protein was attained by adding in the cis side aliquots of protein monomeric solution, from a stock made in 0.5 M KCl. Once the successful insertion of a single αHL heptamer was achieved, either Aβ1−16 human peptide (Asp-AlaGlu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys) or Aβ1−16 rat peptide (Asp-Ala-Glu-Phe-Gly-His-Asp-Ser-Gly-Phe-GluVal-Arg-His-Gln-Lys) were pipetted in the trans side, at a 50 μM concentration from a pre-stock solution made in water at the moment of use (1 mM), from a stock solution (10 mM) made in dimethyl sulfoxide. Throughout experiments, anhydrous copper(II) chloride was added in the same side as the peptides (i.e., the trans chamber) at a concentration ranging between 25 and 200 μM (for experiments 15635

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to what is known as bumping-induced blockages. As revealed further, the Cu2+-mediated alterations of human Aβ1−16 peptide-induced blockage events are fully reversible. When added in excess of Cu2+ in the trans side of the membrane, EDTA led to an almost complete restoration of the blockage activity induced by the Cu2+-free human Aβ1−16 peptide on the ion flow through the α-HL pore (Figure S1, Supporting Information). The kinetic analysis demonstrated that average times corresponding to between current blockage events (τON), caused by either a Cu2+-free or a Cu2+-complexed peptide trapped within the protein lumen (Figure 2a), as well as the

reversible interactions between human Aβ1−16 peptides and the α-HL pore, trans-chamber addition of Cu2+ at incrementally larger concentrations resulted in a change of the blockade currents induced by the peptide itself (Figure 1b−d). Close inspection of the blockage current distribution shows three types of distinct transient current blockages, denoted herein “$”, “#”, and “&” (see also Table 1 for the statistics on the relative blockage amplitude associated with such substates). Table 1. Values of the Current Magnitude Blockage (ΔIblock) Associated with Various Blockage Substates (Denoted “#”, “*”, “&”, and “$”; See Also Figure 1) Induced by the Human Aβ1−16 Peptides upon the Current Flow through a Single αHL Pore, Measured at an Applied Potential Difference ΔV = −100 mV, in the Absence and Presence of Cu2+ at Various Concentrationsa ΔIblockage (pA)

% blockage

0

139.33 ± 2.78#*

89.21

25

129.83 ± 0.84#* 141.65 ± 1.01$*

87.70 95.67

100

146.19 ± 0.12#* 161.07 ± 0.1$* 113.79 ± 0.94&*

88.49 97.50 68.88

200

148.00 ± 1.70$* 99.03 ± 5.95&*

95.46 63.87

[Cu2+] (μM)

a

The blockage percent represents the corresponding relative extent of current flow obstruction, calculated with respect to the absolute value of the ion current measured through the pore in the absence of both peptide and metal.

When Cu2+ is added at low to intermediate concentration values, two forms of peptides are present in the solution, namely, Cu2+-free and Cu2+-complexed peptides. As a result, the transient blockage events of the ion current through the αHL protein can result from the interaction of the pore with either type of peptide. In support of this assertion, histogram analysis of the amplitude of ion current blockages revealed distinct amplitude blockage peaks, corresponding to the cases when the human Aβ1−16 peptide interacted with α-HL in the absence or presence of Cu2+. Due to their rather consistent value seen in experiments performed in the absence or presence of Cu2+, it is reasonable to assume that whereas the blockage amplitude level denoted “#” (Figure 1a, no Cu2+ added) can attributed to the remnant ion current through a transient Cu2+free peptide−α-HL complex, the second type of blockage amplitude, denoted “$” and visible only in the presence of Cu2+, can be assigned to the residual current mediated by an α-HL protein that temporarily traps a Cu2+-complexed peptide. A supplementary argument in favor of this is provided by the fact that in the presence of large values of added Cu2+, the blockages induced by the most likely present peptide species in bulk solution, that is, the Cu2+-complexed human Aβ1−16 peptide, are represented almost solely by “$”-like amplitude events. On the basis of their considerably lower amplitude and faster kinetics, the event currents denoted “&”, seen mostly at intermediate to high concentration values of the added Cu2+, were reckoned to represent the transient collision of the human Aβ1−16 peptide with the pore’s entrance, which precluded a full partitioning of the peptide into the pore lumen, thus giving rise

