Protein Nanopore-Based, Single-Molecule Exploration of Copper

Article pubs.acs.org/Langmuir

Protein Nanopore-Based, Single-Molecule Exploration of Copper Binding to an Antimicrobial-Derived, Histidine-Containing Chimera Peptide Loredana Mereuta,∥,† Irina Schiopu,∥,† Alina Asandei,† Yoonkyung Park,‡ Kyung-Soo Hahm,§ and Tudor Luchian*,† †

Department of Physics, Laboratory of Molecular Biophysics and Medical Physics, “Alexandru I. Cuza” University, Blvd. Carol I, No. 11, Iasi 700506, Romania ‡ Research Center for Proteineous Materials, Chosun University, Gwangju, South Korea § BioLeaders Corp., Daejeon, South Korea S Supporting Information *

ABSTRACT: Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu2+-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu2+bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu2+free peptide is higher than that of a Cu2+-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu2+ on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu2+-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu2+complexed peptide from α-HL pore, as compared to that of Cu2+-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu2+-complexed peptide, which may be indicative of specific Cu2+-mediated contributions to the binding of the Cu2+-complexed peptide within the pore lumen.

■

INTRODUCTION Di- or trivalent ions of iron, zinc, copper, calcium, and magnesium constitute one of the most important cofactors of metalloproteins, for they modulate protein functions for which the stability of the tertiary structure is essential. Previous investigations established that histidine, aromatic amino acids, and cysteine display particularly strong affinity for metal ions and the association between a metal ion and such aminoacids involves electrostatic and coordinative bonds.1 Notably, given the sensitive affinity of proteins and peptides to metal ions, almost four decades ago the “immobilized metal (ion) affinity chromatography” was introduced,2 and subsequent investigation established that metal ion binding influences the aggregation properties, stability of the peptides and proteins, and the energetics of protein folding.3,4 In relation to this and with tremendous clinical implications, it was revealed that the disturbed homeostasis of zinc and copper modulate the pathophysiology of several protein-misfolding induced diseases.5 As a notable example, it was revealed that Aβ binds metal ions,6 and the particular Aβ-Cu2+ and Aβ-Zn2+ © 2012 American Chemical Society

interactions are mainly mediated by three intramolecular histidines (i.e., His-6, His-13, and His-14).7 A relevant physiological outcome of such interactions is an augmentation of the fibrillogenesis of Aβ in vitro,8,9 and metal chelators specific to Cu2+ and Zn2+ were demonstrated to reverse the amyloid aggregation state.10 Results of other investigations have strengthened that the formation and stability of α-helix and β-sheet secondary structures of peptides can be finely tuned by the presence of metals,11,12 and data drawn from work performed on short alanine-based peptides with single Trp-His pairs provided proof in favor of a conformational effect induced by Cu2+,13 whose magnitude is affected by the geometrical spacing and positions of the Trp-His pair.14 In particular cases, the intrinsic tyrosine fluorescence was also employed to successfully pinpoint conformational changes induced by metals on Aβ peptides. Received: September 20, 2012 Revised: November 2, 2012 Published: November 9, 2012 17079

dx.doi.org/10.1021/la303782d | Langmuir 2012, 28, 17079−17091

Langmuir

Article

Figure 1. Typical electrophysiology recordings showing the effect of increasing copper ions concentration on the interaction of a single α-HL protein, with 30 μM CAMA peptide, in symmetrical 2 M KCl solutions buffered at pH 7. In panels a, b, and c are indicated selected traces showing the current fluctuations (downward spikes, from the initially fully open α-HL pore) recorded at +70 mV through a single α-HL pore mediated by its reversible interaction with a single peptide in the absence of Cu2+ (control, panel a), and in the presence of 10 μM (panel b), and respectively of 100 μM (panel c) trans-added Cu2+. On the corresponding bottom-placed panels are shown the scatter plots of the events joint distributions which fingerprints the distribution in the amplitude-time space of the blockage events induced by the peptide alone (panel d), or in the presence of 10 μM (panel e), and respectively of 100 μM (panel f). As evidenced by the inset on panel d, the association of the CAMA peptide with an initially “open” (O) α-HL protein leads to two distinct blockage levels (termed B1 and B2), which are being assigned to the peptide lodged within the pore β-barrel lumen and peptide transitory interaction with the inner region of the α-HL vestibule (see also text). As shown, it is noteworthy that in the presence of Cu2+, a sizable alteration of peptide−protein association and dissociation times ensues, as well as a decrease in the current magnitude block induced by the peptide on the current flow through the protein (ΔIblock).

Tyrosine fluorescence spectra were quenched by Fe3+ and Cu2+, whereas Al3+ and Zn2+ enhanced them, indicating that a structural change in the peptide conformation ensues due to metal ion binding,15 and various buffers were found to act as competitive metal-binding ligands.16 Currently, a rich portfolio of techniques are being employed to characterize the aggregation propensity and folding landscapes of peptides, including gel electrophoresis, size chromatography, electron microscopy,17 electrochemical scanning probe microscopy,18,19 circular dichroism,20,21 surface enhanced Raman spectroscopy,22 infrared reflection absorption spectroscopy,23 and various optical and microscopy techniques.24,25 Apart from conventional techniques relying on the measurement of bulk properties of matter from large ensembles, built on the original resistive-pulse sensing technique and by employing synthetic or natural nanopores that display little or none subconductive states when subjected to a constant transmembrane potential difference, a particularly strong focus was placed on single-molecule approaches capable of revealing kinetic information that is not buried in averaged variables.26−29 Due to its sensitivity, this approach proved to be particularly suited to DNA sensing and sequencing,30−34 revealing the secondary structure of peptides,35−37small molecule detection,38−41 and quantitative investigations of various chemistries at the single molecule level.42−48 When coupled to the resistive-pulse sensing principle and electrophysiology knowledge derived from the patch-clamp technique,49 and with the use of solid-state37,50,51 or protein-based nanopores,52−59 the quantitative investigation of the inter-

actions of peptides and proteins with nanopores became possible at the single-molecule level, to probe and discriminate among conformations of peptides and proteins, examine their folding−unfolding process under various conditions, and to revel molecular insights about their translocation through nanometer scale volumes. Recent work proved that nanopores are useful to learn microscopic details about modulation processes induced by certain divalent metals on peptides and proteins folding processes.60,61 Due to the tremendous impact antimicrobial peptides possess to become, specific and powerful antibiotics, some of which use various protein pores for membrane translocation, it remains of key importance to examine and understand phenomena ruled by weak bonding and molecular recognition, which determine the interactions of such peptides with various protein pores. In this work, we applied electrical detection coupled with a nanopore sensory technique, to investigate and quantify at unimolecular level kinetic details of how divalent copper ions alter the interaction of a synthetic chimera peptide termed CAMA, with the α-HL protein pore, as a function of copper concentration and applied voltage. The construction of the peptide was based on structural features of cecropin A and magainin, and its design was engineered to contain a histidine residue as main target for the metal binding, and a distantly located tryptophan as a useful molecular beacon-like probe to reveal local environment properties changes and ensuing conformational changes in the peptide folding, induced by metal binding. 17080


Langmuir

Article

Figure 2. Quantitative analysis of the average time intervals which reflect the association (τON; panel a) and dissociation (τOFF; panel b) of a single CAMA peptide in interaction with an α-HL protein at pH = 7, measured at various concentrations of Cu2+ (i.e., 0, 3, 5, 10, 20, 50, and 100 μM) added on the membrane trans side. The peptide was present in the trans side of the membrane at a concentration of 30 μM. Data in panel b were constructed by considering the overall time needed for a peptide to completely dissociate from the protein pore, that is, the lumped time interval which describes the states B1 and B2 combined. In panel c, it is revealed the small effect of 3, 5, 10, 20, 50, and 100 μM Cu2+ in added the trans chamber, upon the average duration of the sub-state B2 (τOFF;B2).

Figure 3. Voltage dependence of the association (τON) and dissociation (τOFF) average time intervals which describe the interaction of a single CAMA peptide with the α-HL protein pore in the absence of Cu2+ (panels a and b) and presence of 100 μM Cu2+ added on the trans side of the membrane. By invoking Eyring’s transition state theory (see also text), and considering that within a qualitative kinetic model for the peptide− protein pore interaction the effects of the transmembrane potential can be reckoned as alterations of the association and dissociation activation free energies, the rate constants can be fitted with single decaying exponentials (dashed lines; y(τOFF,τON) = Ae−((x(ΔV))/t) for data presented in panels a, b, and c) and a raising exponential (dashed line; y(τOFF) = Ae((x(ΔV))/t) for data presented in the panel d, see also text). The nonlinear fit of data displayed in panels a and b, in the Cu2+ free buffer, gave time constants of ta = 63.4 ± 4.7 ms (R2 = 0.97) and respectively tb = 67 ± 6.2 ms (R2 = 0.96), whereas data in panels c and d (100 μM Cu2+) fitted with single exponentials resulted in time constants of tc = 43.3 ± 9.2 ms (R2 = 0.86) and respectively td = 36.4 ± 4.3 ms (R2 = 0.95).

