The Role of Lys147 in the Interaction between MPSA-Gold

Article pubs.acs.org/Langmuir

The Role of Lys147 in the Interaction between MPSA-Gold Nanoparticles and the α‑Hemolysin Nanopore Elisa Campos,† Alina Asandei,‡ Colin E. McVey,§ Joaõ C. Dias,†,∥ A. Sofia F. Oliveira,∥ Cláudio M. Soares,∥ Tudor Luchian,‡ and Yann Astier*,†,⊥ †

Single Molecule Processes Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal ‡ Laboratory of Molecular Biophysics and Medical Physics, “Alexandru I. Cuza” University, Boulevard Carol I, N° 11, Iaşi 700506, Romania § Structural Genomics Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal ∥ Protein Modeling Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal S Supporting Information *

ABSTRACT: Single channel recordings were used to determine the effect of direct electrostatic interactions between sulfonate-coated gold nanoparticles and the constriction of the Staphylococcus aureus α-hemolysin protein channel on the ionic current amplitude. We provide evidence that Lys147 of αhemolysin can interact with the sulfonate groups at the nanoparticle surface, and these interactions can reversibly block 100% of the residual ionic current. Lys147 is normally involved in a salt bridge with Glu111. The capture of a nanoparticle leads to a partial current block at neutral pH values, but protonation of Glu111 at pH 2.8 results in a full current block when the nanoparticle is captured. At pH 2.8, we suggest that Lys147 is free to engage in electrostatic interactions with sulfonates at the nanoparticle surface. To verify our results, we engineered a mutation in the α-hemolysin protein, where Glu111 is substituted by Ala (E111A), thus removing Glu111−Lys147 interactions and facilitating Lys147− sulfonate electrostatic interactions. This mutation leads to a 100% current block at pH 2.8 and a 92% block at pH 8.0, showing that electrostatic interactions are formed between the nanopore and the nanoparticle surface. Besides demonstrating the effect of electrostatic interactions on cross channel ionic current, this work offers a novel approach to controlling open and closed states of the α-hemolysin nanopore as a function of external gears.

N

protein nanopore research. While artificial nanopores are the subject of intense research, WT-αHL remains an ideal platform for investigating single-molecular chemistries. Since we know the precise location of each atom from its structure (PDB accession code 7AHL), new chemical functions can be endowed to the protein at desired locations using structurebased mutagenesis techniques.9 We recently discovered that gold nanoparticles coated with 3-mercapto-1-propanesulfonate (MPSA-NP) could be captured within the WT-αHL nanopore cavity (Figure 1), resulting in an ionic current decrease.10 In the present work, we report the importance of nanopore− analyte interactions for residual current intensity over the actual

anopores are emerging as very efficient and sensitive single-molecule sensors.1 Detection of analytes is based on the interaction between a specific analyte and a specific binding site within the pore, causing significant changes in the ionic current passing through the nanopore at an applied potential. The extent and duration of the resulting current block allows for identification of the analyte molecule, whereas the frequency of blocking events reveals its concentration.2,3 The volume of the nanopore is comparable to the size/volume of target analytes. Depending on the bulk concentration, molecules enter the nanopore one at a time, and their kinetics of binding to specific sites can be analyzed individually.2,4 Nanopores constitute a promising technology in the area of biosensors5 with potential applications ranging from medical diagnostics6 to the detection of biological warfare agents.7 Since its structure was resolved,8 α-hemolysin from Staphylococcus aureus (WT-αHL) has been a corner stone in single channel © 2012 American Chemical Society

Received: June 28, 2012 Revised: August 30, 2012 Published: October 8, 2012 15643

