Electroosmotic Trap Against the Electrophoretic Force Near a Protein

May 9, 2016 - (a) The average values of time intervals separating consecutive capture events at the α-HL's lumen opening of trans-added peptides (τo...
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Electroosmotic Trap Against the Electrophoretic Force Near a Protein Nanopore Reveals Peptide Dynamics During Capture and Translocation Alina Asandei,†,# Irina Schiopu,†,# Mauro Chinappi,‡,# Chang Ho Seo,§ Yoonkyung Park,*,∥ and Tudor Luchian*,⊥ †

Department of Interdisciplinary Research, Alexandru I. Cuza University, Iasi 700506, Romania Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Roma, Viale Regina Elena 291, 00161 , Italy § Department of Bioinformatics, Kongju National University, Kongju 314-701, South Korea ∥ Department of Biomedical Science and Research Center for Proteineous Materials, Chosun University, Gwangju 61452, South Korea ⊥ Department of Physics, Alexandru I. Cuza University, Iasi 700506, Romania ‡

S Supporting Information *

ABSTRACT: We report on the ability to control the dynamics of a single peptide capture and passage across a voltage-biased, α-hemolysin nanopore (α-HL), under conditions that the electroosmotic force exerted on the analyte dominates the electrophoretic transport. We demonstrate that by extending outside the nanopore, the electroosmotic force is able to capture a peptide at either the lumen or vestibule entry of the nanopore, and transiently traps it inside the nanopore, against the electrophoretic force. Statistical analysis of the resolvable dwell-times of a metastable trapped peptide, as it occupies either the β-barrel or vestibule domain of the α-HL nanopore, reveals rich kinetic details regarding the direction and rates of stochastic movement of a peptide inside the nanopore. The presented approach demonstrates the ability to shuttle and study molecules along the passage pathway inside the nanopore, allows to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or β-barrel moiety, thus providing convincing proof of a molecule translocating the pore. The kinetic analysis of a peptide fluctuating between various microstates inside the nanopore, enabled a detailed picture of the free energy description of its interaction with the α-HL nanopore. When studied at the limit of vanishingly low transmembrane potentials, this provided a thermodynamic description of peptide reversible binding to and within the α-HL nanopore, under equilibrium conditions devoid of electric and electroosmotic contributions. KEYWORDS: electroosmosis, α-hemolysin, nanopore, peptide transport, single-molecule

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INTRODUCTION The nanoscale visualization and description of biopolymers trafficking through nanopores presents unique opportunities and challenges. Among the main epistemic outcomes of such studies, worth-mentioning are (i) a better understanding of protein and peptide transport across biological membranes, e.g. protein import inside mitochondria and endoplasmic reticulum, protein transport across the chloroplast membrane or the nuclear envelope of eukaryotic cells;1 (ii) RNA, DNA2−4 or peptide sensing and sequencing5−10 (iii) the exploration of peptide and protein folding;9,11−18 (iv) detection of microRNAs and the analysis of epigenetic changes, such as DNA methylation and gene damage;19−23 (v) exploration of the misfolding of peptides and proteins underlying the onset of neurodegenerative diseases24−29 etc. By providing accurate answers to such specific problems, all of the above have the potential of pushing nanopores into the realm of next generation of more sensitive, cheap and portable healthcare devices. © XXXX American Chemical Society

Currently, there is available a generous choice of either solidstate or protein-based nanopores, proven valuable for revealing at the single-molecule level, the microscopic description of the biological processes as those mentioned above.30−34 The physical, working principle of the approach is simple: biopolymers passing through the pore driven usually by an applied potential difference, displace a roughly equivalent volume of solvent and induce a reversible decrease in the ionic electrical current measured across the pore. The subsequent analysis of such volumetric blockade events in terms of duration and magnitude, provide a wealth of information about the physical and chemical properties of the biopolymers. A ubiquitous challenge plaguing the interpretation of such single biopolymers-induced blockade events, is represented by the relatively large translocation speed across the nanopore, Received: March 28, 2016 Accepted: May 9, 2016

A

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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diphytanoyl-sn-glycerophosphocholine (Avanti Polar Lipids, Alabaster, AL) dissolved in n-pentane (HPLC-grade, Sigma-Aldrich, Germany) using the Montal−Muller method.55 As we described before,7 the lipids formed stable bilayers across a ∼120 μm in diameter orifice punctured on a 25 μm-thick Teflon film (Goodfellow, Malvern, MA). The Teflon film that separated the cis (grounded) and trans chambers of the recording cell was pretreated with 1:10 hexadecane/pentane (HPLC-grade, Sigma−Aldrich, Germany). The entrapment of a single α-HL nanopore in the suspended lipid bilayer was attained by adding to the cis (grounded) side aliquots of α-HL protein monomeric solution, from a stock made in 0.5 M KCl. Both cis and trans chambers of the recording cell were filled with 2 M KCl solution buffered at pH = 7 with 10 mM HEPES (Sigma-Aldrich, Germany) or at pH = 2.8 with 5 mM HEPES (Sigma-Aldrich, Germany), depending on the experiment. Once the successful formation of a single α-HL heptamer in the membrane was achieved, the CAMA P6 peptide was pipetted at a bulk concentration of 30 μM in either cis or trans side of the recording cell, from a stock solution of 1 mM made in distilled water. The peptide−induced ionic current fluctuations across the α-HL nanopore were detected and amplified using either an Axopatch 200B (Molecular Devices, U.S.A) set to the voltage-clamp mode, or the eONE miniaturized amplifier (ELEMENTS SRL C/O CesenaLab, Italy), and then low-pass filtered at a corner frequency of 10 kHz. When employing the Axopatch 200B amplifier, data acquisition was performed with a NI PCI 6221, 16-bit acquisition board (National Instruments, USA) at a sampling frequency of 50 kHz, set within an instrument control sequence made in LabVIEW 8.20 (National Instruments, USA). Measurements were carried out near room temperature (Tm ≈ 298.15 K), and the recording cell was housed in a Faraday cage (Warner Instruments, U.S.A), mechanically isolated with a vibration-free platform (BenchMate 2210, Warner Instruments, U.S.A). Numerical analysis and data graphing were mainly done with the help of the Origin6 (Origin Lab, U.S.A) and pClamp 6.03 (Axon Instruments, U.S.A) software. The statistical analysis on the frequency and duration of the CAMA P6 peptide-induced current fluctuations through a single α-HL protein were analyzed within the statistics of exponentially distributed events, as previously described.7 Anion selectivity of the α-HL nanopore was assessed as reported previously32,33 by using an alternative form of the Goldman− Hodgkin−Katz equation