■

RESULTS

In the representative traces shown in Figure 1, panel a, it is seen that under a positively applied potential, the peptide enters the protein pore whose inside β-barrel wall diameter measures ∼20 Å along the lumen region, and induces reversible partial blocks of the ionic current. Close analysis of such traces shows that approximately 75 % of the recorded current events contains two distinct blockage levels, denoted by B1 and B2, which may be associated to the peptide temporarily trapped within the protein pore (as illustrated in Figure 1, panel d), and whose magnitude of the channel current blockade and duration vary with the

In a first set of experiments, we probed the unimolecular interaction between CAMA peptide (Lys-Trp-Lys-Leu-PheLys-Lys-Ile-Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-PheNH2) added at micromolar concentration in the trans side of a membrane and a single cis-added α-HL protein. It has already been extensively proven that the transmembrane pore of α-HL facilitates controlled delivery of ions, small organic compounds, peptides and proteins across a synthetic lipid membrane (vide supra). 17081


Langmuir

Article

Figure 4. Representative current traces showing the effect of 100 μM trans-added Cu2+ upon current fluctuations (downward spikes) recorded at +70 mV, induced by the CAMA peptide (30 μM) through a single α-HL protein at pH = 7 (panels a, b, and c) and pH = 4.6 (panels d and e). The dotted line represents the “open” (peptide-free) state of a single α-HL protein. At pH = 7, the peptide-induced occupancy probability of the protein pore (lumped residency time of the peptide on the pore, corresponding to the summed time intervals encompassing substates B1 and B2, see Figure 1, divided by the total time of observation) was calculated to p = 0.018. Addition of 100 μM Cu2+ led to an increase in the peptide-induced occupancy probability of the protein pore to p = 0.035, and this effect was reversible upon Cu2+ chelation by 200 μM EDTA added in the trans-side of the membrane (panel c; p = 0.015). However, at acidic pH values, addition of the same amount of Cu2+ (100 μM) left the peptide-induced occupancy probability of the pore largely unchanged (p = 0.0033, control condition, panel d, and p = 0.003, 100 μM Cu2+, panel e).

B1 and B2 combined − τOFF, Figure 2, panel b) increased monotonically with the Cu2+ concentration in the trans side of the membrane, whereas average times assigned to the B2 substate alone (τOFF; B2, Figure 2, panel c) remained rather invariant to changes in the Cu2+ concentration. The supplementary analysis of average values of interevents corresponding to blocking events (τON) and times corresponding to the peptide temporarily lodged within the protein pore (τOFF) in the absence and presence of trans-added Cu2+, at various membrane holding potentials, is shown in Figure 3. Data corresponding to control experiments (no Cu2+ added) revealed that both τON and τOFF can be approximated to exponentially decaying functions with increasing potential values (Figure 3, panels a and b). In the presence of 100 μM Cu2+ added in the trans side, τON values retained their exponential-like decay tendency with increasing potential values (Figure 3, panel c), whereas the average value τOFF was found to vary with an approximate raising exponential at similar applied membrane holding potentials (Figure 3, panel d). As we will stress further below, this observation can be perceived as a consequence of conformational changes in the peptide, induced by the bound Cu2+ ions. Within a semiquantitative model for the peptide interaction with the β-barrel pore under an applied electric field, one may resort to the Woodhull-Eyring formalism to understand the capture and dissociation probability of the peptide to and from the protein pore, taking place at various membrane holding potentials.57 Inside the α-HL lumen, a single CAMA peptide is temporarily retained by the constriction formed by Met 113, Lys 147, and Glu 111 residues near the cis end of the protein lumen. If the electrical force acting on it is large enough, then

experimental conditions. We hypothesize that these blockages may reflect the peptide lodged inside the β-barrel lumen (substate B1) and the vestibule of α-HL protein of ∼46 Å in diameter (substate B2, respectively), which by the virtue of the chemical and topological variations on their inner domains,62,63 contribute distinctly to the protein blocking propensity and dissociation processes of the peptide. Although further experimental and molecular dynamics based testing of this hypothesis is still pending, this observation is in line with previous reports detailing the translocation of various polymers and polynucleotides through nanopores.64−66 As we detailed in the Discussion section, a mechanistic analysis applied to the variable magnitude of the protein current blockade allowed the estimation of the excluded atomic volume of a single peptide in the nanopore. When Cu2+ is being added at various concentrations in the trans side of the membrane (Figure 1, panels b and c), the distribution of event parameters (Figure 1, panels e and f) characterizing the recorded ionic current fluctuations induced by the peptide interacting with the protein pore changes. Most notably, the current blockage duration associated to the B1 substate, which we assign to the sojourn time within the α-HL lumen, becomes prolonged and the magnitude of the α-HL current blockade in this substate is diminished from an average value of 96 pA in the absence of Cu2+ to 92.5 pA in the presence of 10 μM Cu2+, and 79 pA when Cu2+ is added at a concentration of 100 μM (Figure 1). Further kinetic analysis revealed that average time values of interevents corresponding to current blocking events (τON) caused most likely by the peptide trapped within the protein pore (Figure 2, panel a) and overall blocking events (substates 17082


Langmuir

Article

data obtained with other peptides (vide supra), this is a reasonable proof of a conformational change undergone by the CAMA peptide, which in the complexation with Cu2+ mostly at His 15 (see also the Discussion section), changes the microenvironment polarity around Trp 2, so that this aminoacid becomes less exposed to the hydrophilic, water environment. To test further the Cu2+ - induced, peptide change of conformation for reversibility, we exposed to EDTA [40 μM] the peptide incubated at pH = 7 in the presence of the maximum amount of Cu2+ added [22 μM]. As presented in Figure 6, panels a and b, the maximum blue-shift of the fluorescence emission spectra of CAMA obtained in the presence of 22 μM Cu2+ is almost completely reversed in the presence of 40 μM EDTA. To better support the Cu2+ effect on CAMA fluorescence spectrum, as being caused mostly by Cu2+ complexation to CAMA’s His 15, supplementary experiments revealed that when incubated in acidic solution, addition of 22 μM Cu2+ produces no alterations in the intrinsic steady-state fluorescence spectra of the peptide (Figure 6, panel c).

the peptide might squeeze past this constriction and diffuse away to the cis side of the membrane. As previously suggested,57,58 we found this scenario consistent with the nonmonotonic voltage dependence of dissociation reaction constants of positively charged peptides that enter and temporarily occlude the β-barrel of the α-HL under the influence of the positive potential (Figure 3). However, for a folded peptide, both partial unfolding at the trans side of the membrane and refolding at the cis chamber side could create additional energetic contributions to translocation. As revealed by further investigations, the Cu2+-mediated alterations of CAMA peptide-induced blockage events, corresponding to the peptide trapping inside the α-HL protein, are fully reversible and not likely to be induced by Cu2+-related changes on the protein pore physicochemical architecture. When added in excess of Cu2+ in the trans side of the membrane, EDTA led to an almost complete restoration of the α-HL blocking probability mediated by the peptide to values seen during control experiments (no Cu2+ added) (Figure 4, panels a, b, and c). In addition, at acidic pH values, when the His 15 from the peptide is most likely protonated, the addition of Cu2+ entailed no visible changes in the probability with which the peptide dwells within the protein pore upon its capture (Figure 4, panels d and e). To further explore in a complementary fashion the effect of Cu2+ on the CAMA peptide conformation, with possible implications in explaining the data seen from single-molecule experiments, we recorded the intrinsic fluorescence spectra changes of the peptide given by its Trp 2, in the absence and presence of various amounts of Cu2+. As shown in Figure 5, steady-state fluorescence experiments revealed a sizable blue shift of the fluorescence emission spectra of Trp 2 residue from the primary structure of the CAMA peptide, corresponding to the change in wavelength of the maximum emission fluorescence signal of the peptide in the presence of variable amounts of Cu2+, as compared to the Cu2+free spectrum. In accordance to the interpretation of similar