dx.doi.org/10.1021/la302613g | Langmuir 2012, 28, 15643−15650

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curves of the different systems (Figure S1 of the Supporting Information). Figure 2 shows single channel recordings of WT-αHL in the presence of MPSA-NP. Figure 2A shows MPSA-NP capture events by WT-αHL at pHs 2.8 and 8.0, at an applied transmembrane potential of +80 mV. We observe that pH 8.0 capture events lead to current block amplitudes of up to 31%, and pH 2.8 capture events lead to blocks up to 100%. These measurements were repeated in 5 independent experiments in two different locations. Further controls were carried out under the same conditions at pHs 4.5, 5.5, 6.9, and 9.9. Whereas 100% current blocks were observed at pHs 4.5 and 5.5, 30% current blocks were observed above pH 5.5 (Table S1 of the Supporting Information). These results show that when Glu111 is protonated, the presence of MPSANP in the protein cavity leads to a full current block. The protonation of Glu111 prevents the formation of a salt bridge with Lys147, which can then engage in salt bridges with MPSANP sulfonates that are still negatively charged at this pH (Table 1). In Figure 2B, we see that MPSA-NP capture events with E111A lead to 89% and 100% block levels at pH 2.8 and 72% and 92% at pH 8.0. At both pH values, the largest block was the most frequent (Table 2). Also at pH 9.9, when the E111A protein cavity is negatively charged (Table 1), a near full block was observed (data not shown). The results observed with E111A confirm that, when Lys147 is not engaged in a salt bridge, the capture of MPSA-NP in the protein cavity systematically yields a near full block of the nanopore. This is regardless of the fact that the MPSA-NP population is not heterogeneous and exhibits slight variations.10,11 The dependence of the single-channel current block on the applied transmembrane potential was also assessed. Table 2 shows current block and dwell-time values for the interaction between MPSA-NP and the WT-αHL nanopore at applied transmembrane potentials +60, +80, and +100 mV, for all pH values studied. At pH 2.8, the number of current block levels is independent of the applied potential. Tables 2 and S1 of the Supporting Information together show that dwell time increases with the applied potential, at all experimental pH values. This stems from the increased stability of MPSA-NP within the protein cavity due to the increased strength of the electrical field. Moreover, the electroosomotic flow of water through the slightly anion-selective WT-αHL protein, from the cis to the trans side of the bilayer, is enhanced as we increase the applied positive potential, increasing also the dwell time.12 For each tested condition, we observed that the dwell time increased with the current block amplitude. Table 2 also shows current block and dwell time values for the E111A nanopore at applied transmembrane potentials of +60, +80, and +100 mV, at both pHs 2.8 and 8.0. Results show that the applied potential did not influence the number of current block levels, at either pH value tested. It was also observed that the dwell time at pH 2.8 was longer than that at pH 8.0. Dwell time for the mutant nanopore was globally shorter than for the WT-αHL nanopore. This result could be explained by the larger positive charge inside the protein cavity at lower pH (Table 1), stabilizing the negatively charged MPSA-NP inside the nanopore. Figure 3 shows that, when a MPSA-NP is trapped in the WTαHL at pH 8.0, the baseline of the event is not always linear and often displays changes in amplitude.

Figure 1. Interaction of MPSA-NP with the WT-αHL nanopore. Molecular model showing a single MPSA-NP inside the WT-αHL nanopore (shown in the cross section; PDB accession code 7AHL). MPSA-NP diameter was determined to be (3.0 ± 0.6) nm.10,11 Residues Glu111 and Lys147 at the constriction region are colored red and blue, respectively. MPSA-NP is emphasized by a surface contour in cyan.

size of the analyte and physical volume it occupies in the pore. We show how MPSA-NP, that results in a 14−31% current block when captured in the WT-αHL cap, can lead to a full current block if electrostatic interactions between the nanoparticle surface groups and the nanopore constriction point are fostered. We studied the nature of the interaction between MPSA-NP and WT-αHL, analyzing current amplitude changes obtained during single-channel recordings. We focus upon Glu111 and Lys147 residues, which are located at the narrowest constriction of the protein nanopore. While these are not the only charged residues in the WT-αHL cap, their positioning at the constriction site enhances their influence on the residual ionic current. On each of the seven WT-αHL monomers, normally, these two amino acids interact with each other via a salt bridge.8 Our recordings reveal small changes in amplitude within single-particle capture events, suggesting that we are able to observe individual Lys147 residues temporarily swapping electrostatic interaction between Glu111 and the MPSA-NP surface sulfonates. Glu111 can be protonated by lowering the pH to prevent formation of the salt bridge with Lys147. As pH is reduced, we observe a transition from partial to full current blocks as MPSA-NP enters the protein. In addition, we have engineered the E111A mutant to show that these full current blocks are due to salt bridges between Lys147 and MPSA-NP sulfonates once the original salt bridge with Glu111 is no longer possible. The mutant exhibits full current blocks in the presence of MPSA-NP at any pH. The interactions between the protein and MPSA-NP surface sulfonates are investigated by characterizing their pH and voltage dependence.

