Electrochimica Acta Electrochemical sensing of 2D condensation in amyloid peptides

The interfacial behavior of the model amyloid peptide octamer YYKLVFFC (peptide 1 ) and two other amyloid peptides YEVHHQKLVFF (peptide 2) and KKLVFFA (peptide 3 ) at the metal | aqueous solution interface was studied by voltammetric and constant current chronopotentiometric stripping (CPS). All three peptides are adsorbed in a wide potential range and exhibit different interfacial organizations depending on the electrode potential. At the least negative potentials, chemisorption of peptide 1 occurs through the formation of a metal sulfur bond. This bond is broken close to − 0.6 V. The peptide undergoes self-association at more negative potentials, leading to the formation of a “pit” characteristic of a 2D condensed ﬁlm. Under the same conditions the other peptides do not produce such a pit. Formation of the 2D condensed layer in peptide 1 is supported by the time, potential and temperature dependences of the interfacial capacity and it is shown that presence of the 2D layer is reﬂected by the peptide CPS signals due to the catalytic hydrogen evolution. The ability of peptide 1 to form the potential-dependent 2D condensed layer has been reported neither for any other peptide nor for any protein molecule. This ability might be related to the well-known oligomerization and aggregation of Alzheimer amyloid peptides. © 2013 Elsevier Ltd.


Introduction
Among human neurodegenerative diseases, special attention has been paid to diseases in which amyloidogenic proteins, when disturbed from their native state, can form oligomeric and polymeric aggregates [1,2]. Such aggregated proteins frequently have a well-defined fibrillar nature termed amyloid. A number of such diseases include type II diabetes, Creutzfeldt-Jakob, Alzheimer's and Parkinson's diseases. The latter two are the most common neurodegenerative disorders among the elderly. The amyloid ␤peptides (A␤) in Alzheimer's disease (AD) can serve as a paradigm for studies of amyloid formation and conformation [1,3]. These peptides derive from cleavage of the transmembrane amyloid precursor protein by secretases. Aggregation of AD and Parkinson's disease peptides and proteins can be induced or inhibited in vitro and in vivo by a number of agents. In vitro the aggregation was studied by different methods, such as circular dichroism, light scattering methods, polarized Raman spectroscopy, X-ray diffraction, thioflavin T or Congo red fluorescence, atomic force microscopy, electron microscopy or solid state NMR, etc. [1][2][3][4]. In recent years also electrochemical methods have been applied in such studies using carbon [5][6][7], Hg [4,6,8], and Au [9] electrodes. Interaction of A␤ peptides and proteins with lipid bilayers at Au and Hg [10,11] electrodes was also studied.
In the past decades electrochemistry of proteins concerned predominantly studies of non-protein components of conjugated proteins yielding reversible chemistry [21]. Oxidation of tyrosine (Tyr) and tryptophan residues in peptides [22] and proteins [23] at carbon electrodes was studied to a lesser extent. Recently it has been shown that using constant current chronopotentiometric stripping (CPS) practically all proteins and many peptides produce the so-called "peak H" (due to the catalytic hydrogen evolution reaction, CHER). This peak has proven to be valuable in protein structure analysis [24,25], and in studies of aggregation of amyloid protein ␣-synuclein [4,8].
In electrochemical techniques, reliable attachment of the peptide or protein to the surface plays a critical role. A recent strategy was proposed involving the reaction of cystamine and 2-iminothiolane with the amyloid fibrils, enabling their covalent linkage to gold surfaces via Au S bonding [26]. Alternatively, cysteine-containing proteins can be attached to some metal surfaces, such as gold or mercury via Au-S or Hg S bonds. This type of binding can however hardly be applied to AD A␤-peptides or to ␣-synuclein (important in Parkinson's disease), lacking cysteine (Cys) residues. Non-covalent electrostatic binding of A␤ peptides to Au electrodes is much weaker and poorly suitable for the peptide immobilization at these electrodes. In A␤-peptides, a Cys residue can be easily added to the given peptide during its synthesis. This was recently recognized by Partovi-Nia et al. [27] who modified the A␤ (12-28) peptide with a Cys residue at the C-terminal to immobilize it onto gold electrodes. With this platform, they were able to monitor electrochemically the interaction of the peptide with Congo Red and with a ␤-sheet breaker peptide.
