Nanopore Analysis of β-Amyloid Peptide Aggregation Transition

Feb 10, 2011 - β-Amyloid 42 (Aβ42) is the predominant form of the amyloid peptide, which is found in the plaques of the brains of Alzheimer's (AD) p...
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Nanopore Analysis of β-Amyloid Peptide Aggregation Transition Induced by Small Molecules Hai-Yan Wang,† Yi-Lun Ying,† Yang Li,† Heinz-Bernhard Kraatz,‡ and Yi-Tao Long*,† †

Shanghai Key Laboratory of Functional Materials Chemistry & Department of Chemistry, East China University of Science and Technology, Shanghai 200237, China ‡ Department of Chemistry, The University of Western Ontario, 1151 Richmond Street, London, Ontario, N5A 5B9, Canada

bS Supporting Information ABSTRACT: β-Amyloid 42 (Aβ42) is the predominant form of the amyloid peptide, which is found in the plaques of the brains of Alzheimer’s (AD) patients and is one of the most abundant components in amyloid aggregates. Information of the Aβ42 aggregation states is essential for developing an understanding of the pathologic process of amyloidoses. Here, we used R-hemolysin (R-HL) pores to probe the different aggregation transition of Aβ42 in the presence of β-cyclodextrin (β-CD), a promoter of Aβ42 aggregations, and in the presence of Congo red (CR), an inhibitor of aggregations. Analyzing the characteristic transit duration times and blockade currents showed that β-CD and CR have opposite effects on the aggregation of Aβ42. Translocation events of the monomeric Aβ42 peptide were significantly lower in amplitude currents than protofilaments, and protofilaments were captured in the RHL nanopore with a longer duration time. CR binds to Aβ42 and its peptide fibrils by reducing the aggregated fibrils formation. In this process it is assumed CR interferes with intermolecular hydrogen bonding present in the aggregates. In contrast to CR, β-CD promotes the aggregation of Aβ42. These differences can readily be analyzed by monitoring the corresponding characteristic blockade events using a biological R-HL nanopore.

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anopore systems have shown to be a sensitive tool for bimolecular detection at the single-molecule level and have been applied to DNA sequencing,1,2 to probe the secondary structure of peptides,3,4 and to monitor protein binding events.5 Briefly, as a molecule translocates through a nanopore which separates two electrolyte-filled chambers, it will prevent ions from passing through the nanopore. The result is a modulation of the ion current. The current response and the time of the modulation are related to the characteristics of translocation events. The analysis of current blockages involving transit events of peptides has allowed us to gain insight into the effects of secondary structure and even get information about folding events.6 Further, nanopores have been used as a nanoreactor to examine chemical reactions at the single-molecule level based on the blockage properties.7 Recently, advances in small molecule detection using biological protein nanopores8 demonstrated the possibility to examine the effects of drugs on pathogenic proteins. A commonly used biological protein nanopore is Rhemolysin (R-HL) which self-assembles in the lipid bilayer from seven identical subunits to form a transmembrane pore with a constriction of about 1.4 nm in diameter.9 Here, we utilized the r 2011 American Chemical Society

R-HL pore to analyze the transient events of the Aβ42. As illustrated in Figure 1, Aβ42 blocks the ion current passing through the R-HL in the presence of a promoter/inhibitor (βcyclodextrin/Congo red) resulting in the characteristic current traces. Aβ42 is a 42 amino acid peptide that appears to be one of the main constituents of amyloid plaques in the brains of people suffering from neurodegenerative disease. Aβ42 readily aggregates into fibrils and plaques, and in its aggregated form, Aβ42 is toxic to neuronal cells.10 Fibrils and plaques are associated with disease states.11 The fibrillogenesis and plaque formation of Aβ in brain is implicated as one of the hallmarks in the pathogenesis of Alzheimer’s disease (AD).12 Results from X-ray crystallography13 and solution-state NMR spectroscopy14 studies substantially showed two stacked, parallel or antiparallel βsheets that perpetuate along the fibril axis existing in Aβ protofilaments.15 Recently, size exclusion chromatography was used to separate monomers, protofibrils, and fibrils of Aβ42.16 Received: November 12, 2010 Accepted: January 22, 2011 Published: February 10, 2011 1746

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Figure 1. The representation of the R-HL pore inserted into the planar lipid bilayer. Aβ42 in the presence and absence of β-cyclodextrin (Aβ42-CD) or Congo Red (Aβ42-CR) were added from the cis chamber, respectively. (a) Representation of the collision behavior of Aβ42-CD. (b) Representation of the blockage behavior of Aβ42. (c) Representation of the blockage behavior of the translocation event of Aβ42-CR. i is equal to the difference between the open pore current and the average amplitude of the blockage current. Similarly, t, the duration time of the blockage, is essential in the current trace.