■

METHODS

Peptide Synthesis. The peptides were synthesized using the solid phase method with Fmoc (9-fluorenyl-methoxycarbonyl)-chemistry.74,75 Peptide purification was then carried out using preparative HPLC on a C18 reverse-phase column. The amino acid compositions of the purified peptides were confirmed using an amino acid analyzer (HITACHI 8500A, Japan). The molecular weights of the synthetic peptides were determined using a matrix-assisted laser desorption ionization (MALDI) mass spectrometer. Electrophysiology Experiments. Single-channel measurements were performed as previously described,40,41 using the Montal− Mueller technique on planar lipid membranes made of 1,2diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids, Alabaster, AL) spread across a 10% (v/v) hexadecane/pentane (HPLC-grade, Sigma−Aldrich, Germany) pretreated, ∼ 120 μm in diameter aperture punctured on a 25 μm-thick Teflon film (Goodfellow, Malvern, MA) that separated the cis (grounded) and trans chambers of the recording cell. The electrolyte used in both cells throughout all measurements contained 2 M KCl, buffered in 10 mM HEPES or 5 mM MES, depending on the needed acidity. All reagents were of molecular biology purity and purchased from Sigma−Aldrich, Germany, unless mentioned otherwise. Insertion of α-hemolysin (α-HL) (Sigma−Aldrich, Germany) heptamer was achieved by adding 0.5−2 μL from a stock solution of the protein monomers made in 0.1 M KCl, to the cis chamber only. Once the successful insertion of a single α-HL heptamer occurred, a 20-residue hybrid peptide termed in what follows CAMA (Lys-TrpLys-Leu-Phe-Lys-Lys-Ile-Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-LysLys-Phe-NH2), incorporating the 1−8 amino acids from cecropin A (CA) and the 1−12 amino acids of magainin 2 (MA)75 was introduced in trans chamber at a concentration of 30 μM. In its deprotonated state, the His 15 residue constituted the main binding site for the added Cu2+ at various concentrations. Anhydrous copper chloride (Sigma−Aldrich, Germany) was added in the same chamber as the peptide (i.e., the trans chamber) at concentrations ranging between 3 and 100 μM. For reversing the metal effect on the added peptide, EDTA (Sigma−Aldrich, Germany) was added at a concentration of 200 μM. Control experiments were carried out with Cu2+ present in the peptide-free buffer, from both sides of the membrane, to exclude a possible Cu2+-induced interference on the ion current blockages through the α-HL protein pore besides peptide-induced ones (see Supporting Information, Figure S1). To better strengthen the Cu2+His interactions as the primary cause for the Cu2+-modulated CAMA peptide interactions with the protein pore, we performed another control experiments in which CAMA peptide was substitute with a

Figure 5. Bulk estimation of the affinity of Cu2+ toward the CAMA peptide measured at pH = 7. Upward triangles represent the blue shift of the fluorescence emission spectra of Trp 2 residue from the peptide (Δλ), corresponding to the change in wavelength of the maximum emission fluorescence signal of the peptide in the presence of various amounts of Cu2+, as compared to the Cu2+-free spectrum. The dashed line represent the nonlinear hyperbolic fit according to eq 9 (R2 = 0.88), which provided an estimate of the dissociation constant of Cu2+ from the CAMA peptide (Kd = 6.3 μM). In the inset are displayed representative, normalized fluorescence emission spectra of the CAMA peptide [1. 5 μM] in the absence of Cu2+ (continuous line) and in the presence of the maximum added Cu2+ ([22 μM], dashed line), to evidence the blue shift of Trp 2 fluorescence emission spectra from the peptide. 17083


Langmuir

Article

Figure 6. (a) Representative, normalized intrinsic fluorescence spectra of Trp 2 from the CAMA peptide [1.5 μM] in the absence (continuous line) and presence of Cu2+ (5 μM, dashed-line, and respectively 22 μM, dotted line). As stated previously, Cu2+ induces a sizable blue shift (Δλ) of the fluorescence emission spectra of the CAMA peptide, as compared to the Cu2+-free spectrum, of Δλ = 4 nm ([Cu2+] = 5 μM) and Δλ = 8 nm ([Cu2+] = 22 μM). (b) Addition of EDTA (dotted line) reverses the Cu2+-induced spectral blue-shift in the emission spectra of the peptide, even when Cu2+ was present at its maximum concentration used in such experiments (dashed line) (Δλ = 8 nm in the presence of Cu2+ [22 μM] and respectively Δλ = 3 nm upon the addition of EDTA [40 μM], as compared to Cu2+- and EDTA-free CAMA spectrum, depicted with the continuous line). (c) At a pH value of 4.6, addition of either Cu2+ [22 μM] or EDTA [40 μM] leaves the normalized intrinsic fluorescence spectra of Trp 2 from the CAMA peptide unchanged with respect to the shift of the fluorescence emission spectra of the peptide alone, when added at a concentration of 1.5 μM in buffer. CAMA peptide, corresponding to the change in wavelength of the emission fluorescence signal maximum of the peptide in the presence of various amounts of the added Cu2+, as compared to the Cu2+-free spectrum (Δλ). To determine the expression that relates dissociation constant Kd, of the reversible interaction between Cu2+ and the CAMA peptide, we considered a simple bimolecular binding case between free peptides (P) and copper ions (Cu2+), in which the two components bind reversibly to form a binary complex (P−Cu2+) with the association kON and the dissociation rate constant kOFF:

functional analogue of it which lacked the His residue (see Supporting Information, Figure S2). All measurements were carried out at room temperature (∼25 °C) and the bilayer chamber was housed in a Faraday cage (Warner Instruments, U.S.), and mechanically isolated with a vibration-free platform (BenchMate 2210, Warner Instruments, U.S.). Current fluctuations reflecting peptides-free or Cu2+-complexed peptides interaction with a single membrane-inserted α-HL protein were detected and amplified with either an EPC 8 patch-clamp amplifier (Heka, Germany) or an Axopatch 200B patch-clamp amplifier (Molecular Devices, U.S.) set to the voltage-clamp mode. Data acquisition was performed with a NI PCI 6221 or a NI 6251 acquisition board (National Instruments, U.S.) at a sampling frequency of 50 kHz, with customized routines written for the LabVIEW 8.20 (National Instruments, U.S.) environment, and before detection and amplification they were filtered at 10 kHz with low-pass Bessel filter. Numerical analysis and graphing were done with the help of the Origin 6 (OriginLab, U.S.) and pClamp 6.03 (Axon Instruments, U.S.) software. Fluorescence Spectroscopy Experiments. The Cu2+-induced changes on the conformation of the CAMA peptide were monitored using the intrinsic fluorescence emission of the Trp 2 residue present in the peptide structure, as described previously.76 All measurements were carried out using a FluoroMax-4 (Horiba Jobin Yvon, U.S.) spectrofluorimeter. The excitation wavelength was fixed at 280 nm and steady state fluorescence emission spectra were recorded between 300 and 450 nm with a 2 nm resolution step. Throughout experiments, the peptide was present at a concentration of 1.5 μM, prepared by diluting a stock solution with an appropriate buffer, 10 mM HEPES (pH = 7) and 5 mM MES (pH = 4.6). The concentrations of the added Cu2+ in these experiments ranged between 0.03 to 22 μM, and were chosen as to match the metal-to-peptide molar ratios used in the electrophysiology experiments. To monitor the concentration dependence of peptide complexation with Cu2+, we followed and analyzed the blue shift of the fluorescence emission spectra of Trp 2 residue from the primary structure of the

k ON

P + Cu 2 + ←→ ⎯ P − Cu 2 +

(1)

k OFF

Under stationary conditions, one can write:

[P]eq [Cu 2 +]eq k ON = [P − Cu 2 +]eq k OFF

(2)

and thereby resulting: [P − Cu 2 +]eq =

k ON [P]eq [Cu 2 +]eq k OFF 2+

(3) 2+

In the expressions above, [P]eq, [Cu ]eq and [P−Cu ]eq represent the free, available concentrations of the Cu2+ - free peptide, Cu2+ ions and the Cu2+-complexed peptide at stationarity. Within the classical Langmuir binding model which is usually employed to evaluate the concentration of the receptor−ligand intermediate, it is introduced as a simplifying assumption that the free ligand concentration remains is equal to the total, initial ligand concentration added to the binding mixture. In this case, however, and given the strong affinity of Cu2+ to the free peptide, such a simplifying assumption may no longer reflect reality. That is, peptide binding of Cu2+ ions which generates (P−Cu2+) binary complexes, leads to a depletion of the free, available concentration of both peptide and Cu2+. To address this properly, an alternative equation must be arrived at, to account for the depletion of concentration of peptide and Cu2+, during the course of (P−Cu2+) complex formation. 17084


Langmuir

Article

Among the main findings of this study, we demonstrated that Cu2+ modulates the ion current fluctuations associated to CAMA reversible association to the α-HL protein. As a first observation, in single-channel recordings with the α-HL pore, a decrease in the extent of transient current blockades produced by the CAMA peptides was seen in the presence of Cu2+ ions. We reasoned that Cu2+ binding to the peptide may have led to a conformational change of it, which diminished the extent to which the peptide was able to partition within the protein pore lumen. In relation to this, in the past studies, electrical recordings through either protein- or solid-state-based nanopores were successfully used to characterize the size and conformational changes of proteins and peptides induced by various physical and chemical factors28,51,54,67−70 The physical ground of this powerful paradigm is very simple: the amplitude of the current blockages caused by a peptide (or protein) in interaction with a nanopore is directly proportional to the volume of peptide region which resides within the nanopore during the interaction. When the peptide or protein is approximated as having a spherical shape, and the inner region of the nanopore can be approximated to displaying a cylindrical topology, particularization of early results of DeBlois and Bean leads to the development of an analytical formalism which relate peptide size to the amplitude of current blockages.71 When this formalism is being applied to our experiments, and given that the length of the peptide used is smaller than the length of the nanopore, the time-dependent changes of the amplitude of ion current blockages (ΔI(t)) mediated by a peptide through a nanopore subjected to a constant potential difference are given by the following:

Denoting by [P0] and [Cu2+ 0 ] the initial concentrations of the peptide and metal, by means of the mass conservation equations and considering a 1:1 stoichiometry16 of binding between Cu2+ ions and the CAMA peptide, one can write:

[P]eq = [P0] − [P − Cu 2 +]eq

(4)

[Cu 2 +]eq = [Cu 02 +] − [P − Cu 2 +]eq

(5)

By replacing eqs 4 and 5 in expression 3, one obtains: [P − Cu 2 +]eq =

k ON ([P0] − [P − Cu 2 +]eq ) k OFF ([Cu 20 +] − [P − Cu 2 +]eq )

(6)

By substituting the ((kOFF)/(kON)) ratio with the dissociation constant Kd, one can rewrite expression 6 as a quadratic equation: 2 [P − Cu 2 +]eq − ([P0] + [Cu 20 +] + Kd)[P − Cu 2 +]eq

+ [P0][Cu 20 +] = 0

(7)

By solving it, this equation leads to two solutions; however, the physically meaningful one, i.e., the one which predicts a saturation value of the [P−Cu2+]eq value at high concentrations of [Cu2+ 0 ] is as follows: [P − Cu 2 +]eq =

([P0] + [Cu 20 +] + K d) −

2

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

(8) When the initially added copper concentration ([Cu2+ 0 ]) goes to infinity, the concentration of the [P−Cu2+]eq intermediate reaches its saturation value, which equals the initial [P0], and this makes perfect sense. Knowing that the blue shift of the fluorescence emission spectra of Trp 2 residue from the primary structure of the CAMA peptide, corresponding to the change in wavelength of the emission fluorescence maximum of the peptide in the presence of Cu2+, as compared to the Cu2+-free spectrum, (Δλ) is proportional to the fractional occupancy ( f) of the peptide species complexed with Cu2+ ( f = (([P − Cu2+)eq)/([Cu2+ 0 ]))), one can monitor the change in (Δλ) as a function of [P−Cu2+]eq.:

ΔI(t ) = −

⎡ ⎛ d peptide lpeptide ⎞⎤ ⎟⎥ •⎢1 + γ ⎜⎜ , ⎢⎣ lpore ⎟⎠⎥⎦ ⎝ d pore (10)

where δ is a shape factor which equals 1 when the peptide inside the pore is viewed as a cylinder aligned parallel to the electric field lines, v(t) (m3) equals to the volume of the peptide lodged within the pore, which may vary with time as the peptide moves across the pore, σ (Ω−1m−1) is the conductivity of the electrolyte, ΔV is the potential difference imposed across the nanopore, and lpore (m) equals the length of the nanopore, and the dimensionless correction factor, γ, which fine-tunes the formula above when the diameter of the peptide (dpeptide) equals the diameter of the pore (dpore), or the length of the peptide (lpeptide) approaches the length of the pore (lpore). As we will detail in what follows, it is clear that mostly at low to intermediate concentration values of the added Cu2+, two forms of peptides are present in the solution, namely Cu2+-free, and Cu2+-complexed. Consequently, the blockage events seen in our recordings may originate from the interaction of the αHL with either peptide. Due to the fact that histogram analysis of the amplitude of ion current blockages (ΔI(t)) failed to evidence distinct amplitude blockage peaks, we concluded that the difference in the extent of α-HL block protein interaction with either peptide may be too small to be resolved in our recordings. However, to provide a quantitative evaluation of the excluded volume of electrolyte within the nanopore caused most likely by the Cu2+-complexed peptide, we applied the formalism described above to the case when the maximum concentration of Cu2+ was added in the buffer solution, namely 100 μM, whereby the most probable species of the peptide present in the buffer would be the Cu2+-complexed one. As it is

Δλ = f Δλmax = Δλmax ([P0] + [Cu 20 +] + K d) −

δ • σ • ΔV • v(t ) 2 lpore

2

([P0] + [Cu 20 +] + K d) − 4[P0][Cu 20 +] 2[P0]

(9) where Δλmax corresponds to the maximum value of the emission fluorescence signal of the peptide, in the presence of the added Cu2+ at saturating concentration values The dissociation constant Kd was extracted from the nonlinear hyperbolic fit according to the eq 9, of the experimentally measured (Δλ) in the presence of various concentrations of the added Cu2+.

■

DISCUSSION In this work, we explored at the single-molecule level the Cu2+ interaction with a model, antimicrobial-derived peptide (CAMA), by using a wild-type α-HL protein pore immobilized in a folded lipid bilayer as a nanoscopic probe. For the sake of simplicity, the peptide was engineered as to contain a single His residue (His 15) as the main target for Cu2+ ions complexation, and in addition we placed a Trp 2 residue to probe local environment properties changes caused by Cu2+ binding, and therefore possible conformational changes in the peptide folding. 17085


Langmuir

Article

Figure 7. Sketched representation of the putative influence exerted by Cu2+ binding to a CAMA peptide, from the viewpoint of the reduction in the excluded volume of electrolyte by the peptide within the α-HL pore, seen as distinct ion current block amplitudes (ΔI) through the protein pore. While in Cu2+-free buffer, peptide is free to fit within the α-HL protein pore (panel a), Cu2+ binding to a peptide at the position His 15 putatively entails a change in the conformation of the peptide (panel b), which precludes the full partition of it into the protein pore. In the insets below we display an oversimplified representation of the free-energy landscape encountered by a Cu2+-free (panel a) or a Cu2+-complexed peptide (panel b) interacting reversibly with the pore, based on the inferred numerical values of the kinetic constants of association/dissociation characterizing the three-states scheme 11 (k1, k2, k3, k4, see also the discussion in the text below). Note that the reaction coordinate on the bottom of the free-energy landscape diagrams does not reflect the moving direction of the dissociating peptide, which can translocate to the cis side of the membrane or return to the trans side ref 57.

By neglecting as a first approximation changes in the electrostatic profile within the α-HL lumen region by the presence of trans-added Cu2+, we believe at this point that the reduction in the excluded volume of electrolyte within the protein pore in the presence of Cu2+, by the residing Cu2+complexed CAMA peptide, is mainly caused by a change in the conformation of the peptide following its interaction with Cu2+ at the position His 15, which subsequently precludes the full partition of the peptide into the protein pore (Figure 7). By comparing the theoretically and experimentally estimated volumes of the CAMA peptide (vide supra), it is noticeable a 13% difference between them, in the sense that the theoretically calculated volume is slightly smaller. By extending this “scaling” factor to the experimentally calculated volumes of the peptide residing in the nanopore in the presence of Cu2+, the ensuing corrected value for the peptide volume occupying a nanopore, when complexed with Cu2+ added at the concentration of 100 μM is being obtained (vCAMA, 100 μM Cu2+,corrected ≈ 2.41 nm3), which is arguably the “closest to theory” estimator of the peptide volume inside the nanopore, in the presence of Cu2+. Taking into account that experiments were performed when a positive potential was applied to the side of peptide addition, and considering the intrinsic dipolar moment a single CAMA peptide, it is conceivable that when captured to the pore mouth a peptide would orient itself most likely with the N-terminal head-on within the pore.37 With these in mind, theoretical estimations based on corrected excluding volume values as calculated above indicate that when complexed with Cu2+ added at 100 μM, blockages induced by the peptide would reflect mainly contributions from amino acids 1−17 alone. That

shown in Figure 2, panel a, and evidenced further below, indepth kinetic analysis of our data suggests that when the concentration of the CAMA peptide was 30 μM, addition of Cu2+ at concentrations larger that 50 μM ensures that the most likely species of peptides remaining in the trans buffer is the Cu2+-complexed one. With applicability to our case and as described in the references above, to take into consideration the access resistance of the nanoporewhich extends from the bulk solution to the mouths of nanoporelpore is replaced by (lpore + 0.8dpore), and as under most experimental conditions, the correction factor γ is considered unity. In the absence of Cu2+ (control conditions), by using estimates for the electrical conductivity of 2 M KCl (σ = 169 mS cm−1), the length of the α-HL pore region (lpore = 5.2 nm) and the average diameter of the α-HL β-barrel stem region (dpore = 1.5 nm), an average ion current blockage of ΔI = 95.9 pA mediated by a peptide partially occluding the pore region of a protein clamped to a constant potential difference ΔV = 70 mV (Figure 1, panels a and d) corresponds to an excluded volume of electrolyte within the pore, by the lodged peptide, of v ≈ 3.3 nm3. This indirectly approximates the value of the CAMA peptide volume, whose theoretically calculated value 3 comes within a close range (vCAMA theory ≈ 2.91 nm ). Following a similar rationale, we estimated that in the presence of 100 μM Cu2+, the excluded volume of electrolyte within the nanopore by a temporarily residing peptide, seen experimentally as an average ion current blockage of ΔI = 79.4 pA measured at the same potential difference (ΔV = 70 mV; Figure 1, panels c and f), equals vCAMA, 100 μM Cu2+ ∼ 2.74 nm3. 17086