We investigated the peptide-based octamer YYKLVFFC (peptide 1) which contains the KLVFF A␤ (16)(17)(18)(19)(20) core domain with additional terminal Tyr and Cys residues to enable possible functionalization. This peptide was de novo designed by one of us (IWH). Cys residue enables not only covalent attachment to Hg and other metal surfaces but also chemical modification via the sulfhydryl moiety, and the two Tyr residues serve as fluorescence tags, and also potentially enable bioconjugation, or responsiveness to enzymes for instance via phosphorylation/dephosphorylation. The solution self-assembly of YYKLVFFC has been investigated using a variety of spectroscopic, scattering and microscopic methods [28]. The formation of ␤-sheet fibrils in peptide 1 was confirmed with a pHdependent change in morphology. Remarkably, this peptide forms a nematic liquid crystal phase at relatively low concentration [28]. This tendency to spontaneously orient was later exploited in a detailed study of the orientation of the alignment of the peptide (in bulk solution, and dried films) using polarized Raman spectroscopy, linear UV dichroism, FTIR, X-ray diffraction and Near-Edge X-ray Absorption Fine Structure (NEXAFS) [29]. This provided detailed information on the orientation of fibrils and of the constituent Tyr fluorophores.
In the present work, we decided to supplement the wealth of information about properties of peptide 1 and other AD peptides obtained by the above methods by applying electrochemical methods, which proved useful in studies of oligomerization and aggregation of ␣-synuclein important in Parkinson's disease [4,6,8]. We focused on the electrochemical and interfacial properties of the above peptide 1 and two other A␤ peptides YEVHHQKLVFF (peptide 2) and KKLVFFA (peptide 3) predominantly at mercury electrodes. All three peptides contain the KLVFF core characteristic for the amyloid AD peptides. Peptide 3 differs from peptide 1 by changes at both termini. Two Tyr residues (YY) at the N-terminus of peptide 1 are replaced by K in peptide 3, and C at the other end of peptide 1 is substituted by A in peptide 3. In peptide 2 the KLVFF core is extended only at the N-terminus by YEVHHQ differing from both peptide 1 and 3 by negatively charged E, additional V and HH. The reduction of the Hg S bond between the Hg surface and the Cys residue was evidenced only in peptide 1. Besides the electroactive properties of the peptides, the interfacial behavior was investigated by differential capacity measurements. Peptide 1 (but not the other two peptides) showed a strong tendency to self-association and formation of 2D condensed layer at the electrode surface, which might be related to tendency of Alzheimer A␤-peptides to aggregation.

Apparatus and electrochemical procedures
Electrochemical measurements were performed using an electrochemical 663 VA Stand multimode three-electrode systems (Metrohm, Herisau, Switzerland) connected with a Autolab Analyzer type III or an Autolab electrochemical system PGSTAT 302 equipped with a frequency-response analyzer module (FRA2) (EcoChemie, Utrecht, The Netherlands). Hanging mercury drop electrode (HMDE, area of drop 0.4 mm 2 ) and basal plane pyrolytic graphite electrodes (PGE, 6.0 mm 2 area) were used as working electrodes, a Ag|AgCl|3M KCl electrode served as the reference electrode and a platinum wire as the auxiliary electrode. Cyclic voltammetry (CV), constant current chronopotentiometric stripping (CPS), differential pulse voltammetry (DPV) and alternating current (ac) voltammetry were employed in conjunction with an accumulation procedure. The chronopotentiometric and ac voltammetric experiments were performed under air. The CV and DPV measurements were done in the absence of oxygen, by passing argon through the solution for 10 min before the measurement, and above the solution during the measurement.
HMDE was held at the potential E A for an accumulation time t A . In all experiments, stirring accompanied the accumulation. The CPS measurements were performed at an applied current of −11.0 A, from an initial potential E i = −0.1 V, if not stated otherwise. In ac voltammetry experiments, the potential perturbation had a frequency of 150 Hz and amplitude of 5.0 mV; the out-of-phase component was recorded at a scan rate of 8.0 mV s −1 , and converted to the differential capacity assuming a simple RC series circuit with a negligible resistance. The peptides were adsorbed for t A = 120 s on PGE from a 20 l drop of peptide solution, followed by washing of the peptide-modified electrode and its transfer to the electrolytic cell containing only the electrolyte (not containing any peptide).

Electroactivity of peptides
The electrochemical activity of the YYKLVFFC (peptide 1) and two additional A␤-peptides not containing Cys was explored both at PGE and at HMDE, using different electrochemical methods. Combining these two electrode materials allows covering of a large potential window encompassing oxidation phenomena at very positive potentials (on carbon) and reduction ones at very negative potentials (on mercury). On the PGE, peaks due to Tyr were clearly evidenced by DPV for peptide 1 at +0.51 V (not shown) and for peptide 2 at +0.53 V while no oxidation peak appeared for peptide 3 not containing Tyr, in agreement with data earlier obtained with peptides [22] and proteins [23] and in studies of a-synuclein oligomerisation and aggregation [4,6,8] and of A␤-peptide aggregation [5,7,20].