Figure 2. Analysis of the average dwell time intervals assigned the association (τON; panel a) and dissociation (τoff; panel b) of a single human Aβ1−16 peptide interacting reversibly with an α-HL pore, measured at various concentrations of the trans-added Cu2+ (i.e., 0, 25, 50, 75, 100, 150, and 200 μM), and the peptide present at a 50 μM concentration. In the inset of panel a is shown a truncated current trace fragment displaying the time between two consecutive peptide association events (τON) and that needed for a peptide to dissociate from the protein pore (τOFF), on the basis of which we constructed the statistics of the distribution of these time intervals.

average duration of blockage events (τOFF; Figure 2b), increased as the concentration of Cu2+ was being raised in the trans chamber. To a first account, these results confirm previous studies which revealed that Cu2+ modulates the ion current fluctuations associated with a His-containing peptide, reversible association to the α-HL protein.22 To probe indirectly the human Aβ1−16 peptide−Cu2+ ion complexation, we investigated next the kinetics of Aβ1−16−αHL reversible interaction as a function of the applied voltage. As for the case of anionic peptides (the net electric charge on the Aβ1−16 peptide was estimated as −1.7 e− at neutral pH), their interaction with a negatively applied potential on the side 15636

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Figure 3. Voltage dependence of the average association (τON) and dissociation (τOFF) dwell times, which describe the reversible interaction of a single human Aβ1−16 peptide (50 μM) with the α-HL protein pore in Cu 2+-free buffer (panels a and b) and the presence of 100 μM Cu2+ added on the trans side of the membrane. In Cu 2+-free experiments (panels a and b), as well as for the data shown in panel c (voltage dependence of the average association (τON) dwell times in the presence of 100 μM Cu2+), we found that the average times can be fitted with single decaying exponentials using the equation f(ΔV) ∼ A exp(−(ΔV)/(ΔV0)), whereas the dissociation (τOFF) dwell times measured in the presence of 100 μM Cu2+ (panel d) were best fitted with a linear function.

of their addition results in a major force to drive the peptides into the α-HL lumen. The analysis of average time intervals between blockage events (τON) and those corresponding to the peptide temporarily trapped within the protein pore (τOFF), in the absence and presence of trans-added 100 μM Cu2+ at various holding potentials, is shown in Figure 3. Data corresponding to control experiments (no Cu2+ added) have revealed that both τON and τOFF can be fitted to exponentially decaying functions with increasing negative potential values (Figure 3a,b). The exponential dependence of average time for peptide capture (τON) on applied voltage suggests that this process is not diffusion-limited. To support this assertion, it is worth recalling that the movement of charged peptides toward the α-HL protein mouth is subjected to an electric force, even before the peptide starts sensing the potential difference across the pore lumen, and numerous studies revealed that an applied potential difference between the cis and trans sides of the membrane containing a nanopore result in electric field lines that extend well out of the pore.34,35 Under simplifying assumptions (i.e., the lipid membrane is being modeled as an ideal insulator containing a cylindrical pore that separates the two electrolyte solutions on the cis and trans side, respectively), a potential difference across the membrane generates an electric field, for which the potential value at a distance r away from the pore mouth is given by

V (r ) =

d2 ΔV 8lr

(1)

where r represents the radial distance from the pore mouth, l the pore length, d its diameter, and ΔV the externally applied potential difference across the membrane. These field lines are generated and maintained at far-fromequilibrium conditions by an ion current across the membrane, and under our experimental conditions (i.e., negatively charged peptides added on the negatively biased trans side of the membrane) they create a potential profile outside the pore mouth that drives peptides toward the α-HL pore. With the help of Smoluchowski theory written for a system with spherical symmetry, describing the peptide electrodiffusion toward the protein mouth that is modeled to capture the incoming peptides within a semispherical region, the peptide diffusion-controlled rate (ratediffusion) is35 ratediffusion =