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RESULTS AND DISCUSSION As shown in Figure 1, Glu111 and Lys147 are found at the narrowest constriction within the interior surface of the WTαHL protein nanopore. At pH 8.0, Lys147 residues form a salt bridge with negatively charged Glu111, since these are both charged at this pH value, as shown by their average pKa values: 3.2 ± 0.5 for Glu111 and 11.7 ± 0.1 for Lys147 in WT-αHL. The average pKa of the MPSA-NP surface sulfonates is 2.9 ± 0.1, while the average pKa of Lys147 in E111A is 10.1 ± 0.1. These values were deduced from the average simulated titration 15644


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Figure 2. Representative ionic current traces mediated by (A) WT-αHL and (B) E111A, in the presence of MPSA-NP (10 μg mL−1) added on the cis side of the lipid bilayer, at pHs 2.8 and 8.0. Current fluctuations reflect the reversible interaction between MPSA-NP and the constriction region of the protein. O, L1, L2, L3, and L4 denote the open pore state and the first, second, third, and fourth level of current block when the MPSA-NP interacts with the protein. On the right, the events histograms show the different levels of current block. These experiments were performed at +80 mV in 2 M KCl solution buffered at pH 2.8 (5 mM MES), and pH 8.0 (10 mM HEPES).

amplitude in percentage of the open pore current. Events of 20% or 30% current block can be explained by the size variation among the MPSA-NP; however, the amplitude changes in the baseline are independent. We suggest that these smaller amplitude changes are due to individual Lys147 temporarily engaging in salt bridge formation with a sulfonate and breaking this interaction to reform its interaction with the Glu111. Therefore, the amplitude and dwell times listed in Table 3 should be due to these salt bridge exchanges between the Glu111 and the sulfonate. Presented in Figure S3 is a summary of the interactions between Lys147 of WT-αHL and the E111A protein with a single MPSA-NP when it is captured inside the protein

Table 1. Charge of the WT-αHL and E111A Protein Cavity and of the MPSA-NP at the Experimental pH Values pH WT-αHL E111A MPSA-NP a

2.8

6.9

8.0

9.9

+8.3 +11.2 −21.2

−1.2 +5.7a −44.0

−3.9 +3.0 −44.0

−8.1 −3.0 −44.0

Experiments not carried out under this pH value.

We have analyzed these events at different voltages and observed that, regardless of the original event amplitude, the changes in amplitude of the baseline always display the same 15645


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Table 2. Summary of the MPSA-NP Capture Events in a Single WT-αHL and E111A Nanoporea,b +60 mV % blockade pH 2.8

81.91 88.27 95.87 17.86

± ± ± ±

0.26 0.62 0.53 0.52

+80 mV

dwell time (s)

N

± ± ± ±

114 99 59 52

0.11 0.5 2.2 0.1

0.02 0.1 1.2 0.1

% blockade 82.81 89.56 96.49 13.84

± ± ± ±

wild-type 1.10 0.3 1.54 0.8 0.21 3.7 0.79 0.004

17.30 ± 2.72 24.81 ± 2.29 30.98 ± 1.84

pH 8.0

+100 mV

dwell time (s) ± ± ± ±

0.1 0.1 1.5 0.001

0.1 ± 0.1 8.6 ± 3.7 13.8 ± 5.5

pH 2.8

99.12 ± 1.83 88.81 ± 2.09

0.29 ± 0.05 0.032 ± 0.004

153 42

E111A mutant 99.74 ± 0.48 0.09 ± 0.01 88.79 ± 1.07 0.07 ± 0.01

pH 8.0

87.29 ± 2.11 69.14 ± 0.92

0.037 ± 0.001 0.008 ± 0.001

818 169

91.82 ± 3.88 72.21 ± 5.33

0.012 ± 0.001 0.006 ± 0.001

N

% blockade

dwell time (s)