Catalytic hydrogen evolution at mercury electrode
Peptide 1 produces two CPS peaks when adsorbed at HMDE at accumulation potential, E A −0.1 V (Fig. 1A). Peak, S centered around −0.65 V (Fig. 1A, inset) responds to the presence of Cys in this peptide and is assigned to the reduction of a Hg S bond [30], indicating that the peptide is chemically immobilized at the electrode surface at potentials positive from the peak. Much higher peak H at very negative potentials is ascribed to CHER. In Fig. 1A a comparison of peaks H of the three peptides obtained under the same conditions is shown. Clearly, all three peptides produce specific peaks differing in their peak potentials, E p and/or in their heights. It has been shown that at neutral pH, accessible lysine (Lys, K), arginine (R) and Cys (C) residues in proteins are involved in CHER [31][32][33]. All three peptides studied in this paper contain K. Peptide 1, which yielded the highest and most negative peak H at E A −0.1 V (Fig. 1A), contains (in addition to KK) one C. The heptameric peptide 3 produces a much smaller step-shaped signal shifted slightly to less negative potentials. Peptide 2 yielded the least negative symmetric peak H close to −1.9 V whose shape and height change only little in dependence on E A (see below). The above data are in agreement with our previous protein studies [24,[34][35][36][37][38] showing great diversity in CPS responses of various peptides and proteins, suggesting that CPS may become useful in proteomics and biomedicine. Here we compared electrochemical behavior of the three A␤-peptides to show the potential of electrochemical methods in amyloid peptide research. In our ac voltammetric studies we observed an unexpected property of peptide 1, i.e. its ability to form a 2D condensed layer at the mercury electrode. To our knowledge such ability has been observed in nucleic bases [39][40][41] and oligonucleotides [42] but not in any peptides or proteins. The rest of this paper addresses therefore this phenomenon.

Peptide interfacial behavior
The adsorption of peptide 1 at the mercury|aqueous solution interface was studied by ac voltammetry, to obtain differential capacity-potential curves. Three adsorption regions can be distinguished ( Fig. 2A), separated by two peaks centered at −0.58 V and −1.53 V. Like many peptides and other molecules bearing a thiol group, the Cys-containing peptide 1 can oxidatively form a Hg S bond [30], and the pseudocapacitive peak at −0.58 V can be attributed to the reduction of this bond, as already evidenced above by CPS (cf. inset in Fig. 1A). At potentials positive of the peak (Region I), the peptide is thus chemisorbed at the interface through the formation of the Hg S bond. In this region, the capacitance is rather insensitive to the potential indicating that the amount of adsorbed peptide does not vary significantly with the potential, as can be expected for a chemisorption phenomenon. By contrast, the capacitance was found to vary with the accumulation time, decreasing progressively with t A until reaching a constant value associated with the formation of a complete monolayer (Fig. 2B). At full coverage, the low value of the capacitance and its potential-independent character suggests the formation of a compact monolayer [43]. This is confirmed by its inhibiting properties toward the electron transfer of an electrochemically reversible redox marker, [Ru(NH 3 ) 6 ] 3+ . Fig. 3 shows that at short accumulation times, the cyclic voltammograms recorded in the presence of [Ru(NH 3 ) 6 ] 3+ in solution have the usual characteristics associated with a reversible (fast kinetics) process. However, at larger values of t A , the reduction wave is significantly shifted in the negative direction while the re-oxidation wave is slightly shifted in the positive direction, indicating a slow electron transfer attributed to the presence of the peptide monolayer at the surface.
While Peptide 1 exhibits a peak around −0.63 V and a roughly constant capacitance at less negative potentials (in agreement with presence of Cys in its molecule) ( Fig. 2A), none of these features is noticeable for peptides 2 and 3 ( Fig. 2A). On the contrary, in the latter peptides the capacitance is highly dependent on the potential and passes through a minimum, identified as the potential of maximum adsorption. Such a classical behavior is typical of the physisorption of a disordered layer [44,45]. At negative potentials the curve of peptide 2 is not below that obtained with blank background electrolyte (in absence of the peptide) suggesting that peptide 2 might be partially or fully desorbed from the surface. Fig. 2A shows that peptides 1 and 3 are still adsorbed on the electrode at potentials more negative than −0.65 V. All three peptides contain hydrophobic residues and basic Lys residues but the presence of glutamic acid (E) close to the NH 2 terminus in peptide 2 can significantly affect the adsorption and orientation of the peptide molecule at the negatively charged surface [33]. It is therefore not surprising that under the given experimental conditions peptides 1 and 3 are still adsorbed at potentials as low as −1.7 V, as evidenced by the low capacitance recorded in this region (Region III) while peptide 2 shows a tendency to desorption already at potentials close to −1.0 V. In spite of this tendency peptide 2 produces CPS peak H (known to be produced by peptides and proteins in their adsorbed states) close to −1.9 V (Fig. 1). This could be due to the large difference between the rate of potential changes in ac voltammetry (scan rate 8 mV s −1 , Fig. 2A) as compared to CPS (∼180 V/s at the given current density, Fig. 1). In CPS, the change in potential from −1.2 V to −1.9 V takes about 31 ms compared to >1 min in ac voltammetry. One can imagine that 31 ms may not be sufficient for complete desorption of a relatively large peptide molecule containing hydrophobic LVFF core in the close neighborhood of a positively charged Lys residue.