Figure 2. Scanning electronic microscopy (SEM) image observation of the different states of peptides: (a) Aβ42-CR staining for 1 h and the arrow indicates the visible protofibrils in Aβ42-CR, (b) mature Aβ42 fibrils, and (c) Aβ42-CD in unordered aggregation morphology.

The protofibril is a structural subunit of a mature fibril, and fibril formation gives rise to the plaque in the AD patients’ brains. Moreover, the protofibrils are proposed kinetic intermediates for fibrils and could bind to Congo red.17 The underlying structural unit of the protofibril is a cross-β crystallite that is 7.6 nm long and 4.2 nm thick and made up of four β-sheet.13 On the other hand, the Aβ42 protofilament is smaller in physical dimension18 (two stacked, parallel β-sheets that perpetuate along the fibril axis with an average spacing of ≈45 Å19), which is similar to the diameter of polyadenine.1 The structure of Aβ42 and its fibrils have been studied by a range of microscopes (atomic force microscopy (AFM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning tunneling microscopy (STM)).20-23 Here, SEM was used to obtain the bulk information of Aβ42 at different aggregation states (Figure 2). Aβ42 adopts a β-sheet conformation in aqueous solution19 and aggregates into amyloid fibrils (Figure 2b) while Congo red (CR) could inhibit the aggregation of Aβ42 (Figure 2a). We assessed the effects of β-cyclodextrin (β-CD) on Aβ42 morphology by SEM images (Figure 2c). The SEM images show that coincubation of Aβ42 with β-CD induces substantial morphological changes in the Aβ42 structure compared to the sample of the Aβ42 alone. β-CD promotes formation of short, thick Aβ42 fibrils with an nonordered aggregation morphology. In addition, circular dichroism spectroscopy24,25 has been used for monitoring the β-sheet-like structure in the aggregated protein26 and shows a negative peak around 217 nm

for CR binding to Aβ42.17 While these results provide a “static” image of Aβ42, information about a structural transition including the aggregation of Aβ42 remains a challenge. In aqueous solution, molecular dynamic simulations provide insight into a conformational change of Aβ4227,28 and indicate that residues 17-42 are involved in sheet formation.19 The work described here focuses on the study fibril aggregation and fibrillogenesis inhibition of Aβ42 employing the R-HL nanopore at the single molecule level. In our investigation, we focus on the effects of β-cyclodextrins (β-CD) as promoters and Congo red (CR) as inhibitors of fibril formation. β-CD possesses seven glucose subunits having the glucopyranoside units locked into a cone-ring structure. The inner diameter of the β-CD cavity is about 0.6-0.65 nm that can be exploited as a drug carrier and has value in foodstuff and medicine.29,30 A recent reports showed β-CD promotes the aggregation of Aβ42,31 but the nature of the interaction between β-CD and Aβ42 remains elusive.32 Similarly, the interactions between Aβ42 and CR, a histological dye for amyloid detection, is not clear.17 What is clear is that CR staining inhibits the aggregation of the protein fibrils and reduces the toxicity of the amyloid. Our own results monitoring the transient translocation events reveal that CR inhibits the aggregation of Aβ42, presumably by weakening the hydrogen bonding interaction leading to β-sheets stacks, which in turn allows translocation of monomeric Aβ42 through the R-HL nanopore. In contrast, βCD is considered to promote the aggregation of Aβ42 and poses a risk for therapeutic applications.33 In essence, these two small 1747

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Table 1. Blockage Events in the Presence and Absence of CR or β-CDa analyte

t1 (ms)

t2 (ms)

τ1 (ms)

Aβ42

0.125 ((0.125) 1.25 ((1.25) 0.51 (0.01)

Aβ42-CR

12.5 ((12.5)

τ2 (ms)

A1/A2

0.28 (0.10)

i2 (pA)

ι1 (pA)

ι2 (pA)

10.07 (0.10) 0.83 ((0.07) 21.25 ((1.25) 96.25 ((1.25) 23.30 (0.60) 96.58 (0.20)