which may preclude the reliable statistics on the blockade duration and amplitude and requires measurements at increasing filter bandwidths. An alternative approach toward improving the statistics based on one biopolymer traversing the nanopore, is to control the molecule capture rate and translocation velocity, or command the same molecule to undergo multiple translocations through successive recapturing at the ends of the nanopore, to achieve optimal time resolution of current blockade signals. To fulfill this goal, various methods have been devised, including: the impact of physical and chemical properties of the solvent on biomolecular motion across nanopores,35−38 exploiting the interaction of the translocated analyte with characteristic antibodies,39 repetitive switching or modulating the transmembrane potential,40,41 using a pressure-voltage biased pore or entropic trapping of biomolecules near nanopores,42,43 altering the nanopore’s physical properties,44 or controlling surface charge in solidstate nanopores.45,46 In recent nonconventional approaches, it was shown that the Coulombic interaction between a charged analyte and a metallic cluster bound to α-HL nanopore plays an important role in the residence time enhancement,47 and by employing engineered polypeptides with opposite charged groups at its termini, elevated values of the applied transmembrane potential enhances the polypeptide capture rate by the α-HL nanopore, and simultaneously increases the peptide’s residence time in the pore.48−50 Another efficient strategy to influence the motion of biomolecules inside nanopores relies on controlling the balance between the electrostatic and electro-osmotic forces acting on them.44,45,51−53 Here we study the capture and translocation behavior of a 20 amino acids short peptide termed CAMA P6 (KWKLFKKIGIGKFLQSAKKF-NH2) across the wild-type α-HL protein pore, at pH = 2.8, and for the first time to our knowledge, we show evidence that electroosmotic flow is able to create an effective absorbing field for peptide capture by the α-HL nanopore against the electrophoretic force. The particular choice of two critical G residues on the peptide (underlined in the sequence above) makes it form a kinked hairpin-like state, which we stress is not a true β-hairpin,8 whose translocation kinetics through the wild-type α-HL protein can be substantially tuned by electroosmotic flow.8,15 We demonstrate that a single peptide can be constrained inside the nanopore, and in a region close to the nanopore mouth, and experimentally visualize the distinct elementary steps associated with the peptide capture, reversible translocation across α-HL’s vestibule and lumen regions, and release from the nanopore. The presented approach provides an important step forward to trap and manipulate individual peptides, and allows the study of physics of a molecule’s capturing and motion process when interacting with a protein nanopore.

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ψ F

( )

rev − [K +]cis [K +]trans exp RT PCl− m = ψrevF P K+ [Cl−]trans − [Cl−]cis exp RT

( ) m

where the activities of potassium ([K ]) and chloride ([Cl−]) ions on the cis and trans side of the membrane are being denoted accordingly, Ψrev is the experimentally measured reversal potential arising under salt gradients across the membrane, Tm is the absolute temperature, and F and R have their thermodynamics meanings. In Figure S1, we show representative I−ΔV diagrams used to calculate the Ψrev at neutral and acidic pH used herein. Molecular Dynamics Simulations. Pore-Membrane Equilibration. The membrane-pore system has been assembled using standard protocols.56,57 In brief, the system was assembled using VMD58 starting from the α-HL crystal structure PDB_ID:7AHL.59 The POPC membrane and the water molecules (TIP3P model) were added using VMD.60 Then the system is minimized and a 60 ps NVT simulation (time step 0.2 fs) was run. During this first step, external forces are added to avoid that water molecules enter the membrane, while α-HL and lipid heads were constrained to their initial position using an harmonic spring acting on the phosphorus atoms (spring constant k = 1 kcal/(mol A2). A second NPT flexible cell simulation (1 ns, time step 1 fs) was run to compact the membrane. During these runs the lipid heads were free to move. The third, and last, equilibration step consists in a NPT constant area simulations (2 ns, time step 2 fs). During this last step all the atoms are free to move. The resulting periodic box after the equilibration has the following dimensions: Lx = 12.649 nm, Ly = 12.461 nm, and Lz = 21.75 nm, and the number of atoms is 344 749. +

EXPERIMENTAL SECTION

Peptide Synthesis. The CAMA P6 peptide (KWKLFKKIGIGKFLQSAKKF-NH2) was synthesized using the solid-phase method with Fmoc (9-fluorenyl-methoxycarbonyl)-chemistry.8,54 To purify the peptide, HPLC on a C18 reverse-phase column was employed, and the primary sequence of the purified peptides was confirmed using an amino acid analyzer (HITACHI 8500A, Japan). The molecular weights of the synthetic peptides were determined using a matrixassisted laser desorption ionization (MALDI) mass spectrometer. Electrophysiology. Single−molecule experiments were performed using a single channel-forming α-HL protein (Sigma-Aldrich, Germany) inserted into a planar lipid membrane made from 1,2B