Langmuir

Article

ring of His and the indole ring of Trp are good chelating ligand to Cu2+, the decisive implication of the His 15 residue in this conformational change of the peptides was demonstrated during experiments carried out at low pH values, in which little or no changes in the intrinsic fluorescence emission spectra of CAMA peptides occurred (Figure 6, panel c), thus probing the lowered propensity of Cu2+-peptides complexation events. We posit that such observations may be complemented by further CD or FTIR experiments, whereby the effect of Cu2+ on the secondary structure of peptides would be better pinpointed, and the dependence of the conformational effects of Cu2+ on the geometrical spacing and position of Trp-His pair better explored. Dissociation constants for the CAMA peptide−Cu 2+ complexes were calculated by nonlinear least-squares fitting and statistical analysis the titration data shown in Figure 5 to the eq 9, and found in good agreement with data presented in literature for other His-containing peptides.16 In addition to changes in the electrolyte excluded volume by the CAMA peptide within the α-HL nanopore caused by varying concentrations of Cu2+, our single-molecule data analysis revealed a sizable Cu2+-dependent change in the association (τON) and dissociation (τOFF) time average values (Figure 2, panels a and b), which were used to describe the kinetics of peptide−protein nanopore reversible interaction. To account for this result, we resorted to a minimalist model able to describe the interaction of CAMA peptide with the protein pore, Cu2+-free or complexed with Cu2+ ions. In the absence of interaction with a CAMA peptide, we propose that the α-HL protein assumes the so-called open state, denoted by “O” (see eq 11). In this state, the protein can interact with either a “Cu2+-free peptide” (denoted by P), thus entering a first type of “closed-state” (C1), or with a “Cu2+complexed peptide”, (denoted by P−Cu2+), giving rise to a second type of blockages (C2). In mathematical terms, the overall kinetic scheme described above writes:

is, a conformational change in the peptide structure ensues following its complexation with Cu2+ ions, so that the very last aminoacids Lys 18-Lys 19-Phe 20 are putatively excluded from partitioning within the pore lumen. From the voltage-dependence studies of the peptide binding to the α-HL protein, in the absence and presence of Cu2+ ions, we noted that the kinetics of α-HL−peptide interaction is voltage-dependent, and as described in many details before,57 the Eyring’s transition state theory coupled to the Woodhull formalism can be successfully employed to capture the influence exerted by an applied potential difference upon the free-energy landscape of the interacting CAMA peptide. However, we observed a remarkable difference between the average dwell time intervals associated to the peptide bound to the α-HL protein (τOFF) in the absence or presence of 100 μM Cu2+ (Figure 3, panel b and d). In contrast to the average duration of such events when no Cu2+ was added in the buffer solution, which turned out to exponentially decrease with increasing potential difference values, in the presence of 100 μM Cu2+, the average τOFF durations displayed an exponentiallike increase with increasing holding potentials. We posit that in the Cu2+ free solution, and as described before,57,65 peptide dissociation events most likely reflect successful translocation instances of the peptide through the protein. In the presence of 100 μM Cu2+, and even though blockage times used to construct a statistics of the τOFF contain lumped together events in which either a “Cu2+-free” or a “Cu2+-complexed” peptide blocks the pore (vide infra), we propose that most of the blockage events seen result from “Cu2+-complexed” peptides, which cannot pass through the protein and are being released backward to the trans side, against the applied potential. This scenario may be further supported by the fact that an ensuing conformational change in the peptide which follows its complexation with Cu2+ might restrict the peptide from entering the protein lumen or squeeze past the constriction site of the α-HL, to diffuse to the cis side of the membrane. To account for the existence of the distinct blockage levels corresponding to the B2 substate recorded in the presence of added Cu2+, which presumably reflect the transitory interaction of a peptide with the inner region of the α-HL vestibule (vide supra), we suggest that they reflect the interactions between the still present Cu2+-free peptides and the protein pore. Even at high concentrations of Cu2+ one cannot completely dismiss the existence of such interactions. It still remains an open issue to understanding why the blockage amplitude of the ion current through an α-HL pore, entailed by the interaction with a Cu2+-free peptide in the presence of added Cu2+ and associated to blocking events which display the levels corresponding to the B2 substate, is smaller than the blockage amplitude given by the peptide in the absence of Cu2+. As demonstrated above (Figure 5), by examining the Trp 2 emission spectra obtained when incrementing amounts of Cu2+ were added in the buffer solution which incubated the peptide, the effect of metal was evidenced as a strong blue-shift of the emission maximum. It is therefore suggested that the peptide complexation with Cu 2+ at the His 15 residue mainly, and additional carbonyl groups of peptide backbone,72 give rise to a conformation change of CAMA peptides, which translate into a less exposed Trp 2 to the solvent polar environment, and the effect of Cu2+ on the conformation change of peptides is concentration dependent. Despite the fact that both His 15 and Trp 2 residues may be implicated in complexing Cu2+ ions, since both the imidazole

r3

r1

k4

k2

C2 ↔O ↔ C1

(11)

In this model, r1 and r3 reflect association rates of a single αHL protein with either a “Cu2+-free peptide”, or a “Cu2+complexed peptide”, which are dependent upon the free, available concentrations of (P), and (P−Cu2+) complexes, respectively, whereas k2 and k4 represent dissociation rate constants of the (C1) and respectively (C2) intermediates. To arrive at the expressions which describe the equilibrium concentrations values of the available “Cu2+-free peptide” (P) and “Cu2+-complexed peptide” (P−Cu2+), and as described in details above, we considered a simple bimolecular reaction scheme between free peptides (P) and copper ions (Cu2+) in which the two components bind reversibly to form a binary complex (P−Cu2+), with the association kON and the dissociation rate constant kOFF: k ON

P + Cu 2 + ←→ P − Cu 2 + k OFF

(12)

As we described in the Materials and Methods section (expression 8), the [P−Cu2+]eq. value predicted at intermediate concentrations of [Cu2+ 0 ] under stationary conditions writes: 17087


Langmuir

Article

In a similar way, the probability (PC2(t)) of the α-HL pore to still be found in the C2 state at a given time (t), assuming that at the initial time (i.e., when one start measuring the duration of τOFF event), the protein was being blocked by a “Cu2+complexed peptide” (P−Cu2+), writes: r3 −k4t PC2(t ) = e r1 + r3 (19)

[P − Cu 2 +]eq =

([P0] + [Cu 20 +] + K d) −

2

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

(13)

Returning now to the kinetic model (11), which describes in simplest terms the interaction of the CAMA peptide with a single α-HL protein, in the presence added Cu2+, by virtue of a formalism described extensively in previous references,49,73 the theoretical average value of τON time interval in-between peptide-generated blocking events, writes: τON =

1 1 = r3 + r1 k 3[P − Cu 2 +]eq + k1[P]eq −1

With these in mind, the probability with which the α-HL pore is found in the blocked state at the time (t), which can represent physically either the C1 or C2 substates, becomes: r3 −k4t r1 −k 2t Pblocked(t ) = PC1(t ) + PC2(t ) = e + e r1 + r3 r1 + r3

(14)

(20)

−1

where r1 (s ) and r3 (s ) have the meaning of association rates of a single α-HL protein with either a “Cu2+-free peptide”, or a “Cu2+-complexed peptide”, and k1 (M−1s−1) and k3 (M−1s−1), respectively, are the corresponding bimolecular rate constants of association for the two possible pathways of α-HL protein blockage. By the use of [P − Cu2+]eq value deduced above, and taking into account the law of mass conservation: 2+

[P − Cu ]eq + [P]eq = [P0]

Consequently, the probability that at the given time (t) the α-HL pore leaves the blocked state, thus returning to its peptide-free substate (Rblocked(t)) becomes: R blocked(t ) = 1 − Pblocked(t ) r3 −k4t r1 −k 2t =1− e − e r1 + r3 r1 + r3

On the basis of the grounds of basics statistics, the time derivative of (Rblocked(t)) equals the probability density function of the distribution of τOFF events duration (pdf τOFF), which integrated over the (0,∞) time interval equals unity. By resorting again to basic statistics, one can infer that the theoretical estimation of the average of τOFF values is then given by the following:

(15)

one obtains an analytical expression for the average τON values: τON = =

1 k 3[P − Cu ]eq + k1([P]eq ) 2+

2+

k 3[P − Cu ]eq

1 + k1([P0] − [P − Cu 2 +]eq )