Of particular interest is the interfacial behavior of peptide 1. The occurrence of three different regions of low capacitance indicates a potential-dependent adsorption behavior. Fig. 2B presents the capacity curves recorded for different accumulation times at the accumulation potential E A = −0.5 V. It can be seen that for accumulation times up to 30 s, the capacitance is not much lower than for the bare electrode. At longer t A , however, when larger quantities of peptide are adsorbed on the electrode, the capacitance suddenly drops to its minimum value, the curve forming a "capacitance pit" whose width progressively increases, in the negative direction, until reaching a constant value at times longer than 120 s (Fig. 2B). The capacitance value at the bottom of the pit expressed as C (Fig. 2C) corresponds to 3.85 F cm −2 . A larger width of the pit is indicative of a higher stability of the monolayer, provided by the attractive interactions between adsorbed molecules. The fact that the width significantly increases only in one direction is correlated with the chemisorption through the sulfur bond, which takes Our results show that it is not necessary to adsorb the peptide in the potential region where the Hg S bond is formed (region I) to observe the pit. This was demonstrated by performing a set of experiments where the accumulation potential was located within the potential range of the pit, and the capacitance variation was monitored as a function of the accumulation time (not shown). Fig. 4 shows the capacity vs. time curves obtained at E A = −0.83 V. In this set of experiments, a new drop was formed at the time t A = 0 s, then the current was recorded while stirring the solution at a given rate. All the curves start from the same initial value corresponding to the capacitance of the bare electrode|electrolyte interface. A monotonic decrease of the capacitance is then observed, followed by a discontinuity in the form of a steep variation of the capacitance, then a final stabilization to a common value corresponding to the pit bottom. For every curve, the discontinuity occurs at a similar value of the capacitance or, conversely, a similar peptide coverage. Upon increasing the stirring rate, the initial decrease of the curves becomes faster, indicating a control by the mass transport of the peptide from the solution. However, if only mass transport were involved in the kinetics, the capacitance would continue to decrease in a monotonic fashion. By contrast, the shape of the curves demonstrates that another phenomenon takes place at a given threshold coverage. Such a shape is characteristic of systems undergoing 2D condensation [43,46], and is associated with the ordered phase formation kinetics. This experiment shows that the low capacitance recorded in region II corresponds to a real equilibrium state, and not to a possible metastable state. Fig. 2C shows that capacity of the pit bottom remains constant when varying the temperature. On the other hand the width of the pit increases with the temperature lowering. This behavior is typical of 2D condensed monolayers, confirming that a self-association process takes place in the potential range of the pit [47].
We were interested in whether the formation of the 2D layer will influence the CPS response of peptide 1. We adsorbed peptide 1 at E A −0.9 V, i.e. within the pit, for t A 180 s, followed by recording of CPS peak H. Compared to peak H obtained after adsorbing this peptide at E A −0.1 V (Fig. 1A), the former adsorption resulted in four peaks H (I-IV) (Fig. 1B). Adsorption at E A −1.3 V negative to the pit resulted in a single peak close to E p of peak H-III obtained at E A −0.9 V (Fig. 1C). Similar experiments were performed with the two other peptides not forming any pit in their ac voltammograms ( Fig. 2A). Adsorption of peptide 2 at the above E A values resulted in peaks H differing little in their heights but showing shifts in their E p s to less negative values at more negative E A s (Fig. 1). In contrast adsorption of peptide 3 at these three E A s produced only a step-shaped signal at E A −0.1 V, while at E A −0.9 V and −1.3 V well developed peaks were obtained (Fig. 1). These results suggest that all three peptides can adsorb even at −1.3 V and during the fast potential changes in CPS, they still remain at the surface (probably attached by their hydrophobic parts) taking part in CHER close to −1.9 V (Fig. 1C), under conditions where their presence at the surface is not detectable by the slow scan capacitance measurement ( Fig. 2A). No doubt, CPS peaks are highly sensitive to changes in the peptide layers adsorbed at the charged Hg surface. At this stage we are not able to explain the large number of CPS peaks obtained for peptide 1 after adsorption at E A −0.9 V. We can only speculate that during the fast potential changes in CPS from −0.9 V to about −2 V, the 2D layer is disturbed, yielding differently oriented peptide molecules at the surface, in which the CHER is more or less difficult, resulting in peaks at different potentials. More work will be necessary to understand better the relationship between the structure of the peptide 2D layer and its CPS responses.