1.25 ((1.25) 24.38 (0.05) 4.35 (0.02)

Aβ42-CD (16.5 h) 0.125 ((0.125) n/a

i1 (pA)

n/a

1.11 ((0.04) 18.25 ((1.25) 26.25 ((1.25) 18.06 (0.07) 25.47 (0.30) n/a

23.25 ((1.25) n/a

26.82 (0.40) n/a

Values of Aβ42 were measured at 25.0 ( 0.5 °C. t1, t2, i1, and i2 in parentheses are represented by the half value of the bin width in the histogram. The values in parentheses for τ1, τ2, ι1, and ι2 are the errors of fit measurements. A1 and A2 are the number of current events forming the population, and the values in the parentheses are based on three separate experiments. n/a means not available. a

molecules cause the opposite results with respect to Aβ42 aggregation. In our study, we analyze the frequencies and current amplitude of the blockade/translocation events of Aβ42 in the presence of the aggregation inhibitor or promoter and interpret differences in these event terms of structural changes of Aβ42 occurring at the single molecule level, which are impossible to obtain by bulk spectroscopic techniques. Our results demonstrate that we can distinguish between different states of aggregation and show the utility of the nanopore for the evaluation of peptide conformations at the single molecule level.

’ METHODS R-Hemolysin (R-HL) was purchased from Sigma-Aldrich (St. Louis, MO) and used without purification. Aβ42 with a purity of 98% by HPLC was purchased from GL Biochem (Shanghai, China) Ltd. and used as received. The amino acid sequence of Aβ42 is NH2-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-GluVal-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-GlySer-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-ValIle-Ala-COOH. β-CD and CR were purchased from the Sinopharm Chemical Reagents Co., Ltd., (Shanghai, China) and were used as received. In the assay of exploring the self-aggregation tendency of Aβ42, Aβ42 was placed at 4 °C in the refrigerator. Diphytanoyl-phosphatidyl-choline in CHCl3 was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Unless otherwise noted, all other chemicals were of reagent grade. All solutions were prepared by using ultrapure water (18.2 MΩ cm-1; Mili-Q, Millipore). The lipid bilayers were created by applying 30 mg/mL diphytanoyl-phosphatidyl-choline in decane (g99, Sigma-Aldrich St. Louis, MO) to a 150 μm orifice in a 1 mL Delrin cup integrated into a lipid bilayer chamber (Warner Instruments, Hamden, CT) filled with 1 M KCl and 10 mM phosphate buffer (pH = 8.0).34,35 A bilayer was deemed stable by monitoring its resistance and capacitance. The two compartments of the bilayer cell are termed cis and trans chambers, where the trans chamber is defined as virtual ground. The potential was applied at 100 mV from the cis chamber by the Ag/AgCl electrode. The experiments were run under a voltage-clamp condition using a Chem-Clamp amplifer (Dagan Corporation, Minneapolis, MN). Currents were filtered at 10 kHz by DigiData 1440A (Axon Instruments, Forest City, CA) hardware and recorded by a PC running PClamp 10.2 (Axon Instruments, Forest City, CA). The R-HL was injected adjacent to the aperture in the cis chamber, and pore insertion was determined by a well-defined jump in current value. The electrolyte solution used throughout all bilayer measurements was 1.0 M KCl in 10 mM phosphate buffer (pH = 8). Once a stable single-pore insertion was detected, 10 μL of analyte was added to the cis chamber, proximal to the aperture. The concentration of CR used is 200 μM, and the

Figure 3. (a) Representative traces of Aβ42 added from the cis chamber. (b) The scatter plot of Aβ42 with full blockage events. (c) The histogram of blockade currents of Aβ42 shows two populations. P1 with the high-amplified current concentrated at 96.58 ( 0.23 pA and P2 related to the currents of bumping events concentrated at 23.41 ( 0.08 pA. The histogram is fitted into a Gaussian function. (d) The histogram of duration time of Aβ42 at a high-amplitude current (P2) and can be fitted by a exponential function. All experiments were recorded at an applied transmembrane potential of 100 mV.