[C]peptide πd 2μΔV 4l

(2)

where [C]peptide represents the concentration of the peptide on the bulk trans side, μ is the peptide electrophoretic mobility, and the rest of the parameters have the same meaning as above. Therefore, if the peptide capture is a diffusion-controlled process, its rate should depend linearly upon the applied voltage and, conversely, the inverse of the rate, quantified in our experiments as being proportional to the average of time values 15637

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measure between blockage events (τON), should vary as ∼ ΔV−1. However, as Figure 3a shows, the average time-tocapture of the Cu2+-free Aβ1−16 peptide by the α-HL (τON) varies exponentially on voltage. We used Akaike’s Information Criterion Test (AIC) to establish that τON versus ΔV more likely fits with an exponential function of ΔV than a ΔV−1, and this result suggests that the mouth of the protein lumen creates a free energy barrier to peptide capture. According to classical Kramers theory, the exponential rate enhancement of peptide capture results most likely from the fact that the applied potential lowers the height of this trans energy barrier.14 Voltage dependence of average values corresponding to current blockages dwell times, reflecting peptide interaction with the protein lumen (τOFF; excluding bumping events), were well described by a decreasing exponential function versus hyperpolarization of the membrane trans side (Figure 3b). In the general case, blockage events with lifetimes described by τOFF do not necessarily reflect translocation events entirely. On the basis of oversimplified physical considerations, increasing the trans-side negative potential may create conditions for the transto-cis electrophoresis to dominate diffusion of a human Aβ1−16 peptide situated within the protein lumen, and this can be seen as correspondingly lower τOFF average values. On the basis of our data, it is therefore safe to conclude that current blockageinduced human Aβ1−16 peptides most likely reflect translocation events. In the presence of 100 μM Cu2+ added in the trans side of the membrane, the average of τON values retained their exponential-like decay tendency with increasing negative potential values (Figure 3c), whereas the average of τOFF values was found to increase almost linearly with increasing negatively trans-applied potentials (Figure 3d). As we will stress further below, this observation is indirectly indicative of conformational changes of the human Aβ1−16 peptide induced by the bound Cu2+ ions, which has been previously suggested by different techniques.26,36 Under the influence of an external potential difference, a Cu2+-complexed peptide can move toward the protein pore and plug it in a voltage-dependent manner rather than translocate to the cis side, as the Cu2+induced folding precludes its squeezing past the constriction region of the protein pore. In this scenario, the voltage dependence of average τOFF values indicated by Figure 3d would reflect the increasingly higher free energy barrier encountered by a Cu2+-complexed peptide to return to its originating trans side of the membrane, upon dissociation from the protein pore. When added on the trans side of the membrane at similar concentrations, the rat Aβ1−16 peptide displayed a larger propensity to interacting with the protein pore as compared to human Aβ1−16 peptide, as it is supported by the enhanced number of current blockages induced by incoming rat Aβ1−16 peptides (Figure 4). In contrast to the data recorded with the human Aβ1−16 peptide (Figure 1), increasing concentrations of Cu2+ added on the trans side of the membrane led to a higher frequency of blockage events associated with the rat Aβ1−16 peptide interaction with the lumen of the protein pore (Figure 5). This observation is also counterintuitive, because Cu2+ coordination to primary binding sites on the peptide, especially at higher metal concentrations, would be expected to stabilize it on a folded conformation that prevents it from entering the pore.

Figure 4. Representative ion current traces measured at ΔV = −100 mV through a single α-HL protein, showing blockages induced by the human (left column) and rat (right column) Aβ1−16 peptide added on the trans chamber at a concentration of 50 μM. Due to the shorter blockage events induced by the rat Aβ1−16 peptide, the cutoff frequency of the low-pass filter (fc) was set at 20 kHz, as opposed to the case of the experiments involving the human Aβ1−16 peptide, where fc was set at 10 kHz, and this is reflected by the increase in the noise of the traces showing the interaction between the rat Aβ1−16 peptide and the protein pore.