τOFF =

(16)

in which [P − Cu2+]eq represents the solution of the quadratic eq 7 arrived at above (eq 8). In order to deduce an analytical expression for the average value of τOFF time intervals at stationarity, describing the kinetics of α-HL pore dissociation from either a “Cu2+-free peptide” (P) or a “Cu2+-complexed peptide” (P−Cu2+), it should be recalled that for the experimental reasons described before, one cannot reliably discern between the two types of blocking events based solely on the amplitude of ion current blockages (ΔI(t)). Our strategy was therefore to employ a statistical approach, toward deriving first the probability density function which characterizes the distribution of lumped τOFF events, based on the general theory of single-channel kinetics analysis of a three-state Markov model.73 To do so, we first derived the probability (PC1(t)) of the α-HL pore to still be found in the C1 state at a given time (t), assuming that at the initial time (i.e., when one start measuring the duration of τOFF event), the protein was being blocked by a “Cu2+-free peptide” (P). Simple kinetics analysis shows that within the frame of our model: PC1(t ) = PC1(0)e−k 2t

(21)

∫0

∞

τOFF(pdfτOFF)dt

(22)

By computing the integral 22, with the use of the analytical result for (pdf τOFF), we deduced the calculated expression for the average of τOFF values: τOFF =

k4r1 + r3k 2 r1k 2k4 + k 2k4r3

(23)

Once again, considering that the analysis takes place under stationary conditions, by replacing the values for the r1 and r3 reactions rates as a function of the reaction constants (k1 and k3, respectively) and intermediate concentrations values of the corresponding substates, one obtains: τOFF = 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 ) (24)

in which all terms have the explained meaning, and [P − Cu2+]eq represent to the solution of the quadratic equation calculated above (expression 8). By inspecting the expression of [P − Cu2+]eqwhich enters as a common parameter in the analytical values of both τOFF and τON one can see that τOFF and τON come to depend upon parameters of association−dissociation of the kinetics underlying the three-states model (i.e., k1, k2, k3, k4), the initial concentration values of the CAMA peptide, [P0], initial concentration of the metal added, [Cu2+ 0 ], as well as the dissociation constant Kd of the peptide-Cu2+ reversible interaction.

(17)

The probability of finding the protein at t = 0 in that C1 state (i.e., Pc1(0), when one starts measuring the duration of the corresponding τOFF event) is given within our three-state model by ((r1)/(r1 + r3)), so the expression above becomes: r1 −k 2t PC1(t ) = e r1 + r3 (18) 17088


Langmuir

Article

Upon the replacement of [P0] with its value (30 μM), as it was used in the experiments used to arrive at data shown in Figure 2, and the experimentally estimated value of Kd (vide supra, Figure 5, Kd = 6.3 μM), the derived expressions 16 and 24, allowed us to estimate values for the kinetic parameters characterizing the entire reaction scheme, (i.e., k1, k2, k3, k4). That is, by nonlinear fitting of the τON data shown in Figure 2, panel a by expression 16 (Figure 8, panel a), in which the independent parameter remained the initially added concen3 −1 −1 tration of metal (Cu2+ 0 ), we inferred that k1 = 125 × 10 M s 3 −1 −1 and k3 = 25 × 10 M s .

the CAMA peptide, the resulting complex encounters a higher energy barrier toward its association with a protein pore (Figure 7). This would probably be due, but not restricted to, to the extra entropy cost needed to fit an “overcoiled” peptide within the narrow domain of the α-HL lumen region. At the same time, the observed ∼ten times higher value of the dissociation rate constant of the Cu2+-free peptide from α-HL protein pore (k2), as compared to that of the Cu2+-complexed peptide (k4), indicates the existence of a deeper free energy well for the protein interaction with a Cu2+-complexed peptide (Figure 7), which seem to support at least in part (vide infra) the involvement of specific Cu2+-mediated contributions to the binding enthalpy of the Cu2+-complexed peptide within the pore lumen. By resorting to transition state theory to model the reversible interaction between a Cu2+-free peptide and the protein pore, whose oversimplified free-energy landscape is depicted in Figure 7, panel a, numerical values inferred for k1 and k2 (vide supra) resulted in standard, free-energy barriers levels for association and dissociation of ΔGassociation,free−peptide * = 10.5 kcal mol−1 andΔGassociation,free−peptide * = 14.7 kcal mol−1, respectively . In a similar fashion, the values of k3 and k4 (vide supra) resulted in standard, free-energy barriers levels for association and dissociation of the a Cu2+-complexed peptide interacting with a single α-HL pore (Figure 7, panel b) of ΔGassociation,Cu2+−peptide * = 11.4 kcal mol−1 and respectively ΔG*association,Cu2+−peptide = 16.1 kcal mol−1. It should be noted however, that these energies contain lumped an electrical contribution from the peptide interacting with the applied potential difference (ΔV = 70 mV), maintained along the lipid membrane during kinetic experiments. In future studies, and with contributions from large-scale molecular dynamics simulations able to map the electrostatic potential within the protein pore and transport of peptides through α-HL, we plan to refine and extend this approach, as to decipher the microscopic details which govern the interaction of peptides with the α-HL in the presence of various metals. In conclusion, our work demonstrates the use of the α-HL protein as a robust single-molecule tool to unravel metaltriggered conformational changes in short peptides with metalbinding sites, and quantify simultaneously the kinetics of peptide-free and peptide-bound metals to the protein pore. With the complementary use of molecular dynamics simulations and atomistic characterization of peptide interactions with protein pores, this approach holds the promise of a more insightful and versatile exploration of metals-mediated peptides or proteins folding-unfolding processes. This presents important biophysical and biotechnological potential, as it may add a stepping stone to envisioning powerful strategies for understanding activity modulation of metal-binding, membrane active peptides. In addition, the presented approach may offer a new perspective to unravel at the unimolecular level the dynamic molecular recognition events at biological membrane receptors or protein translocases, which selectively respond to peptides or small proteins.

Figure 8. Nonlinear fitting of the τON (upward triangle) and τOFF (downward triangle) data shown in Figure 2, panels a and b, with expressions 16 and 24 (see also text), to derive numerical values of the association rate constants k1 and k3, as well as dissociation rate constants k2 and k4, which characterize the interaction of a single α-HL protein with either a “Cu2+-free peptide” or a “Cu2+-complexed peptide” (see model 11 in the main text).

The fact that beyond ∼50 μM of the trans-added Cu2+, the τON values seem to reach a Cu2+ concentration-independent plateau, serves as an indication that under such circumstances the most likely peptide species present in the trans solution is the “Cu2+-complexed peptide” (P−Cu2+), whose concentration attains its maximum value, and therefore remains invariant with further augmentation of the trans-added Cu2+. Following a similar route of thinking, and with the use of expression 24, nonlinear fitting of the τOFF data shown in Figure 2, panel b, resulted in k2 = 106.3 s−1, and k4 = 9.96 s−1, respectively (Figure 8, panel b). From our results, it thus appears that the association rate constant between an α-HL protein pore and a Cu2+-free peptide (k1) is approximately five times higher than that with a Cu2+-complexed peptide (k3). This observation seems to reconcile well with the scenario proposed above, in which, due to its conformational changes caused by the bound Cu2+ on

■

ASSOCIATED CONTENT

S Supporting Information *

This information is available free of charge via the Internet at http://pubs.acs.org/.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 17089