concentration of β-CD is 2 mM. Data were collected using a threshold level that was at least 10 pA from the baseline open pore current. Since the blockage current lever under 10 pA could be decoupled with the noise baseline, analysis of all data was performed by ClampFit 10.2 (Axon Instruments, Forest City, CA) and OriginLab 8.0 (OriginLab Corporation, Northampton, MA). The SEM images were performed by an FEI-Sirion 200 field emission scanning electron microscope (FEI Co., Hillsboro, OR) with 20 kV operating voltage. The data were recorded such that events (ionic current blockages) that were larger than a threshold value of 10 pA less than the nonobstructed pore were saved to memory.36 These events, defined by any current blockage that exceeded the threshold value,37 were strung together in series and each event was analyzed.38 The raw current data events were analyzed by measuring both the amplitude of the ionic current blockage and the duration of the current blockage. Thus, each event consists of two data points described as blockage current amplitude and duration. On the basis of previous literature,39 the blockage of ionic flow under applied potentials is the result of large linear biopolymers transiting the pore, resulting in a restriction in ionic 1748

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Figure 4. (a) Representative current traces of Aβ42-CR adding from the cis chamber. (b) The scatter plot of Aβ42-CR; the green circled area indicates events at currents of less than 20 pA, defined as long-lived events. The corresponding green fitting line is carried out by exponential function shown in part d. The red circled area indicates events with currents larger than 20 pA, defined as short-lived events. An exponential function is used to fit the corresponding durations and gives a red fitting line as shown in part d (inset figure). These events are responding to two different states of Aβ42 inhibited by CR. (c) The histogram of blockade currents of Aβ42-CR shows two populations with a high-amplitude current concentrated at 25.47 pA and the low-amplitude current concentrated at 18.06 pA. (d) The histograms of the duration times are fitted by the exponential functions as the green line for the blockage current centered at 18.06 pA and the red line for blockage current centered at 25.47 pA. All experiments were performed at an applied transmembrane potential of 100 mV.

flow. Each event consists of two parameters, namely, blockage current and duration time; thus, a convenient method to view the data is via a scatter plot. In addition, the population distribution of blockage currents can be determined from a histogram and the curve is fitted into a Gaussian function which describes the population of events. In this manuscript, the peak of the fitted Gaussian distribution is defined as ι while the peak current of the histograms is defined as i. Likewise, a histogram of the blockage duration time can be fit into an exponential decay function representing the population lifetime. The exponential fit of the decay provides the lifetime, τ (milliseconds) and the time of the most probable translocation defined as time of peak t (milliseconds). The errors of i and t are represented by the half value of the bin width in the histogram. The event currents lower than 10 pA were judged to represent the transient collision of Aβ42 and ignored in our data analysis. Additionally, A1 and A2 are the number of events forming the population and used as the frequency behavior of analyzed peptides.

’ RESULTS AND DISCUSSION The reported 3D structure of Aβ42 fibrils indicates that the residues 17-42 formed a intermolecular core structure and the hydrophobic surface formed by residues 17-20.40 The monomer of Aβ42 is composed of antiparallel β-sheets with residues

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18-26 and 31-42 forming β-strands, which interact through H-bonding interactions.19 Aβ42 could be electrophoretically driven into the pore from the cis chamber by the applied transmembrane potential of 100 mV, resulting in a shift of blockade current for Aβ42 as a function of different aggregation states. The charge transport characteristics are dominated by the narrowest constriction, but the vestibule may help to orient the Aβ42 molecules entering the pore. The addition of a solution of Aβ42 (1.55 μg/mL in phosphate buffer) results in transit events that cause a significant reduction of the ion current through the R-HL pore from about 100 to 5 pA (Figure 3a). The analysis of the current distribution shows two types of transient current blockages, defined as P1 and P2 (Table 1 and Figure 3b,c). As illustrated in Figure 3b, the events with low-amplitude current have shorter durations while the events with a high-amplitude current have longer durations. The former events are not voltage dependent and are interpreted as collisions of monomeric Aβ42 or self-aggregated fibrils with the cis entrance of the R-HL pore. Curiously, a few of the bumpinginduced blockages last approximately 120 ms. It is presumably caused by monomeric Aβ42 which might self-organize within the vestibule. Since the cis entrance of the R-HL nanopore is about 2.6 nm in diameter and the constriction is about 1.4 nm in diameter, we speculate that the Aβ42 protofilaments may be captured for a few microseconds by horizontal access into the vestibule of the R-HL pore and block the pore entrance and thus with a characteristic full amplitude blockage. Our results are consistent with the dimension of Aβ42 protofilaments, which is larger than the diameter of the R-HL’s constriction. The events with high-amplitude current might be attributed to the unfolded Aβ42 protofilaments traversing through R-HL or Aβ42 protofilaments entering the vestibule and remaining there for tens of microseconds to several milliseconds (Figure 3d). The Aβ42 monomers may enter into the cis entrance in a parallel fashion by its negative charged surface41 and then pass through the pore in a few milliseconds. Upon addition of an equimolar amount of Aβ42 and CR, which was premixed for 1 h in the cis chamber, there is an increase in translocation events and corresponding long duration times are observed (Figure 4a). In the presence of Aβ42-CR, the amplitude of the translocation current decreased. On the basis of the traces shown in Figure 4a, the characteristic current observed for these events is about 29 pA with shorter duration times. Figure 4b shows that the number of captured events (i2 = 96.58 pA) due to fibrils dramatically decreases in the presence of CR. Two main models for the molecular interactions of CR with Aβ42 were proposed before.17,42-45 One model suggests electrostatic interactions result from the negatively charged sulfate group of CR interacting with the positively charged amino acid residues of Aβ42.44 In addition, hydrophobic contributions were proposed due to insertion of CR into the groove on the β-sheet surface of the protofilaments.45 Another model postulated that CR intercalates between two antiparallel β-sheets in a parallel fashion. Residues 15-23 are suggested as the location of CR binding to antiparallel β-sheet strands,42 and particularly, it was proposed, interactions between the aromatic rings of Phe19 and the two phenyl rings of CR. According to the R-HL pore analysis, both models are reasonable. Thus, when CR is inserted into the antiparallel β-sheets, it potentially enlarges the distance between the carbonyl oxygen and the amide nitrogen to about 3.6 Å,42 which is larger than the native distance of 1.9 Å and disrupts the 1749