The blockade currents mediated by a Cu2+-free rat Aβ1−16 2+ ]=0 peptide temporarily confined within the protein pore (ΔI[Cu blockage = 128.3 ± 1.25 pA) also differ from that of the human Aβ1−16 2+ [Cu ]=0 peptide (ΔIblockage = 139.33 ± 2.78 pA; see Table 1). Moreover, and similarly to the tendency seen in the case of the human Aβ1−16 fragment behavior in the presence of Cu2+, the amplitude histograms of blockages ensued by the interactions of the protein pore and the Cu2+-complexed rat Aβ1−16 peptide revealed an elevated value of the relative [Cu 2+ ]=100 μM blockage amplitude (ΔIblockage = 133 ± 1.15pA; 2+

]=400 μM = 142 ± 0.67pA). We conjecture that the ΔI[Cu blockage increase in the extent of ion current blockage caused by ‘Cu2+complexed’ human and rat Aβ1−16, as compared to situation seen in the absence of Cu2+, may be a consequence of a tighter topological fit of the Cu2+-complexed’ peptides to the inner space of protein pore. Thus, an overall decrease in ‘Cu2+complexed’ peptide flexibility inside the pore and augmented steric hindrance, leads to a more prominent reduction of the effective diameter of α-HL. A statistical analysis of single-channel current traces showing the interaction between a rat Aβ1−16 peptide and a α-HL protein in Cu2+-free conditions, or when metal was added at various concentrations on the same membrane side as the peptide, was performed to separate bumping events from those representing peptide interactions with the protein inside the lumen, and for the latter type of events the average blockade

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Figure 5. Representative single-channel current traces showing the interaction between the trans-added rat Aβ1−16 peptide (50 μM) with an α-HL protein, in the absence of Cu2+ (panel a) and presence of trans-added Cu2+ (panel b, 100 μM; panel c, 400 μM), at an applied potential difference ΔV = −100 mV. In panels below we show the outcome of the analysis on the average dwell time intervals assigned the association (τON; panel d) and dissociation (τOFF; panel e) of a single rat Aβ1−16 peptide interacting reversibly with an α-HL pore, measured at various concentrations of the transadded Cu2+ (i.e., 0, 100, 200, 300, and 400 μM). The inset of panel d shows a short original current trace displaying the time between two consecutive peptide association events (τON), and dissociation time from the protein pore (τOFF), in the absence and presence of Cu2+.

absence of Cu2+ (see Table 1). It appears that the Cu2+complexed peptide, which most likely is unable to translocate the pore (Figure 3), enters the protein lumen and assumes a tighter geometrical fit than the Cu2+-free peptide, giving rise to larger blockage events than Cu2+-free peptide. Interestingly, the same augmenting effect by the added Cu2+ upon the peptideinduced ion current blockage through the protein pore was also visible in the experiments involving the rat Aβ1−16 peptide (see above). The kinetic activity assay carried out at similar concentrations of both human and rat Aβ1−16 peptides, in the absence and presence of trans-added Cu2+, revealed two noteworthy results. In the absence of Cu2+, the trans-side-added rat Aβ1−16 peptide was prone to a larger number of captures by the protein pore as compared to the human Aβ1−16 peptide (Figure 4), and this is further confirmed by the quantitative analysis of the average value of time intervals between blockage events. We must stress that there is no simple explanation for this phenomenon, as the overall charge, hydrophobicity, and physical size show very small differences for both peptides (see Table S1, Supporting Information). Even more strikingly, we observed that whereas addition of Cu2+ led to a decrease in the likelihood of human Aβ1−16 association events to the protein pore (Figure 2), the opposite occurred when Cu2+-complexed rat Aβ1−16 peptides interacted with the protein pore (Figure 5). For the interpretation of this result, we performed the kinetic analysis of averaged time intervals of interevents separating consecutive current blockages (τON) caused by the peptide trapped within the protein pore and pore blockage events (τOFF), as they were measured in the absence and presence of increasing concentrations of trans-added Cu2+. As a minimalist kinetic model and as discussed previously,22 we propose that in the absence of interaction with a peptide, the α-HL protein is found in its fully open state, denoted “O” (see eq 3). In this state and in the presence of added Cu2+, the protein can associate

times (τOFF) and durations between consecutive blockage events (τON) were derived (Figure 5d,e). These data confirmed that Cu2+ complexation by the rat Aβ1−16 peptide led to a sizable alteration of the kinetics, describing its interaction with the protein pore, yet distinct in terms of kinetic constants from that seen in the case of the human Aβ1−16 peptide.