N

75 65 43 94

86.28 91.52 96.46 13.85

± ± ± ±

0.70 0.38 0.15 0.26

0.7 1.9 4.4 0.005

± ± ± ±

0.1 1.2 2.1 0.001

21 24 26 46

34 16 12

17.30 22.32 26.22 31.43

± ± ± ±

0.02 0.75 0.53 0.72

0.1 3.8 14.6 17.8

± ± ± ±

0.1 1.0 5.4 11.3

42 12 12 9

102 86

98.13 ± 5.02 79.32 ± 7.09

0.06 ± 0.01 0.03 ± 0.01

115 38

1423 682

90.39 ± 4.83 71.92 ± 8.86

0.005 ± 0.0001 0.003 ± 0.0002

1084 384

a Experiments were performed applied potentials of +60 mV, +80 mV, and +100 mV in 2 M KCl solution buffered at pH 2.8 (5 mM MES) and pH 8.0 (10 mM HEPES). bValues are the average of N events and are presented as the mean ± standard deviation.

Figure 3. Representative capture event showing changes of current amplitude between defined amplitude levels, while the MPSA-NP is inside the single WT-αHL nanopore. (1) Empty pore current amplitude of 155 pA. (2) Nanopore current amplitude (115 pA) with a single MPSA-NP trapped inside, while Lys147 interacts with Glu111 through a salt bridge. (3) Nanopore current amplitude (110 pA) with a single MPSA-NP trapped inside, while Lys147 interacts electrostatically with MPSA-NP sulfonate ligands. A histogram of the amplitude changes (2 and 3) is plotted on the right. Recordings were made at +80 mV in 2 M KCl, 10 mM HEPES (pH 8.0), in the presence of 10 μg mL−1 MPSA-NP added on the cis side of the lipid bilayer.

MPSA-NP in the constriction region of the WT-αHL nanopore. The increase of dwell time with increasing applied potential was possibly due to the stability of the MPSA-NP in the nanopore, since the previously mentioned interactions were favored by the orientation and the intensity of the electric field. The dwell time of the interaction between MPSA-NP and the E111A mutant nanopore was shorter than that determined for WT-αHL, under the same experimental conditions. Dwell time was observed to increase with applied potential in WT-αHL, but increased applied potential resulted in a decrease in dwell time for the interaction between MPSA-NP and E111A (Figure 4). In fact, the decrease in dwell time with stronger interactions between the analyte and the protein nanopore was already observed previously.13 Simulation results showed that, in WTαHL, Cl− ions have a 4-fold higher concentration than K+ ions at the constriction region,14 demonstrating that this region is a strong binding site for Cl− ions. Other simulation studies15 showed that the passage of Cl− ions is favored through the narrowest part of the constriction, with this slight anion selectivity of WT-αHL being attributed to Lys147. Moreover, theoretical studies of ion permeation16 determined that cation/ anion current ratio decreased 43% relative to the wild type when Glu111 is neutralized, resulting in an anionic conductivity that is 3-fold larger. We hypothesize that the shorter dwell times for MPSA-NP observed with the E111A at higher

Table 3. Current Amplitude Variation (ΔI), Percentage of ΔI Relative to Open Pore Current (Io) [% (ΔI/Io)], and Dwell Time (τoff) Values for Current Block Levels Observed During Capture Events of a Given MPSA-NP by a WTαHLa,b Vm

N

ΔI ± SE (pA)

% (ΔI/Io)

τoff ± SE (s)

+80 mV +90 mV +100 mV

251 21 27

5.72 ± 0.18 7.29 ± 0.11 8.48 ± 0.24

3.71 4.39 4.19

0.089 ± 0.023 0.164 ± 0.039 0.243 ± 0.032

a

Experiments were performed at applied potentials of +80 mV, +90 mV, and +100 mV in 2 M KCl and 10 mM HEPES (pH 8.0). bΔI and τoff values are the average of N events and are presented as the mean ± standard error (SE).

nanopore, illustrating the influence on the likely gating mechanism of the nanopore. Figure 4 represents ionic current traces from a single E111A nanopore, in the presence of MPSA-NP, at applied transmembrane potentials of +40, +60, +80, and +100 mV. At pH 8.0, we observed that the dwell time decreased as the applied transmembrane potential increased. Further recordings carried out at pH 8.0 showed that no current blocks occurred at a potential of +20 mV (data not shown). We observed that the applied transmembrane potential influenced the values of the current block and the dwell time of 15646