Langmuir

Article

(17) Sachse, C.; Fandrich, M.; Grigorieff, N. Paired β-Sheet Structure of an Aβ(1−40) Amyloid Fibril Revealed by Electron Microscopy. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7462−7466. (18) Adamcik, J.; Jung, J. M.; Flakowski, J.; De Los Rios, P.; Dietler, G.; Mezzenga, R. Understanding Amyloid Aggregation by Statistical Analysis of Atomic Force Microscopy Images. Nat. Nanotechnol. 2010, 5, 423−428. (19) Batchelor-McAuley, C.; Dickinson, E. J. F.; Rees, N. V.; Toghill, K. E.; Compton, R. G. New Electrochemical Methods. Anal. Chem. 2012, 84, 669−684. (20) Chen, Y. R.; Huang, H. B.; Chyan, C. L.; Shiao, M. S.; Lin, T. H.; Chen, Y. C. The Effect of Aβ Conformation on the Metal Affinity and Aggregation Mechanism Studied by Circular Dichroism Spectroscopy. J. Biochem. 2006, 139, 733−740. (21) Won, A.; Pripotnev, S.; Ruscito, A.; Ianoul, A. Effect of Point Mutations on the Secondary Structure and Membrane Interaction of Antimicrobial Peptide Anoplin. J. Phys. Chem B. 2011, 115, 2371− 2379. (22) Chou, I. H.; Benford, M.; Beier, H. T.; Cote, G. L.; Wang, M.; Jing, N.; Kameoka, J.; Good, T. A. Nanofluidic Biosensing for βAmyloid Detection Using Surface Enhanced Raman Spectroscopy. Nano Lett. 2008, 8, 1729−1735. (23) Hoernke, M.; Falenski, J. A.; Schwieger, C.; Koksch, B.; Brezesinski, G. Triggers for β-Sheet Formation at the Hydrophobic− Hydrophilic Interface: High Concentration, In-Plane Orientational Order, and Metal Ion Complexation. Langmuir 2011, 27, 14218− 14231. (24) Munishkina, L. A.; Fink, A. L. Fluorescence as a Method to Reveal Structures and Membrane-Interactions of Amyloidogenic Proteins. Biochim. Biophys. Acta 2007, 1768, 1862−1885. (25) Harriman, O. L. J.; Leake, M. C. Single Molecule Experimentation in Biological Physics: Exploring the Living Component of Soft Condensed Matter one Molecule at a Time. J. Phys.: Condens. Matter 2011, 23, 503101. (26) Kasianowicz, J. J.; Robertson, J. W. F.; Chan, E. R.; Reiner, J. E.; Stanford, V. M. Nanoscopic Porous Sensors. Annu. Rev. Anal. Chem. 2008, 1, 737−766. (27) Gu, L.-Q.; Shim, J. W. Single Molecule Sensing by Nanopores and Nanopore Devices. Analyst 2010, 135, 441−451. (28) Majd, S.; Yusko, E. C.; Billeh, Y. N.; Macrae, M. X.; Yang, J.; Mayer, M. Applications of Biological Pores in Nanomedicine, Sensing, and Nanoelectronics. Curr. Opin. Biotechnol. 2010, 21, 439−476. (29) Howorka, S.; Ziwy, S. Nanopore Analytics: Sensing of Single Molecules. Chem. Soc. Rev. 2009, 38, 2360−2384. (30) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Characterization of Individual Polynucleotide Molecules Using a Membrane Channel. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13770− 13773. (31) Clarke, J.; Wu, H.; Jayasinghe, L.; Patel, A.; Reid, S.; Bayley, H. Continuous Base Identification for Single-Molecule Nanopore DNA Sequencing. Nat. Nanotechnol. 2009, 4, 265−270. (32) Branton, D.; Deamer, D. W.; Marziali, A.; Bayley, H.; Benner, S. A.; Butler, T.; Di Ventra, M.; Garaj, S.; Hibbs, A.; Huang, X.; et al. The Potential and Challenges of Nanopore Sequencing. Nat. Biotechnol. 2008, 26, 1146−1153. (33) Deamer, D. W.; Akeson, M. Nanopores and Nucleic Acids: Prospects for Ultrarapid Sequencing. Trends Biotechnol. 2000, 18, 147−151. (34) Meller, A.; Nivon, L.; Brandin, E.; Golovchenko, J.; Branton, D. Rapid Nanopore Discrimination between Single Polynucleotide Molecules. Proc. Natl. Acad. Sci. U.S.A. 2006, 97, 1079−1084. (35) Zhao, Q.; de Zoysa, R.; Wang, D.; Jayawardhana, D.; Guan, X. Real-Time Monitoring of Peptide Cleavage Using a Nanopore Probe. J. Am. Chem. Soc. 2009, 131, 6324−6325. (36) Wang, H.-Y.; Ying, Y.-L.; Li, Y.; Kraatz, H.-B.; Long, Y.-T. Nanopore Analysis of β-Amyloid Peptide Aggregation Transition Induced by Small Molecules. Anal. Chem. 2011, 83, 1746−1752. (37) Talaga, D.; Li, J. Single-Molecule Protein Unfolding in Solid State Nanopores. J. Am. Chem. Soc. 2009, 131, 9287−9297.

Author Contributions ∥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors acknowledge the financial support offered by postdoctoral grants POSDRU/89/1.5/S/49944, POSDRU/89/ 1.5/S/63663 (Communicating Science), and PN-II-ID-PCCE2011-2-0027.

■

REFERENCES

(1) Ueda, E. K. M.; Gout, P. W.; Morganti, L. Current and Prospective Applications of Metal Ion−Protein Binding. J. Chromatogr. A. 2003, 988, 1−23. (2) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Metal Chelate Affinity Chromatography, a New Approach to Protein Fractionation. Nature 1975, 258, 598−599. (3) Wilson, C. J.; Apiyo, D.; Wittung-Stafshede, P. Role of Cofactors in Metalloprotein Folding. Q. Rev. Biophys. 2004, 37, 285−314. (4) Tsang, D.; Tsang, Y. S.; Ho, W. K.; Wong, R. N. Myelin Basic Protein Is a Zinc-Binding Protein in Brain: Possible Role in Myelin Compaction. Neurochem. Res. 1997, 22, 811−819. (5) Butterfield, S. M.; Lashuel, H. A. Amyloidogenic Protein− Membrane Interactions:Mechanistic Insight from Model Systems. Angew. Chem., Int. Ed. 2010, 49, 2−29. (6) Zatta, P.; Drago, D.; Bolognin, S.; Sensi, S. L. Alzheimer’s Disease, Metal Ions and Metal Homeostatic Therapy. Trends Pharmacol. Sci. 2009, 30, 346−355. (7) Minicozzi, V.; Stellato, F.; Comai, M.; Serra, M. D.; Potrich, C.; Meyer-Klaucke, W.; Morante, S. Identifying the Minimal Copper- and Zinc-Binding Site Sequence in Amyloid-Beta Peptides. J. Biol. Chem. 2008, 283, 10784−10792. (8) Bush, A. I.; Pettingell, W. H.; Multhaup, G.; Paradis, M. D.; Vonsattel, J. P.; Gusella, J. F.; Beyreuther, K.; Masters, C. L.; Tanzi, R. E. Rapid Induction of Alzheimer Aβ Amyloid Formation by Zinc. Science 1994, 265, 1464−1467. (9) Atwood, C. S.; Scarpa, R. C.; Huang, X.; Moir, R. D.; Jones, W. D.; Fairlie, D. P.; Tanzi, R. E.; Bush, A. I. Characterization of Copper Interactions with Alzheimer Amyloid Beta Peptides: Identification of an Attomolar-Affinity Copper Binding Site on Amyloid Beta 1−42. J. Neurochem. 2000, 75, 1219−1233. (10) Cherny, R. A.; Legg, J. T.; McLean, C. A.; Fairlie, D. P.; Huang, X.; Atwood, C. S.; Beyreuther, K.; Tanzi, R. E.; Masters, C. L.; Bush, A. I. Aqueous Dissolution of Alzheimer’s Disease Aβ Amyloid Deposits by Biometal Depletion. J. Biol. Chem. 1999, 274, 23223−23228. (11) Ghadiri, M. R.; Choi, C. Secondary Structure Nucleation in Peptides. Transition Metal Ion Stabilized Alpha-Helices. J. Am. Chem. Soc. 1990, 112, 1630−1632. (12) Schneider, J. P.; Kelly, J. W. Synthesis and Efficacy of Square Planar Copper Complexes Designed to Nucleate β-Sheet Structure. J. Am. Chem. Soc. 1995, 117, 2533−2546. (13) Zou, J.; Sugimoto, N. Effect of Copper Ion on Conformations of Short Alanine-Based Peptides with Single Trp/His Stacking Interactions. Prot. Peptide Lett. 1999, 6, 373−378. (14) Zou, J.; Sugimoto, N. Complexation of Peptide with Cu2+ Responsible to Inducing and Enhancing the Formation of α-Helix Conformation. BioMetals 2000, 13, 349−359. (15) Chen, W.-T.; Liao, Y.-H.; Yu, H.-M.; Cheng, I. H.; Chen, Y.-R. Distinct Effects of Zn2+, Cu2+, Fe3+ and Al3+ on Amyloid-β Stability, Oligomerization, and Aggregation. Amyloid-β. Destabilization Promotes Annular Protofibril Formation. J. Biol. Chem. 2011, 286, 9646− 9656. (16) Tougu, V.; Karafin, A.; Palumaa, P. Binding of Zinc(II) and Copper(II) to the Full-Length Alzheimer’s Amyloid-β Peptide. J. Neurochem. 2008, 104, 1249−1259. 17090