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DISCUSSION To interrogate the sensitizing effect histidine residues have on peptide coordination of Cu2+ ions, we employed herein the human and rat Aβ1−16 peptide isoforms, capitalizing on the fact that His-13, a critical residue in coordinating the Cu2+ ion, is lacking in the rat Aβ1−16 peptide. The interpretation of our data is facilitated by previous knowledge suggesting that the Cu2+− Aβ1−16 stoichiometry is 1:1; that is, there is a single binding site for Cu2+ on the peptide.26,36−39 Structural modifications induced by Cu2+ coordination were suggested by X-ray absorption spectroscopy and circular dicroism studies, which have shown that Cu2+ intrapeptide binding to human Aβ1−16 can induce the formation of a regular structure, and gives rise to a rather compact peptide structure.39,40 Our results indicate that Cu2+ complexation alters the topology of the human Aβ1−16 peptide, which is partially precluded from association to the protein pore lumen. We also demonstrated that this phenomenon depends upon the nature of the coordinated metal, as Zn2+ addition was less effective in modulating the human Aβ1−16 peptide−α-HL reversible interactions (Figure S2 in the Supporting Information), and we plan to approach this more thoroughly in a forthcoming work. As presented above, in the presence of trans-added Cu2+, the amplitude blockage peak denoted “$” is indicative of Cu2+-bound peptide interacting with the lumen of the protein pore. However, considering that the conformational flexibility of the human Aβ1−16 is reduced by the bound Cu2+,28 it is worth noting that the amplitude of “$”-like events is actually slightly higher than that seen in the 15639

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reversibly either with a Cu2+-free peptide (denoted “P” in what follows), giving rise to blockage events belonging to a first type of “closed-states” (C1), or with a Cu2+-complexed peptide (denoted “P−Cu2+”), thus favoring blockage events related to a distinct, second type of closed-states (C2). The overall kinetic scheme described above is written

in Figures 2 and 5d,e with eqs 5 and 6. However, this procedure is highly sensitive to the value one inputs for Kd. As mentioned herein, there is extensive data in the literature suggesting Kd values for Cu2+ binding ranging from ∼nM31,38 to ~ μM41 for the truncated Aβ peptides, and attomolar to μM for the full length Aβ peptides.29,30 It is therefore obvious that a relatively large degree of bias would be imposed on the values of rate constants derived from the fit, depending on whether nanomolar or micromolar values are used for Kd (Figures S3 and S4, Supporting Information). We must also stress that in the case of our experimental conditions, Kd values for Cu2+ binding to Aβ1−16 fragments in the range of nanomolar are hardly reconcilable with the data shown in Figures 2 and 5d,e. As shown in Figure S5 in the Supporting Information, an extremely effective binding of Cu2+ to Aβ1−16 peptides (e.g., Kd = 0.95 nM) would lead to a saturation plateau when [Cu2+] reaches ∼50 μM; that is, beyond this concentration most of the peptide would be in the Cu2+-complexed form. In turn, and as a relevant example, this would entail that average τON and τOFF values reflecting the reversible interaction of a single human Aβ1−16 peptide with an α-HL pore in the presence of various Cu2+ concentrations saturate when the trans-added metal reaches ∼50 μM, because the pool of Cu2+-complexed Aβ1−16 peptide reaches its maximum value and almost no Cu2+-free Aβ1−16 peptides are available in the solution, on average terms. As Figure 2 shows, however, this is hardly the fact, and if it was, the τON and τOFF dependence versus [Cu2+] would pretty much resemble the continuous lines shown in Figure S4 (Supporting Information). In our numerical estimations we considered a simplified approach, so as to not implicate the actual value of Kd when evaluating the k1, k2, k3, k4 rate constants, and our rationale went as follows: in the absence of Cu2+, only interactions between the Cu2+-free peptide and the protein pore are seen, meaning that eqs 5 and 6 would become

k 3[P−Cu 2 +]

k1[P] C2 ←⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯→ O ←⎯⎯→ C1 k2 k4