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Peptide capture at the α-HL’s lumen entrance by the electroosmotic flow. At neutral or acidic pH, positively applied ΔV values drive the trans-added, cationic peptide into the α-HL’s β-barrel, through the electrophoretic force (Felp). Peptide−pore interactions are seen as downward spikes, reflecting the stochastic reduction of pore current induced by the reversible association of a peptide with an open protein pore (panel A, subpanels a and c; panel B, subpanels a and d). As expected for a cationic peptide, in pH-neutral electrolytes, negative ΔV values drive the peptide away from the α-HL values β-barrel as Felp is cis-to-trans oriented, precluding peptide-pore interactions events (panel A, subpanels b and d). By stark contrast, at pH = 2.8, negative ΔV values were seen to facilitate peptide-pore interactions against the Felp force (panel B, subpanel b), seen as transient blockade events oriented upward. The enhanced electroosmotic flow of solvent across the α-HL nanopore at low pH’s and negative ΔV’s generate an equivalent electroosmotic force acting on the peptide (Felo), opposite to Felp, and the net result of these forces acting on a single peptide near the α-HL’s lumen, constitutes the main driving force of peptide partitioning inside the pore’s β-barrel (panel B, subpanel e). Under such conditions, the summation of Felo and Felp slows down the peptide kinetics inside the pore sufficiently enough, so that the metastable residence of a single peptide inside α-HL values lumen and vestibule are visible through the distinct blockade events denoted by IL and IV, respectively, from the open−pore current (IO) (panel B, subpanel c; see also text). In these experiments, the trans-added peptide concentration was 30 μM, and 2 M KCl was added symmetrically in both cis (grounded) and trans chambers.

monitored over time. A positively applied ΔV creates attractive, funnel-shaped electric field lines near the trans opening of the pore, which act electrophoretically on the randomly diffusing in solution cationic peptides, and squeeze them one at a time inside the α-HL’s lumen during the capture process (Figure 1, panel A, subpanels a and c). It should be remembered that for a peptide brought in the vicinity of the pore’s entrance, the actual excursion of it inside the pore and subsequent transit, requires climbing across a free energy barrier. This involves the entropy penalty for proper orientation of the peptide chain at the pore’s mouth and partial unfolding, and an enthalpy energy component stemming mainly from electrostatic interactions of the peptide with fixed charges lining the pore’s entrance. It has previously been shown that entry of an unfolded protein or peptide into a nanopore can be described reasonably well by the van’t Hoff−Arrhenius relationship.5,11 Negatively applied ΔV values prevent cationic peptides from entering the α-HL nanopore from the trans side, at neutral pH (Figure 1, panel A, subpanels b and d), as the electrophoretic force acts outwardly on the peptides, driving them away from the pore mouth. Surprisingly, the drop in solution acidity to pH = 2.8 led to single peptides entering the protein β-barrel against the outward electrophoretic force at negatively applied ΔV values (Figure 1, panel B, subpanels b and e). A similar phenomenon was seen in experiments in which the peptide was added from the cis side of the membrane (Figure 2). Namely, imposing

Protonation State. The protonation state of ASP, GLU and HIS residues was calculated using H++ server (see Supporting Information). The server provides a pKa value for each single residues. We protonated all the residues for which pKa > pH using standard psfgen tools included in VMD.58 The list of the protonated residues is reported in the Table S1. In Figure S2 we display charged residues exposed toward the α-HL’s interior, at pH 7 and pH 2.8. Ionic and Electroosmotic Flow Measurement. Ions were added via autoionize VMD plugin58 and a further short NPT constant area simulation (0.5 ns, time step 2 fs) was run to allow small adjustments of Lz. A homogeneous and constant electric field E = (0, 0, Ez) acting along the z direction is applied. This is equivalent to the application of a constant voltage ΔV = EzLz.61 Snapshots are saved every 20 ps, and average currents are estimated as in ref 56. During the production runs, the lipid heads were constrained via an harmonic spring acting on the phosphorus atoms (spring constant k = 1 kcal/(mol A2). All the simulations have been performed with NAMD62 with CHARMM3663 force field, including the NBFIX64 parameters for ions.

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RESULTS AND DISCUSSION

Peptide capture against the Electrophoretic Force. Figure 1 shows the CAMA P6 interaction with a single α-HL nanopore in neutral (pH = 7.0) and acidic (pH = 2.8) buffer solutions, as they are described in Experimental Section. In these experiments, the peptide was added to the solution on one side of the membrane (trans side) at a concentration of 30 μM. Positive or negative transmembrane potentials (ΔV) were then applied and the ion current across the nanopore was C

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. Peptide capture at the α-HL’s vestibule entrance by the electroosmotic flow. At neutral or acidic pH, negatively applied ΔV values drive the cis-added, cationic peptide into the α-HL’s vestibule, under the influence of the electrophoretic force (Felp). The stochastic reductions of the open pore current caused by the reversible association of a peptide with an open protein pore, are seen as upward spikes (panel A, subpanels a and c; panel B, subpanels a and d). At neutral pH, positive ΔV values generate trans-to-cis oriented electric forces acting on the cationic peptide, driving it away from the α-HL’s vestibule, as indicated by the lack of peptide−pore blockade events (panel A, subpanels b and d). Notably, at pH = 2.8, positively applied ΔV values resulted in vigorous peptide−pore interactions, seen as stochastic blockade events oriented downward, despite the fact that the Felp force still tends to drive the cis-added peptide away from the pore (panel B, subpanel b). This is due to the enhanced electroosmotic flow of solvent across the α-HL nanopore at low pH, which at positive ΔV values generate an equivalent electroosmotic force cis-to-trans oriented (Felo) acting on the peptide, opposite to Felp, and the net result is the cis-added peptide passage from bulk to α-HL’s vestibule (panel B, subpanel e). The net result of Felo and Felp vectorial summation influences the peptide passage inside the pore, to an extent that the transient residence of a single peptide inside α-HL’s vestibule and lumen are visible through the distinct blockade events denoted by IV and IL, respectively, from the open−pore current (IO) (panel B, subpanel c; see also text). Peptide concentration on the cis chamber was 30 μM, and 2 M KCl was added symmetrically in both cis (grounded) and trans chambers throughout the experiments.