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Figure 4. Single-channel recordings of E111A nanopores after the addition of 10 μg mL−1 MPSA-NP to the cis compartment, at applied potentials of +40, +60, +80, and +100 mV. The right-handed charts are event histograms showing the dwell-time distribution for the most frequent block at each potential. Statistical data from the exponential curve fitting retrieved mean dwell-time values of 115 ± 13 ms (number of events = 34) for +40 mV, 37.12 ± 1.13 ms (number of events = 818) for +60 mV, 11.52 ± 0.40 ms (number of events = 1423) for +80 mV, 4.65 ± 0.12 ms (number of events = 1084) for +100 mV. Experiments were performed in 2 M KCl and 10 mM HEPES at pH 8.0. TGG-3′ (antisense). Mutated codons are underlined. The mutagenesis product was used to transform XL1-Blue supercompetent cells (Stratagene, La Jolla, CA). The cells were grown overnight at 37 °C in Luria broth medium containing ampicillin (100 μg mL−1, LB-amp). The sequence of the mutated gene was verified by DNA sequencing. Expression and Purification of E111A. The E111A αHL mutant was expressed and purified based on a previously described procedure.19 Briefly, the plasmid pT7-αHL-E111A was transformed into Escherichia coli BL21 (DE3) (Novagen, EMD Chemicals, Gibbstown, NJ) using a heat-shock technique. A small overnight culture (10 mL) grown at 37 °C in LB-amp was used to inoculate a 500 mL culture. Cells were grown at 37 °C to an OD600 of 0.7 in the same culture medium. Expression of the recombinant protein was then induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, Apollo Scientific, Cheshire, UK) to a final concentration of 0.5 mM. Following the addition of IPTG, cells were grown at 28 °C for another 4 h and harvested by centrifugation at 11300g at 4 °C for 20 min. The cell pellet was chemically lysed by resuspending in 25 mL of lysis buffer containing 50 mM HEPES, 25% w/v sucrose, 5 mM MgCl2, 1% w/v Triton X-100, 0.1 mg mL−1 lysozyme, and 10 units mL−1 Benzonase Nuclease (Novagen, EMD Chemicals) and incubated at 4 °C for 2 h with shaking. The cell debris was centrifuged at 16000g at 4 °C for 20 min. The supernatant was recovered, and the protein was fractionated by ammonium sulfate precipitation. After 1 h at 4 °C with stirring, the suspension was centrifuged at 27000g at 4 °C for 30 min. The pellet, which contained the E111A monomer, was recovered and resuspended in buffer A (25 mM MES, pH 6.0). The solution was dialyzed first against buffer A and then against buffer B (25 mM MES, 50 mM NaCl, pH 6.0), at 4 °C for 1 h, in Slide-A-Lyzer G2 Dialysis Cassettes (3.5 kDa MWCO, Thermo Scientific, Waltham, MA). The suspension was centrifuged at 20000g at 4 °C for 30 min, and the clear

voltages could arise from the fact that the E111A has a higher anion affinity at the constriction region. The higher voltage may lead to increased competition between sulfonates and Cl− ions to interact with Lys147, reducing the dwell time of the MPSANP. Moreover, according to previous theoretical studies,17,18 it is expected that the increase in analyte density in the nanopore leads to a decrease in the dwell time of the same analyte in the nanopore.

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CONCLUSIONS Here, we have demonstrated the formation of salt bridges between Lys147 and MPSA-NP and have observed these interactions at the single molecule level. The formation of salt bridges by the seven subunits of αHL with MPSA-NP led to a full block of the cross channel ionic current. Our results suggest that such interactions could be exploited to control opening and closing of cross-membrane channels for selective cell membrane permeation applications.