(3) 2+

In the scheme above, [P] and [P−Cu ] denote the available concentrations of the Cu2+-free peptide and Cu2+-complexed peptide, respectively, k1 and k3 stand for the association rate constants of the Cu2+-free peptide and Cu2+-complexed peptide with a protein pore initially found in the open state, whereas k2 and k4 represent the corresponding dissociation rate constants. As we described in detail before,22 at any given initial concentration of the free peptide [P0]) and Cu2+ ([Cu2+ 0 ]), the equilibrium value of the binary complex (P−Cu2+) is given by [P−Cu 2 +]eq = ([P0] + [Cu 02 +] + Kd) −

([P0] + [Cu 20 +] + Kd)2 − 4[P0][Cu 02 +] 2

(4)

whereby Kd is denoted the dissociation constant of the reversible peptide−Cu2+ ion interaction. To arrive at an analytical expression for the average value of τOFF time intervals at stationarity, one must recall that τOFF intervals reflect times while the α-HL pore lumen is associated with either a Cu2+-free peptide (P) or a Cu2+-complexed peptide (P−Cu2+). We undertook a statistical analysis on τOFF time intervals reflecting either type of dissociation reaction and implicated a statistical approach able to describe the distribution of lumped τ OFF events (i.e., τ OFF values corresponding to a either a protein pore dissociating from a Cu2+-free peptide (P) or a Cu2+-complexed peptide (P−Cu2+)), through the k1−k4 reaction constants. On the basis of the general theory of single-channel kinetics analysis of a three-state Markov model,22 the theoretical average value of the lumped τOFF time intervals is k4k1 × ([P0] − [P−Cu 2 +]eq ) + k 2k 3[P−Cu 2 +]eq k 2k4 × (k1([P0] − [P−Cu 2 +]eq ) + k 3[P−Cu 2 +]eq ) (5)

Similarly, the theoretical average value of lumped, association time intervals measured between peptide-generated (i.e., either a Cu2+-free peptide (P) or a Cu2+-complexed peptide (P− Cu2+))) blockage events of the current through the protein pore (τON) equals

=

1

τON([Cu2+] → 0) =

1 k1([P0])

(7)

(8)

τOFF([Cu2+] →∞) =

1 k4

τON([Cu2+] →∞) =

1 k 3[P0]

(9)

(10)

On the basis of eqs 7−10, the kinetic constants characterizing the reversible interactions between the human Aβ1−16 peptides and the α-HL protein pore in the presence of added Cu2+ were found to be k1 (human Aβ1−16) = (26 ± 1.7) × 103 s−1 M−1, k2 (human Aβ1−16) = (3.1 ± 0.2) × 103 s−1, k3 (human Aβ1−16) = (4.1 ± 0.3) × 103 s−1 M−1, and k4 (human Aβ1−16) = 131.6 ± 9.5 s−1. Following a similar line of reasoning, the kinetic constants describing the reversible interactions between the rat

k 3[P−Cu 2 +]eq + k1([P]eq ) 1 k 3[P−Cu 2 +]eq + k1([P0] − [P−Cu 2 +]eq )

1 k2

Conversely, in the presence of the highest concentration of the added Cu2+, when almost all Cu2+-free peptides are depleted and the concentration of the Cu2+-complexed peptide reaches its maximum value (i.e., [P−Cu2+]eq = [P0]), the current blockages seen originate mostly from the interaction between the protein pore and a single Cu2+-complexed peptide, so that eqs 5 and 6 become

τOFF =

τON =

τOFF([Cu2+] → 0) =

(6)

To arrive at numerical estimates for the kinetic parameters characterizing the entire reaction scheme of eq 3 (i.e., k1, k2, k3, k4), a nonliner fit would be needed on experimental data shown 15640

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In conclusion, we highlighted herein the potential of the αHL as a tool to investigate the reversible binding of various metals to physiologically relevant Aβ fragments. With the use of specific tailored peptides, our findings may pave the way to an easy-to-use methodology able to detect and recognize ions or other small molecules in bulk, e.g., peptide inhibitors of Aβ oligomerization, with protein nanopores.