Table 1. Electric Conductance Values of a Single α-HL Nanopore (Gopen) Measured in Symmetrically Added KCl (2 M) Across the Nanopore, in the Range of Positive and Negative ΔV values, at pH = 7 and 2.8a pH = 7 pH = 2.8

Gopen (+ΔV) (nS)

Gopen (−ΔV) (nS)

1.8 ± 0.01 2.4 ± 0.07

1.7 ± 0.02 2.1 ± 0.02

+ΔV (nS)

−ΔV (nS)

GL = 0.5 ± 0.006 GV = 1.1 ± 0.02

GL = 0.4 ± 0.01 GV = 0.9 ± 0.03

The analysis blockade events shown in Figure 1 and 2, panels B, sub-panels c, allowed the estimation of electric conductance of a single α-HL nanopore with a peptide present in either the lumen or vestibule region of the pore, at positive and negative ΔV values, associated to the IL and IV current levels generated by peptide entry into the pore aided by the electroosmotic force at pH = 2.8. In Figures S3 and S4, we present the original I−ΔV diagrams from which we extracted the electric conductance values shown herein. a

negative transmembrane potentials across the membrane at neutral pH, determined electrophoretic funneling of a peptide toward the pore’s vestibule, whereas no such interactions were seen at positive ΔV values (Figure 2, panel A). In contrast, by working in solutions buffered at pH = 2.8 and positive ΔV values, resulted in the emergence of vigorous blockade events associated with α-HL-peptide interactions, despite the fact that the electrophoretic force action on the peptide would drive it away from the vicinity of α-HL’s vestibule (Figure 2, panel B, subpanels b and e). The open pore α-HL conductance depends upon the sign of the transmembrane potential across it, indicative of an electrical rectification behavior, and is pH dependent, as found previously.65

Previously, we reported on the discovery that peptide dynamics through the α-HL nanopore is largely affected by the electroosmotic flow, which greatly alters the peptide translocation particularly in low pH values, by slowing down the peptide drift velocity along the protein pore.8 It should be noted that by lowering the pH buffer around 2.8 results in almost tripling of the α-HL’s anion-selectivity as compared to neutral pH (PCl−/PK+ ≈ 1.1, at pH = 7, and PCl−/PK+ ≈ 3.6, at pH = 2.8, where by PCl− and PK+ we denote the α-HL’s distinct permeabilities for Cl− and K+). A crucial observation reported herein, is that when the peptide was added on the trans side, any of the peptide-induced current blockade events seen at acidic pH and negative ΔV values started with a more prominent reduction in the open D

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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where κ represents the inverse Debye length and a the particle diameter), the electrophoretic velocity (velp) of a particle in an εε external electric field E writes velp = 0η r ξpeptideE , where ε0, εr,

pore current (denoted by IL), followed by shallower blockades (denoted by IV) (Figure 1, panel B, subpanel c). In contrast, when the peptide was added on the cis side, peptide-induced current blockade events seen at acidic pH and positive ΔV values, always started with the shallower blockade level (denoted by IV), preceding a subsequent more prominent one (denoted by IL) (Figure 2, panel B, subpanel c). Note that the IL and IV blockade events are associated with a peptide present in either the lumen or vestibule region of the α-HL nanopore (vide infra) and reflect the distinct extent of obstruction of ionic current across the α-HL by a peptide, caused by the nonequal effective volumes of the α-HL’s lumen and vestibule nanocavities. On the other hand, the visualization of IL and IV blockade events is possible as peptide’s motion across the nanopore gets slowed down, as a result of the interplay between the oppositely oriented electrophoretic and electroosmotic forces acting on the peptide at acidic pH.8 The experimentally estimated values of the distinct α-HL’s conductance states recorded in the range of either positive or negative ΔV values, corresponding to the blockade levels denoted by IL and IV visible at low pH, are shown in Table 1. We propose that experimental results embodied by Figures 1 and 2, panels B, are consistent with the manifestation of the counterflow by electroosmosis, altering of the direction of peptide’s drift motion, otherwise ruled by the sign of the electrophoretic force. That is, the increased anion selectivity of the α-HL nanopore at pH = 2.8 than at neutral pH, results in the augmentation of the net motion of the electrolyte solution inside the nanopore, which is oriented toward the positively biased side of the nanopore. As a result, the electro-osmotic flow generated at pH = 2.8 through the α-HL nanopore held at a negative potential on the trans side, extends outside the pore interior, and is able to create an absorbing region for a transadded peptide, driving it toward the pore’s β-barrel thus giving rise to the IL blockade level (Figure 1, panel B). Similarly, the augmented electroosmotic flow manifested at pH = 2.8 across a positively biased α-HL nanopore, overtakes the motion of peptides under the influence of the oppositely oriented electrophoretic force, and helps capture the cis added peptides in the vicinity of the protein’s vestibule, generating as expected (the α-HL’s vestibule has a larger volume than the β-barrel) the shallower, IV blockade level (Figure 2, panel B). These results emphasize, for the first time to our knowledge in protein nanopores, not only the ability to harness the electroosmotic flow to drive analytes inside the nanopore against the electrophoretic motion, but also help identify the precise entry region of the analyte inside the α-HL nanopore, that is, through either lumen or vestibule moiety. Complementary and as we will explore below, this finding is crucial to also determine the precise exit region of a peptide transiently captured inside the nanopore. This allows to rigorously follow a single molecule translocation through the pore and study the kinetic behavior of the molecule inside the α-HL nanopore in deeper details (vide infra). Classical Perspective of the Electroosmotic-Driven, Peptide Interaction with the α-HL Nanopore. In an attempt to qualitatively rationalize the findings shown in Figures 1 and 2, we resort to approximate models able to describe the net drift velocity of peptides (vdrift) in the vicinity of the nanopore’s mouth, in the absence of pressure or concentration gradients, and under the collective action of electrophoresis and electroosmosis.51,66,67 Under the Helmholtz−Smoluchowski limit for high ionic strength (κa ≫ 1,