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EXPERIMENTAL SECTION

Materials. All chemicals used were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO), unless otherwise stated. Genetic Constructs and Mutagenesis. The E111A mutant was constructed by site-directed mutagenesis (QuickChange II SiteDirected Mutagenesis Kit, Stratagene, La Jolla, CA) using the WTαHL gene cloned into a T7 vector as a template. The mutagenic PCR primers were 5′-CCAAGAAATTCGATTGATACAAAAGCGTATATGAGTACTTTAACTTATGGA-3′ (sense) and 5′-TCCATAAGTTAAAGTACTCATATACGCTTTTGTATCAATCGAATTTCT15647


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pCLAMP 6 (Molecular Devices). With the E111A mutant, the number of events was sufficient. Therefore, the dwell time was determined by fitting the dwell-time histogram to a single exponential on data gathered on three experiments. Event searches were performed to obtain current amplitude histograms for each experimental condition. Blocked-pore current amplitudes were the peaks of the Gaussian fit to the amplitude histogram from each experimental condition. The traces were displayed in Origin 6.2 (OriginLab). Construction of MPSA-NP Model. MPSA-NP model was built based on the gold core of the X-ray structure of p-mercaptobenzoic acid (p-MBA)-modified gold nanoparticle,23 since both cores had similar average dimensions. The sulfonate ligands were built in Chem3D (CambridgeSoft Corporation, Cambridge, MA) and simulated with MM2 (Tripos Inc., St. Louis, MO). They were connected to the gold core by keeping the geometry of the Au−S1− C1 original vector of the p-MBA nanoparticle. The rest of the sulfonate was built from here using a Monte Carlo (MC) simulated annealing procedure of the first dihedral angle, while keeping the others in the lowest energy conformation (extended). This allowed the generation of an optimized packing of the sulfonate ligands at the surface of the MPSA-NP. Forty-four sulfonate ligands were connected to the gold core, as in the original p-MBA nanoparticle. Each MC step consisted of the random rotation of the dihedral S1−C1 of a random sulfonate ligand, and in the calculation of the energy (considering van der Waals interactions between ligands and the energy of the S1−C1 dihedral; parameters were taken from the GROMOS 54A6 force field24). A total of 104 MC runs of 250000 accepted or rejected MC steps were preformed in order to accomplish an annealing process. These MC steps were carried out at a different temperature per run, starting at RT = 20000 kJ mol−1 and applying a reduction factor of 0.9 per run. The initial temperature was selected in order to have a better convergence. Three replicas of this process were run with different random number seeds, which return all the same final results with minimal differences. The C++ program that was used to implement this process used double precision, given that single precision gave larger differences between replicas. pKa Calculations. The methodology used for the simulation of protonation equilibrium was previously described in detail.25 The methodology uses the Poisson−Boltzmann (PB) equation to compute the individual and pairwise terms in order to obtain the free energies of protonation changes, which are then used to perform MC sampling of protonation states; midpoint pKa's for all sites are then obtained from the respective titration curves. All calculations are done considering the alternative proton positions (tautomers) for all titration sites, as well as the nontitratable serine and threonine sites. Details on the chosen positions, model compound pKa, etc. were those previously described,25,26 except for the sulfonate ligands of the MPSA-NP (see below). The WT-αHL protein was inserted in a model mimicking a membrane. The atomic charges and “doubly thermal” radii used in the PB calculations were derived from the 53A6 force field of GROMOS,24 except for the MPSA-NP (see below for details). All PB calculations were performed with MEAD, version 2.2.9,27,28 using a temperature of 300 K, a solvent probe radius of 1.4 Å, and a Stern (ion exclusion) layer of 2.0 Å. The ionic strength was set to 2 M, and the dielectric constant of the solvent was set to 80, while those of WTαHL and MPSA-NP were set to 20.26 This value was found to give good results in the current parametrization.26 For the MPSA-NP, a two-step focusing cycle was used with consecutive grid spacings of 1.0 and 0.25 Å, while for the protein, a three-step focusing cycle was used with consecutive grid spacings of 2.0, 1.0, and 0.25 Å. The MC simulations were done using PETIT, version 1.5,25 which performs multisite titrations by performing trial changes of state of both single sites and pairs of sites. MC runs were performed at intervals of 0.2 pH units, each using 105 MC cycles, one cycle consisting of sequential state changes over all individual sites and also all pairs of sites with at least one interaction term above 2.0 pKa units. Set-up of Proteins and MPSA-NP for pKa Calculations. The WT-αHL protein conformation was taken from the PDB entry 7AHL. The atoms missing from the crystallographic structure were