Alignment of peptide 1 in bulk and at a solid silicon surface was studied by numerous methods [3,28,29]. Linear dichroism provided a picture of alignment of peptide strands and showed initial association of phenylalanine (F) residues followed by subsequent registry and orientation of Tyr residues. X-ray diffraction data from aligned stalks yielded orientation order parameters. Comparison of the results of polarized Raman spectroscopy with results from NEX-AFS spectroscopy prepared as films on silicon indicated that fibril are aligned parallel to the surface with phenyl rings perpendicular to the surface. Formation of disulphide bridging was excluded by Raman spectroscopy and dityrosine formation probed by fluorescence experiments was found only at alkaline conditions but not at neutral pH. Peptide secondary structure was probed by Fouriertransform infrared (FTIR) and circular dichroism spectroscopies [28]. FTIR data showed features in the amide I region indicating antiparallel ␤-sheet structure. Taking the above results together we can conclude that they do not show any feature displaying striking difference between peptide 1 and other amyloid peptides and do not allow us at this stage to explain the unique ability of this peptide to form the 2D condensed layer. We can only speculate that it is the combination of the hydrophobic region (commonly present in amyloid peptides) with Cys SH group (normally absent in amyloid peptides), and its strong affinity to mercury surfaces, which is involved in the 2D condensation of peptide 1. On the other hand, our data (Fig. 2) and our previous experience [22,25,31] suggest that neither the hydrophobic region alone nor the SH group alone was sufficient to induce formation of the peptide 2D layer.

Conclusions
We show that a model amyloid peptide 1 displays at the electrified interface electrochemical characteristics associated with the formation of a 2D condensed self-assembled monolayer (Fig. 2). Our results obtained with this peptide represent the first example of a potential-dependent 2D layer at an electrified interface in peptides and perhaps the second one among the biologically important oligomers. Earlier it was shown that interactions of hydrophobic bases with hydrophobic Hg electrodes play a critical role in DNA adsorption [48,49] at moderate ionic strengths. Similarly, it can be expected that hydrophobic residues in peptide 1 will be important in adsorption of this peptide at HMDE and might be involved in the ability to form a pit in the capacitance-potential curves. In peptides 2 and 3 the amyloid sequence KLVFF is identical with that of peptide 1 but peptide 2 does not show any sign of pit formation while peptide 3 displays only a small tendency to form a pit close to −0.2 V, greatly differing from the usual pits observed with peptide 1 (Fig. 2) or with DNA bases [40,41,50] and oligonucleotides [42]. Hydrophobic interactions play a critical role in A␤-peptide oligomer and fibril formation [3]. Compared to in vitro solution experiments, studies on surfaces appear more biologically important because of its relevance to pathological processes occurring on various surfaces in cells and particularly in cell membranes. Some in vitro investigations indicated that presence of artificial or biological surfaces can greatly accelerate structural transition of A␤ peptides from random coil to ␤-sheet structure and to formation of heterogeneous A␤-species [3]. Our finding of 2D condensed layer at negatively charged hydrophobic surface (Fig. 2) suggests that peptide 1 formed a densely packed ordered layer, which adhered strongly to the surface and removed small ions and water molecules from it. It is tempting to suggest that formation of this layer is related to the ability of peptide 1 to oligomerize and form larger aggregated species. We also demonstrate that peptide 1 can be covalently bound to HMDE thus providing a new way to immobilize A␤-peptides to mercury surfaces (Fig. 1A, inset), suitable for studies on the growth of amyloid fibrils covalently attached to a surface. Moreover, we show for the first time that peak H can reflect formation of the condensed 2D layer (Fig. 1). Electrochemical methods and particularly the CPS peak H, which is highly sensitive to local changes in protein structures [36,51] can become very useful in A␤ peptide studies. Replacing HMDE by solid amalgam electrodes, which can easily be incorporated into peptide chips [52], may help to create a new tool for parallel analysis of A␤ peptides.