and η represent the vacuum permittivity, relative permittivity of the buffer, and the solution viscosity, respectively, and ξpeptide is zeta potential of the peptide. By solving the Navier−Stokes equation for the motion of the electrolyte solution inside a uniformly charged nanopore under the influence of an external electric field (E), imposing the condition of small surface potentials of the nanopore wall and the assumption of the noslip boundary condition, and considering that the Debye layer is small compared to the nanopore’s diameter, one arrives at an approximate expression for the solvent velocity (velo), εε velo = 0η r ξnanoporeE , where the meaning of all parameters is as

described above, and ξnanopore is zeta potential of the nanopore inner wall. To fully describe the velocity profile of a particle, the zeta potential of the nanopore’s inner wall (ξnanopore) must be known, which unlike the zeta potential of the peptide is more laborious to infer, and usually involves measurements of the streaming potential. A more practical approach toward evaluating velo, especially useful for the case of largely inhomogeneous charged nanopores and nonuniform geometry, as the α-HL nanopore, proceeds indirectly, via the estimation of the water flux from the ion current through the pore within the simplifying assumptions of the Goldman−Hodgkin−Katz formalism for the independent movement of K+ and Cl− ions and no concentration gradient.8,53,68 In analogy to our previous description,8 we propose that the net drift velocity of a cationic peptide with the electrophoretic mobility μ situated in bulk solution at the mouth of the nanopore of length lpore and crosssectional area Spore, whose anion selectivity is given by (PCl−/ PK+) and is subjected to a potential difference ΔV giving rise to an open-pore current I, writes as the algebraic difference between an electrophoretic and electroosmotic term

vdrift

NI ΔV =μ − −h lpore |e |

where μ =

z |e−| D , kBTm

( (

PCl− P K+ PCl− P K+

) + 1) S −1

1 pore[H 2O]

(1)

z, and D are the peptide’s valence number

and diffusion coefficient, respectively, e− stands for the elementary charge, kB is Boltzman’s constant, Tm the absolute temperature, Nh is the number of water molecules carried by each ion which moves through the pore (Nh = 10) and [H2O] the concentration of water (3.4 × 1028 m−3). Turning attention to Figure 1, panel A, we recall that the velocity of the electroosmotic flow through the anion selective α-HL nanopore clamped at negative ΔV values is trans-to-cis oriented, it extends in the pore’s opening vicinities, and within simplifying assumptions, at distance r from the pore varies as ∼r−2,69 and thus is prone to influence the net local velocity of trans-added peptides. If the nanopore is only slightly anion selective or it lacks it (PCl−/PK+ → 1), the peptide’s drift velocity at the nanopore’s mouth acquires only the electrophoretic term in the electric field sensed across the nanopore, ΔV vdrift = velp = μ l . However, low pH values augment the pore

anion selectivity ratio of the pore and beyond a critical threshold, the electroosmotic velocity of a bulk peptide at the mouth nanopore of cross-sectional area Spore E

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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velo

NI = −h |e |

( (

PCl− P K+ PCl− P K+

pore’s surface, water viscosity close to the charged surface of the pore, etc. However, the simplified electrokinetic mechanism described above captures the essence of the process in which, under proper conditions (i.e., increased anion selectivity of the α-HL nanopore), a cationic peptide transitions from a spatial region away from the pore, toward the α-HL’s inner volume, against the electrophoretic force. In relation to this findings, in future studies we plan to also explore the influence played by different ionic strengths of the electrolyte on the electroosmotic capture of peptide at low pH’s, by the α-HL nanopore. Further evidence to support the role of electroosmotic flow comes from all-atom molecular dynamics (MD) simulations. We performed simulations at pH = 2.8 and pH = 7 of the α-HL nanopore under the action of an external voltage ΔV and we directly measured the ion and the electroosmotic flows. Figure 3 reports the electroosmotic flow expressed as number of water

) + 1) S −1

1 pore[H 2O]