supernatant was loaded onto a Mono S 5/50 GL column (GE Healthcare, Buckinghamshire, UK), attached to an Ä KTA Explorer system (GE Healthcare) pre-equilibrated with buffer C (25 mM MES, 30 mM NaCl, pH 6.0). All purification procedures were carried out at room temperature. The E111A protein was eluted with a linear gradient from 30 to 500 mM NaCl, beginning with buffer C and ending with buffer D (25 mM MES, 500 mM NaCl, pH 6.0). The fractions containing the E111A monomer were identified by SDS− PAGE analysis and applied on a Superdex 75 10/300 GL gel-filtration column (GE Healthcare) pre-equilibrated with 50 mM HEPES (pH 7.4) containing 150 mM NaCl. Fractions containing the E111A monomer were identified by SDS−PAGE, pooled, and concentrated with the buffer exchange to 10 mM HEPES and 2 M KCl (pH 8.0) using a Corning concentrator (Spin-X UF, 10 kDa MWCO, Corning, Amsterdam, The Netherlands). Sample purity was estimated by SDS− PAGE with the Coomassie Blue staining method. The resulting samples were stored frozen in aliquots with an additional 10% glycerol at −80 °C. MPSA-NP Synthesis. MPSA-NP was synthesized and characterized as previously described.10 Single-Channel Current Recordings. Single-channel current recordings were performed with planar lipid bilayers generated by the Montal and Mueller method,20 as described before.10,12 Briefly, the chamber used had two compartments, cis and trans, separated by a 25μm-thick Teflon film (Goodfellow Cambridge Ltd., Huntingdon, UK) containing an aperture 120 μm in diameter. The aperture was pretreated with 10% v/v hexadecane/n-pentane (HPLC-grade). 1,2Diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids, Alabaster, AL) in n-pentane (10 mg mL−1) was added to both compartments, filled previously with electrolyte solution containing 2 M KCl buffered at pH values of 2.8 in 5 mM MES, 6.9 and 8.0 in 10 mM HEPES, and 9.9 in 10 mM CAPS, depending on the experimental condition. After evaporation of n-pentane, the lipid bilayer was formed by lowering and raising the electrolyte level in one or both sides of the aperture. WT-αHL and E111A protein were added to the cis compartment in its monomeric form from a stock solution of the protein monomers made in distilled water and in storage buffer (10 mM HEPES and 2 M KCl (pH 8.0), 10% v/v glycerol), respectively. After a single protein nanopore was formed in the lipid bilayer, MPSANPs were added to the cis compartment at a concentration of 10 μg mL−1. Positive potentials of +60, +80, and +100 mV were applied across the lipid bilayer with Ag/AgCl electrodes. The cis compartment was grounded so that a positive potential indicates a higher potential in the trans compartment, meaning that positive charges are moving from the trans to the cis side of the lipid bilayer. Current fluctuations reflecting MPSA-NP interactions with a single membrane-inserted WT-αHL protein were recorded using an Axopatch 200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) connected to Ag/AgCl electrodes working in the whole-cell mode and filtered at 10 kHz with the built-in low-pass Bessel filter. Data acquisition was performed with a NI PCI 6221, 16-bit acquisition board (National Instruments, Austin, TX) at a sampling frequency of 30 kHz in LabVIEW 8.20 (National Instruments). Numerical analysis and graphing were preformed with the help of Origin 6.2 (OriginLab, Northampton, MA) and pCLAMP 6 (Molecular Devices). For the MPSA-NP interaction with the E111A mutant, current fluctuations were recorded with a patch clamp amplifier (EPC8, HEKA Elektronik, Lambrecht/Pfalz, Germany) connected to Ag/AgCl electrodes, through which positive potentials of +20 mV, +40 mV, + 60 mV, + 80 mV, and +100 mV were applied. Data was acquired with PATCHMASTER software (HEKA Elektronik) and analyzed with pCLAMP 10 (Molecular Devices). Recordings were collected using a 10 kHz low-pass Bessel filter at sampling frequencies of 30 kHz with a computer equipped with a LIH 8 + 8 A/D converter (HEKA Elektronik). All measurements were performed at room temperature. Data Analysis. The number of interaction events between the MPSA-NP and WT-αHL available with several types of experimental conditions was small. In this case, the statistical inference of rate constants was implemented using an alternative procedure to dwelltime histograms, as done previously.21,22 Data was analyzed with 15648