Aβ1−16 peptide and the protein pore in the presence of added Cu2+ are k1 (rat Aβ1−16) = (54.3 ± 4.3) × 103 s−1 M−1, k2 (rat Aβ1−16) = (4.4 ± 0.6) × 103 s−1, k3 (rat Aβ1−16) = (73 ± 5.6) × 103 s−1 M−1, and k4 (rat Aβ1−16) = (2.04 ± 0.17) × 103 s−1. We stress that these values describing the kinetics of rat and human Aβ1−16 peptides interacting with the α-HL protein, in either ‘Cu2+-free’ or ‘Cu2+-complexed’ form represent approximate estimates, given the assumptions made in relations (9) and (10). The association reaction constant of the Cu2+-free rat Aβ1−16 fragment is almost double compared to the Cu2+-free human Aβ1−16 peptide, and we suggest a scenario that involves indirectly the preferential orientation of the peptides at the pore’s mouth. Given their intrinsic dipole moment, and considering that our experiments were performed with the trans side of the membrane more negative electrically than the cis side, peptides are likely to enter the lumen of the protein pore from the C terminus,42 aided in their preferential orientation by the converging electric field gradient. This is relevant because although the mean hydrophobicity values of the N terminus for both human and rat Aβ1−16 peptides are similar (−0.11; see also Table S1 in the Supporting Information), the C terminus mean hydrophobicity for the rat Aβ1−16 fragment was estimated at −0.98, whereas that of the human Aβ1−16 fragment equals −0.64 (Table S1). Therefore, the slightly higher hydrophilic manifestation at the C-terminus of the rat Aβ1−16 fragment can be advantageous for funneling it into the α-HL pore mouth, by comparison to the human Aβ1−16. An intriguing issue which we aim at addressing in forthcoming work, is to explain why the ‘Cu2+-complexed’ rat Aβ1−16 associates considerably more favorably with the α-HL pore as compared to the Cu2+-free’ peptide, while the ‘Cu2+complexed’ human Aβ1−16 associates less favorably with the αHL pore, as compared to its Cu2+-free’ counterpart (vide supra, the comparison between k1 and k3 for both human and rat ̈ approach, one would expect that Cu2+ Aβ1−16). In a naive binding leads to a consistent decrease in the propensity of both types of ‘Cu2+-complexed’ peptides for interacting with the protein pore. We posit the distinct manifestation of Cu2+induced conformational changes on the human and rat Aβ1−16, so that an increase in hydrophilic residues exposure occurs for the case of the rat Aβ1−16, making it better predisposed towards a favourable interaction at the entrance of the α-HL lumen. Equally challenging for future investigations remains, there remain to be established molecular determinants supporting that Cu2+ coordination by the peptide augments substantially the dissociation (k4) rate constant which characterizes the Cu2+-complexed’ rat Aβ1−16 peptide interaction with the protein pore, as compared to the case of the human Aβ1−16 fragment. Previous results are somewhat contradictory with respect to Cu2+ affinity towards human and rat amyloids.27 From our results howevercompare for instance Figure 1 (panels a and c) and Figure 5 (panels a and b)is clear that Cu2+ induces a more appreciable effect on human Aβ1−16−α-HL interactions as compared to the case of rat Aβ1−16, in the presence of similar concentrations of the metal. The dissimilarities seen in this respect may reflect variations in experimental conditions and preparation of peptides. Nevertheless, our approach may constitute a viable alternative to assessing the affinity of various metals (or other agonists, for that matter) towards peptide constructs which do not possess aromatic aminoacids, usually employed to report on such estimations.30

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures and table. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*(T.L.) E-mail: [email protected]. Author Contributions ∥

A.A. and I. S. contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We acknowledge the financial support offered by grants PN-IIID-PCCE-2011-2-0027, PN-II-PT-PCCA-2011-3.1-0595, and PN-II-PT-PCCA-2011-3.1-0402.

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REFERENCES

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