Langmuir

Article

Membrane Protein OmpF from E. coli and an Analogue of the HP(2− 20) Antimicrobial Peptide. J. Bioenerg. Biomembr. 2010, 42, 173−180. (60) Stefureac, R.; Waldner, L.; Howard, S. P.; Lee, J. S. Nanopore Analysis of a Small 86-Residue Protein. Small 2008, 4, 59−63. (61) Baran, C.; Smith, G. S. T.; Bamm, V. V.; Harauz, G.; Lee, J. S. Divalent Cations Induce a Compaction of Intrinsically Disordered Myelin Basic Protein. Biochem. Biophys. Res. Commun. 2010, 391, 224− 229. (62) Gouaux, E. α-Hemolysin from Staphylococcus aureus: An Archetype of β-Barrel, Channel-Forming Toxins. J. Struct. Biol. 1998, 121, 110−122. (63) Aksimentiev, A.; Schulten, K. Imaging α-Hemolysin with Molecular Dynamics: Ionic Conductance, Osmotic Permeability, and the Electrostatic Potential Map. Biophys. J. 2005, 88, 3745−3761. (64) Butler, T. Z.; Gundlach, J. H.; Troll, M. Ionic Current Blockades from DNA and RNA Molecules in the α-Hemolysin Nanopore. Biophys. J. 2007, 93, 3229−3240. (65) Wong, C. T. A.; Muthukumar, M. Polymer Translocation Through α-Hemolysin Pore with Tunable Polymer-Pore Electrostatic Interaction. J. Chem. Phys. 2010, 133, 045101. (66) Kowalczyk, S. W.; Tuijtel, M. W.; Donkers, S. P.; Dekker, C. Unraveling Single-Stranded DNA in a Solid-State Nanopore. Nano Lett. 2010, 10, 1414−1420. (67) Fologea, D.; Ledden, B.; David, S. M.; Li, J. Electrical Characterization of Protein Molecules by a Solid-State Nanopore. Appl. Phys. Lett. 2007, 91, 053901. (68) Han, A.; Creus, M.; Schurmann, G.; Linder, V.; Ward, T. R.; de Rooij, N. F.; Staufer, U. Label-Free Detection of Single Protein Molecules and Protein−Protein Interactions Using Synthetic Nanopores. Anal. Chem. 2008, 80, 4651−4658. (69) Han, A.; Schurmann, G.; Mondin, G.; Bitterli, R. A.; Hegelbach, N. G.; de Rooij, N. F.; Staufer, U. Sensing Protein Molecules Using Nanofabricated Pores. Appl. Phys. Lett. 2006, 88, 093901. (70) Yusko, E. C.; Prangkio, P.; Sept, D.; Rollings, R. C.; Li, J.; Mayer, M. Single Particle Characterization of Aβ Oligomers in Solution. ACS Nano 2012, 6, 5909−5919. (71) DeBlois, R. W.; Bean, C. P. Counting and Sizing of Submicron Particles by the Resistive Pulse Technique. Rev. Sci. Instrum. 1970, 41, 909−916. (72) Stöckel, J.; Safar, J.; Wallace, A. C.; Cohen, F. E.; Prusiner, S. B. Prion Protein Selectively Binds Copper(II) Ions. Biochemistry 1998, 37, 7185−7193. (73) Molecular and Cellular Biophysics; Jackson, M. B., Ed.; Cambridge University Press: New York, 2006. (74) Merrifield, R. B. Chemistry of Amino Acids. Science 1976, 232, 341−347. (75) Lee, D. G.; Park, Y.; Jin, I.; Hahm, K.-S.; Lee, H.-H.; Moon, Y.H.; Woo, E.-R. Structure−Antiviral Activity Relationships of Cecropin A-Magainin 2 Hybrid Peptide and its Analogues. J. Peptide Sci. 2004, 10, 298−303. (76) Schiopu, I.; Mereuta, L.; Apetrei, A.; Park, Y.; Hahm, K.-S.; Luchian, T. the Role of Thryptophan Spatial Arrangement for Antimicrobial-Derived, Membrane-Active Peptides Adsorption and Activity. Mol. BioSyst. 2012, DOI: 10.1039/c2mb25221j.

(38) Gu, L.-Q.; Cheley, S.; Bayley, H. Capture of a Single Molecule in a Nanocavity. Science 2001, 291, 636−640. (39) Gu, L.-Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Stochastic Sensing of Organic Analytes by a Pore-Forming Protein Containing a Molecular Adapter. Nature 1999, 398, 686−690. (40) Asandei, A.; Mereuta, L.; Luchian, T. The Kinetics of Ampicillin Complexation by γ-Cyclodextrins. A Single Molecule Approach. J. Phys. Chem. B. 2011, 115, 10173−10181. (41) Asandei, A.; Apetrei, A; Luchian, T. Uni-molecular Detection and Quantification of Selected β-Lactam Antibiotics with a Hybrid ΑHemolysin Protein Pore. J. Mol. Recognit. 2011, 24, 199−207. (42) Shin, S. H.; Bayley, H. Stepwise Growth of a Single Polymer Chain. J. Am. Chem. Soc. 2005, 127, 10462−10463. (43) Jayawardhana, D.; Crank, J.; Zhao, Q.; Armstrong, D.; Guan, X. Nanopore Stochastic Detection of a Liquid Explosive Component and Sensitizers Using Boromycin and an Ionic Liquid Supporting Electrolyte. Anal. Chem. 2009, 81, 460−464. (44) Jaikaran, D. C. J.; Woolley, G. A. Characterization of Thermal Isomerization at the Single Molecule Level. J. Phys. Chem. 1995, 99, 13352−13355. (45) Kasianowicz, J. J.; Bezrukov, S. M. Protonation Dynamics of the α-Toxin Channel from Spectral Analysis of pH-Dependent Current Fluctuations. Biophys. J. 1995, 69, 94−105. (46) Luchian, T.; Shin, S.-H.; Bayley, H. Single-Molecule Covalent Chemistry with Spatially Separated Reactants. Angew. Chem., Int. Ed. 2003b, 42, 3766−3771. (47) Luchian, T.; Shin, S.-H.; Bayley, H. Kinetics of a Three- Step Reaction Observed at the Single Molecule Level. Angew. Chem., Int. Ed. 2003a, 42, 1926−1929. (48) Shin, S.−H.; Luchian, T.; Cheley, S.; Braha, O.; Bayley, H. Kinetics of a Reversible Covalent−Bond Forming Reaction Observed at the Single Molecule Level. Angew. Chem., Int. Ed. 2002, 41, 3707− 3709. (49) Single Channel Recording, 2nd ed. Sakmann, B., Neher, E., Eds.; Plenum Press: New York, 1995. (50) Yusko, E. C.; Johnson, J. M.; Majd, S.; Prangkio1, P.; Rollings, R. C.; Li, J.; Yang, J.; Mayer, M. Controlling Protein Translocation Through Nanopores with Bio-Inspired Fluid Walls. Nat. Nanotechnol. 2011, 6, 253−260. (51) Freedman, K. J.; Jürgens, M.; Prabhu, A. S.; Ahn, C. W.; Jemth, P.; Edel, J. B.; Kim, M. J. Chemical, Thermal, and Electric FieldInduced Unfolding of Single Protein Molecules Studied Using Nanopores. Anal. Chem. 2011, 83, 5137−5144. (52) Wolfe, A. J.; Mohammad, M. M.; Cheley, S.; Bayley, H.; Movileanu, L. Catalyzing the Translocation of Polypeptides through Attractive Interactions. J. Am. Chem. Soc. 2007, 129, 14034−14041. (53) Pastoriza-Gallego, M.; Rabah, L.; Gibrat, G.; Thiebot, B.; van der Goot, F. G.; Auvray, L.; Betton, J.-M.; Pelta, J. Dynamics of Unfolded Protein Transport Through an Aerolysin Pore. J. Am. Chem. Soc. 2011, 133, 2923−2931. (54) Payet, L.; Martinho, M.; Pastoriza-Gallego, M.; Betton, J.-M.; Auvray, L.; Pelta, J.; Mathé, J. Thermal Unfolding of Proteins Probed at the Single Molecule Level Using Nanopores. Anal. Chem. 2012, 84, 4071−4076. (55) Meng, H.; Detillieux, D.; Baran, C.; Krasniqi, B.; Christensen, C.; Madampage, C.; Stefureac, R. I.; Lee, J. S. Nanopore Analysis of Tethered Peptides. J. Pept. Sci. 2010, 16, 701−708. (56) Sutherland, T. C.; Long, Y.; Stefureac, R. I.; Bediako-Amoa, I.; Kraatz, H.; Lee, J. S. Structure of Peptides Investigated by Nanopore Analysis. Nano Lett. 2004, 4, 1273−1277. (57) Movileanu, L.; Schmittschmitt, J.; Scholtz, J. M.; Bayley, H. Interaction of Peptides with a Protein Nanopore. Biophys. J. 2005, 89, 1030−1045. (58) Asandei, A.; Apetrei, A.; Park, Y.; Hahm, K.-S.; Luchian, T. Investigation of Single-Molecule Kinetics Mediated by Weak Hydrogen-Bonds within a Biological Nanopore. Langmuir 2011, 27, 19−24. (59) Apetrei, A.; Asandei, A.; Park, Y.; Hahm, K.-S.; Winterhalter, M.; Luchian, T. Unimolecular Study of the Interaction Between the Outer 17091


Protein Nanopore-Based, Single-Molecule Exploration of Copper

Recommend Documents