may override the electrophoretic term, and facilitates the capture of peptide from the trans volume close to the β-barrel mouth, toward the pore (Figure 1, panel B). Simple calculations assuming that, qualitatively, the net charge of the peptide is reduced to ∼4 |e−| due to the screening effect of the 2 M KCl buffer,5 and replacing the length of the αHL nanopore, lpore = 10 nm, the cross-sectional area of the αHL’s lumen Spore = 3.8 × 10−18 m2, the bulk translational diffusion coefficient of the peptide D ≈ 1 × 10−11 m2 s−1,8 and using I = GΔV, where the unitary conductance of an open αHL nanopore in a 2 M KCl buffer in the range of negative ΔV’s was estimated at G = 1.7 nS (see Table 1), show that around neutral pH (PCl−/PK+ ≈ 1.1) and room temperature Tm = 298.15 K, the net velocity of the peptide near the α-HL’s lumen entrance at ΔV = −80 mV is vdrift ≈ −9.3 × 10−3 ms−1. With the vectorial conventions considered, the negative sign of vdrift is interpreted as the peptide moving away from the pore’s entrance. A similar estimation reveal that at pH = 2.8 (PCl−/PK+ ≈ 3.6), and with the calculated unitary conductance of an open α-HL nanopore, G = 2.1 nS (see Table 1), the net velocity of the peptide at the α-HL’s lumen entrance at ΔV = −80 mV (Figure 1, panel B) is vdrift ≈ 3.3 × 10−2 ms−1, whose positive sign indicates that at such low pH the peptide moves toward the pore’s lumen and is likely to enter the nanopore, driven by the electroosmotic flow. Similar as above, at a positive ΔV = 80 mV, the net drift speed of a peptide added on neutral buffer (pH = 7) on the cis side of the membrane, prone to get captured by the α-HL’s vestibule with a cross-sectional area of Spore = 5.3 × 10−18 m2 (Figure 2, panel A), was estimated at vdrift ≈ 1 × 10−2 ms−1. For this estimation we used the value of unitary conductance of an open α-HL nanopore in the range of positive ΔV values, in a neutral buffer, G = 1.8 nS (see Table 1). By the virtue of same conventions as above, the positive sign of velocity indicates that the peptide moves away from the vestibule entrance, under the prevalent action of the electrophoretic force. At pH = 2.8, the unitary conductance of an open α-HL nanopore in the range of positive ΔV values was estimated at G = 2.4 nS (see Table 1), and the net velocity of the peptide at the α-HL’s vestibule entrance at ΔV = 80 mV (Figure 2, panel B) was estimated at vdrift ≈ −2.5 × 10−2 ms−1. The negative sign indicates that the peptide moves toward the pore’s vestibule, driven by the electroosmotic flow. Note that the absolute value of this net drift speed at pH = 2.8 and ΔV = 80 mV (|vdrift| = 2.5 × 10−2 ms−1, vide supra) is slightly lower as compared to the case when the peptide gets captured on the lumen opening, at pH = 2.8 and ΔV = −80 mV (|vdrift| = 3.3 × 10−2 ms−1, vide supra). As expected based on water’s incompressibility property, this reflects in part a lower absolute velocity of the net flow of solvent at the α-HL’s vestibule as compared to its lumen entrance, caused by the slightly larger vestibule diameter as compared to the lumen region. At this stage, we stress that the exact values of the peptide velocity still remain subject of further refinement, considering that peptide’s physical properties (net charge, diffusion coefficient), as well as local gradients of the electric and electroosmotic forces action on it near the pore, depend on the geometry of the pore, peptide−pore longrange interactions or transient adsorption processes to the

Figure 3. Molecular simulation data of electroosmotic flow through the α-HL nanopore. The graph reports the electroosmotic flow (number of water molecules/nanoseconds) as a function of the applied potential obtained via atomistic simulations. The flux is assumed positive from lumen to vestibule. Red circles refer to pH = 2.8, while gray squares refer to pH = 7. It is apparent that for pH = 2.8 an electroosmotic flow sets-in. For positive ΔV, that is, electrical field directed from lumen to vestibule, the electroosmotic flow is negative, while the opposite happens for negative ΔV. For pH = 7, the large relative statistical errors do not allow to drag definitive conclusion on the presence of an electroosmotic flow.

molecules per nanosecond as a function of ΔV. It is apparent that at pH = 2.8 an electroosmotic flow sets in. The flow is directed as the negative ions, that is, it is opposite to the direction of the electrical field E. This is, again, easily explained considering the strong anionic selectivity of the α-HL nanopore at pH = 2.8 also confirmed by the different contribution of the positive and the negative ions fluxes to the total ionic currents, see Table S2. As usual for nonequilibrium all-atom MD simulations to reduce the statistical errors, the applied voltage is quite larger that the experimental one. Also employing this higher voltage, despite the quite long simulations (∼100 ns for each data point), the relative errors are very large. This occurrence does not allow us to draw strong conclusion on the presence of electroosmotic flow at pH = 7, although previous experimental studies52 indicates that also in this case an electroosmotic flow sets is. Nevertheless, the electroosmotic flow at pH = 7, if any, is much smaller than the one at pH = 2.8. Since the net charge of the CAMA P6 peptide remains invariant when changing the pH from neutral to acidic values,8 it will be interesting to address in future experiments and F

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 4. Asymmetry of voltage-dependence of peptide capture by the α-HL nanopore at low pH. (a) The average values of time intervals separating consecutive capture events at the α-HL’s lumen opening of trans-added peptides (τon (trans → lumen)), under the influence of the electroosmotic force (Felo) at negative ΔV values (the direction of the electric field E⃗ across the membrane is shown). The sketched free energy diagram at the lumen opening illustrates qualitatively the energy height (ΔFtrans) a peptide must surpass to enter the pore from the trans side. (b) The average values of time intervals separating consecutive capture events at the α-HL’s vestibule opening of cis-added peptides (τon (cis → vestibule)), under the influence of the electroosmotic force (Felo) at positive ΔV values. The sketched free energy diagram at the vestibule opening illustrates qualitatively the energy height (ΔFcis) a peptide must surpass to enter the pore from the cis side. The trans- or cis-added peptide concentration was 30 μM, and 2 M KCl was added symmetrically in both cis (grounded) and trans chambers buffered at pH = 2.8.