Langmuir reconstructed using Modeler 9v3.29 The protein was inserted into an association of equally spaced atoms (2.5 Å), with a diameter of 200 Å, trying to model a lipid bilayer with a thickness of about 40 Å.30 These atoms had a radius large enough (2.2 Å) to guarantee that the membrane was water-free. For the MPSA-NP, each terminal oxygen of the sulfonate ligand could have one of two low energy positions for placing the proton, as evidenced by quantum chemical calculations preformed with Gaussian 0931 (data not shown). These minima correspond to approximately 120° torsions of the C−S−OH dihedral, in the same direction of the neighboring oxygen atoms. (The other 120° torsion corresponds to higher energy and was not considered.) Thus, considering these two positions of the proton per sulfonate oxygen, each ligand has six tautomers to consider. In these tautomers, by approximation, the torsions of the above-mentioned dihedral were considered 120° exactly. The gold doubly thermal radius was calculated according to 12−6 Lennard-Jones parameters for face-centered cubic metal that reproduce densities, surface tensions, and interface properties with water and (bio)organic molecules, and that can be applied in several force fields, previously published.32 The sulfonate ligands and the sulfonate bound to gold mimicking the MPSA-NP, in each protonation state, were energy optimized using B3LYP/6-31(d) and PCM, considering the molecule inside a dielectric constant of four. For the gold atoms, the SDD effective core potential was considered. Single-point calculations were performed with B3LYP/cc-pVTZ and PCM on the geometry of sulfonate ligands and MPSA-NP. Electrostatic potentials in space were calculated in order to input then in the RESP fitting. The sulfonate ion-moiety partial charges calculated for the isolated ligands were imposed in the fitting of the sulfonate bound to two gold atoms. Additionally, the carbon C2 charge was set to zero, and the charge of the terminal sulfonate groups were constrained to 0 and −1, for the protonated and deprotonated form, respectively. The RESP fitting was made using a united atom approach, where only polar protons were considered. Since gold atoms are not homogeneous in the MPSA-NP (some are bound to two sulfonate ligands and others are bound to just one), their charges were set to zero, as an approximation. This approximation was confirmed to be reasonable by other RESP fitting procedures (data not shown). To run PB/MC calculations, we needed to have the pKmod for the tautomers of the sulfonate ligand. This was obtained by fitting the simulated global titration curve of a free sulfonate (with the same number of carbons as the one used in the MPSA-NP) to the experimental pKa (2.4) of this sulfonate. By changing the pKmod of the tautomers, the fitting can be achieved.

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ACKNOWLEDGMENTS

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REFERENCES

The MPSA-NP was a kind gift from the Supramolecular Nanomaterials and Interfaces Laboratory, École Polytechnique Fédérale de Lausanne (EPFL), Switzerland, headed by Dr. Francesco Stellacci. We acknowledge James Yates (ITQB) for proofreading the manuscript and for help with the artwork. We thank the reviewers for their remarks, which helped us to improve the original manuscript. This work was supported by Fundaçaõ para a Ciência e a Tecnologia (FCT) through Grant PEst-OE/EQB/LA0004/2011 and Project PTDC/QUI-QUI/ 099599/2008. E.C. was supported by the FCT Ph.D. Grant SFRH/BD/40431/2007. A.A. was supported by Grant POSDRU/89/1.5/S/49944. J.C.D. was supported by the FCT postdoctorate Grant SFRH/BPD/21873/2005. A.S.F. Oliveira was supported by Grant PTDC/QUI-BIQ/113446/ 2009. T.L. was supported by Grant PN-II-ID-PCCE-2011-20027.

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ASSOCIATED CONTENT

S Supporting Information *

Average titration curves, ionic current traces, and a summary of the interactions of Lys147 with MPSA-NP. This material is available free of charge via the Internet at http://pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail:[email protected]. Present Address ⊥

IBM Watson Research Center, Yorktown Heights, NY 10598.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. E.C. and A.A. contributed equally. Notes

The authors declare no competing financial interest. 15649


Langmuir

Article

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The Role of Lys147 in the Interaction between MPSA-Gold

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