In addition, we note that at pH = 2.8, the 14 aspartic acids (D127 and D128 from all seven monomers of the homoheptameric α-HL) on the lumen are protonated, so that the opening mouth of the lumen behaves as a highly positively charged ring containing 7 lysines (K131 on each monomer of the homoheptameric α-HL), with a net charge ∼7|e−|.53 On the vestibule end of α-HL nanopore, protonated residues D13, D2, D4, D227 on each of the seven monomers of the homoheptamer, at acidic pH, leave a net positive charge on residues K8, R56, R104, K154 from each monomer of the protein, rendering the α-HL’s vestibule positively charged at acidic pH (net charge ∼28|e−|). Knowing that by definition the

molecular simulations, the implication of the effective screening of peptide charge by the electroosmotic flow, which may also contribute to the effects reported herein, as proposed previously.70 Asymmetry of Electroosmotic-Driven Peptide Capture at the α-HL’s Vestibule or Lumen Entry. An additional interesting finding is related to the capture dynamics of the peptide by the α-HL nanopore at pH = 2.8, when the peptide-pore collisions driven mainly by the electroosmotic flow take place either from the trans or cis side of the membrane. As we show in Figure 4, the average time of peptide association with the pore (τon), estimated from measuring time intervals separating consecutive peptide-induced α-HL current blockade events (see also Figure 5, panel a) is almost 1.5 orders of magnitude larger when the peptide is being captured from the lumen opening (Figure 4, panel a), as compared to the case when a peptide enters the α-HL nanopore through the vestibule (Figure 4, panel b). A plausible explanation for this phenomenon can be constructed on at least one physical argument: distinct enthalpy and entropy barriers for the peptide excursion into the α-HL nanopore from either side. Subsequent to peptide capture at the pore’s mouth under the prevailing influence of the electroosmotic flow, the molecule must partially unfold and confine itself within the narrow α-HL nanopore, to generate a successful capture event. Such a relative decrease in entropy is currently accepted to work as a barrier to inclusion of the molecule inside the pore.11,17,66,67 Given the larger diameter at the entrance of the α-HL’s vestibule (d ≈ 2.6 nm), and the even larger mean diameter of the vestibule interior (d ≈ 3.6 nm), as compared to the relatively uniform diameter of the lumen (d ≈ 2.2 nm),59 we posit that the entropic component necessary for peptide confinement within the α-HL’s lumen is larger than that posed by peptide entry inside its vestibule.

Debye length κ −1 =

ϵr ϵ0 kBTm 2 |e−|2 NAI1000

, where εr and ε0 represent

the relative permittivity of electrolyte (εr ≈ 70) and vacuum permittivity, respectively, NA is Avogadro’s number, e− stands for the elementary charge, kB is Boltzman’s constant, Tm the absolute temperature, and I represents the ionic strength of electrolyte in molar units, the Debye length in our experiments is κ−1 ≈ 1.9 Å. Thus, the localization of the net positive charge on the vestibule toward the internal surface of the pore alleviates to a certain extent the contribution of an enthalpic barrier (electrostatic in nature), that would hinder the peptide capture from the cis side. In contrast, a net positive charge distribution on the lumen mouth at low pH (vide supra) lines the pore’s entrance facing the trans buffer, and therefore is prone to impede the cationic peptide partitioning inside the lumen. On the framework of Eyring’s transition state theory applied to the peptides interaction with the α-HL nanopore,5 the net results of these contributions reflect as a larger activation free energy (ΔF) of a cationic peptide entering the αHL nanopore from the lumen than the vestibule opening, at low pH (see Figure 4, free energy diagram insets). G

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 5. Distinct microscopic substates characterizing the peptide dynamics inside the α-HL nanopore at low pH. (a) Representative trace showing blockade events reflecting the cis-added, peptide-α-HL interactions at ΔV = 80 mV, under the collective influence of electroosmotic (Felo) and electrophoretic (Felp) forces, at pH = 2.8. The open−pore current is denoted by “free α-HL”, and the association time for peptide capture which separates consecutive blockade events is shown (τon). Peptide capture at the α-HL’s vestibule and subsequent passage across the nanopore generate mainly three distinct types of events. In the first type of blockade events, a peptide captured at the α-HL’s vestibule by the prevalent electroosmotic force, moves reversibly between the nanopore’s vestibule and lumen, as indicated by the corresponding current states in panel b, where “■” denotes the vestibule → lumen transitions and “●” denotes the lumen → vestibule transitions. Eventually, the peptide moves irreversibly from the lumen to the vestibule region, and exits the pore on the cis side, denoted “★” associated with a vestibule → cis transition (panel b). The second type of blockade events reflects the unidirectional movement of a peptide captured on the nanopore’s vestibule to the lumen region, depicted as a vestibule → lumen transition (■), and then released on the trans side, depicted as a lumen → trans transition (▼) (panel c). The third type of blockade events illustrates the peptide capture on the nanopore’s vestibule and then return to the cis side, without movement across the nanopore, associated with a vestibule → cis transitions (★) (panel d). The sketchy representation of molecular states and microscopic transitions associated with the type I, II and III events described above, are shown in panels e (type I), f (type II) and g (type III). E⃗ represents the electric field across the α-HL nanopore.

we do not have a reasonable explanation for this finding. We can only speculate that although prevalent at pH = 2.8, the electroosmotic force driving the peptide into the α-HL’s vestibule from the cis side, varies less rapidly with ΔV as compared to the electrophoretic counterpart, beyond a certain threshold. As a result, the net velocity of the peptide, although still directed toward pore’s vestibule, would decrease with an increase in ΔV. Dynamics of a Transiently Trapped Peptide Inside the α-HL Nanopore under the Influence of Electroosmotic Flow. Advantages stemming from exploring a single peptide dynamics inside the nanopore under controlled velocity, include addressing crucial issues regarding molecular trafficking along nanodomains, such as (i) precise identification of the peptide residence on distinct regions of the nanopore, and subsequent volumetric, kinetic and thermodynamic analysis of transition steps between such regions, and (ii) providing convincing evidence regarding the direction of movement of a peptide inside the nanopore, up to the peptide exit through either opening. For the sake of concreteness, in the analysis below we will focus only on data embodied by Figure 2, panel B (peptide added in cis), although the rationale can be easily

A puzzling discovery refers to the voltage dependence of the peptide capture by the α-HL nanopore at pH = 2.8, when driven by electroosmotic flow, on either lumen or vestibule opening (Figure 4). Considering the lumped electrophoretic and electroosmotic forces effect in setting the direction of peptide transport through the nanopore (expression 1), it is expected that once the magnitude of the electroosmotic force becomes prevalent, a linear increase in the net velocity of the peptide versus ΔV should occur. Thus, the capture rate of the peptide by the protein pore is expected to increase with ΔV, so that the average time of peptide association with the pore (τon) should decrease. While this prediction is nicely met with the peptide added on the trans side, and negative ΔV values (Figure 4, panel a), the exact opposite tendency was seen when the peptide entered the α-HL nanopore through the vestibule opening, at positive ΔV values (Figure 4, panel b). That is, despite the fact that under such conditions the peptide excursion toward the pore’s vestibule is critically determined by the electroosmotic force, which is proportional to the ΔV and points inwardly to the pore opposite to the electrophoretic component, the average time of peptide association with the pore (τon) increases with an increase in ΔV. At the present stage H

DOI: 10.1021/acsami.6b03697 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 6. Electroosmotic-captured, peptide dynamics inside the α-HL nanopore at pH = 2.8. (a) The voltage-dependence of the peptide reaction rate from the α-HL’s vestibule to the cis side of the membrane (k(vestibule → cis)). The microscopic transitions underlying this process are embodied by the “★”-like blockade events, illustrated in Figure 5, panels b and d. (b) The voltage-dependence of the peptide reaction rate from the α-HL’s vestibule to the lumen region (k(vestibule → lumen)). The microscopic transitions associated with this phenomenon consist of the particular “■”-like blockade events, illustrated in Figure 5, panels b and c. (c) The voltage-dependence of the reaction rate of a peptide from the α-HL’s lumen to the vestibule region (k(lumen → vestibule)), which correspond to the “●”-like blockade events shown distinctly in Figure 5, panel b. (d) The voltage-dependence of the reaction rate of a peptide from the α-HL’s lumen to the trans side (k(lumen → trans)), which correspond to the “▼”-like blockade events shown distinctly in Figure 5, panel c. The distinct values of the applied transmembrane potential were positively biased on the trans side of the membrane, in contact with the α-HL’s lumen. For clarity, in the cross-section representations of the α-HL nanopore are illustrated the specific molecular transitions associated with the reaction rates calculated herein. The arrows indicate the direction of the electric field across the ⎯→ ⎯ ⎯→ ⎯ membrane (E⃗ ), and electroosmotic ( Felo) and electrophoretic force ( Felp ) acting on the peptide.

with spatiotemporal accuracy the peptide movement along αHL’s vestibule or lumen domains (Figure 5, panels b−d), allowed us to rationalize the presented data in kinetic terms (see Supporting Information for a full description of the kinetic mechanism). We started by estimating the number of each of the transitions in a given time T (the total observation time), the total time spent by the peptide inside the vestibule (TV) and the total time spent by the peptide inside the lumen (TL). For all the of events types described above, we then counted the number (M) of distinct peptide transitions along the α-HL nanopore during the observation time T, and measured their corresponding dwell times, as follows: (i) for the vestibule → lumen transitions (■), we counted the M(vestibule → lumen) transitions whose corresponding dwell times were denoted by τvestibule→lumen; (ii) for the lumen → vestibule transitions (●), we counted the M(lumen → vestibule) transitions whose corresponding dwell times were denoted by τlumen→vestibule; (iii) for the vestibule → cis transitions (★), we counted the M(vestibule → cis) transitions whose corresponding dwell times were denoted by τvestibule→cis; (iv) for the lumen → trans transitions (▼), we counted M(lumen → trans) number of transitions whose corresponding dwell times were denoted by τlumen→trans. Subsequently, for the calculus of the total time spent by the peptide in the vestibule (TV) we added up τvestibule→lumen and τvestibule→cis intervals, and the total time spent by the peptide in the lumen (TL) resulted from the addition of the τlumen→vestibule and τlumen→trans intervals. In Figure S6, we show representative histograms of time intervals associated with the transitions made by a peptide inside the α-HL nanopore between the lumen and vestibule regions, collected at pH = 2.8 and ΔV = +80 mV, demonstrating the exponential distribution of such durations.

extended for the case when the peptide is added on trans compartment (Figure 1, panel B). Inspection of the current traces shown in Figure 5 reveals mainly three distinct types of molecular events following the peptide capture by the α-HLs on the vestibule opening, under the influence of the electroosmotic force at transmembrane positive ΔV values. By correlating the magnitude of open-pore current blockade signatures with the distinct, transient presence of the peptide inside α-HL’s vestibule or lumen region (vide supra), we posit that type I of such events illustrates a single peptide moving back and forth between the vestibule and lumen region, the type II events reflects the unidirectional movement of a peptide captured on the nanopore’s vestibule to the lumen region, followed by its release on the trans side, and the type III of events illustrates the peptide capture on the nanopore’s vestibule and then its return to the cis side, without movement across the nanopore. Particularly interesting, note that by virtue of this analysis, one can precisely pinpoint occurrences associated with true peptide translocations across the nanopore, as reflected for instance by the type II events (Figure 5, panels c and f). We mention that a very rare number of events related to type II were recorded, showing a subsequent peptide recapture from the trans side of the membrane (not shown here), but their scarcity (