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Figure 5. (a) Representative typical current traces of Aβ42-CD. (b) The scatter plot of Aβ42-CD (incubated at 16.5 h) showing the bumping events only. (c) The histogram of blockade currents of Aβ42-CD fitted by a Gaussian function. (d) The histograms of the duration time of Aβ42-CD fitted by an exponential function. Experiments were performed at an applied transmembrane potential of 100 mV.

hydrogen bonding between two β-sheets of Aβ42.25 Since the length of the β-sheet strand of Aβ42 and CR are about 1.9 nm,17 this large complex could not translocate through the R-HL nanopore by horizontal access to the pore and is supported by our observations of short-lived events that might be due to dissociated monomeric Aβ42 upon the addition of CR. In the case of Aβ42 protofilaments, a full current amplitude reduction was observed when the protofilaments occupied the RHL pore. For Aβ42-CR, two populations of events were observed. On the basis of a previous study,46 two populations were also observed for R-helical peptide translocating through the RHL pore. For Aβ42-CR, the major population has an i1 of 18.06 pA (defined as long-lived events), and the minor population has an i2 of 25.47 pA (defined as short-lived events) as shown in Figure 4c. A scatter plot shows the distribution of blockage events and gives a visual indication of the relative sizes of the two populations (Figure 4b). The distribution histograms of the duration time are fitted by exponential functions that result in lifetimes of τ1 of 24.38 ms and τ2 of 4.35 ms, respectively (Figure 4d). The short-lived events with τ2 might be assigned to monomeric Aβ42 to translocate through the pore, as the scatter plot distribution is more concentrated than the long-lived events. The long-lived events could be attributed to the horizontally captured Aβ42 monomer organizing itself in the vestibule before transit occurs. The process of long-lived events is believed to occur through the interaction between CR and protofilaments in the second model. As shown in Figure 3b, this type of longlived event was also observed when Aβ42 translocates through the nanopore. However, the number of these long-lived events in the Aβ42 assay was much fewer than that of the Aβ42-CR assay. In addition, it could be further confirmed that CR inhibits the Aβ42 peptides’ aggregation. In order to exclude the influence of CR giving rise to these events, experiments were carried out with CR under the same

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Figure 6. Blockade events vs interaction time curve of β-CD with Aβ42 at equimolar concentrations. The results indicate aggregation of Aβ42. The count number of events is the occurrence of blockade events within 1 min. The curve was fitted to a simple polynomial equation.

conditions. Current traces and duration time distributions are shown in the Supporting Information and give rise to the distinctly different i-t behaviors with different ion currents and time distributions. It is necessary to investigate the effects of β-CD on the R-HL pore before studying the effects of β-CD on Aβ42. β-CD is a cyclic molecule with a hydrophobic cavity. At micromolar concentrations β-CD could enter the R-HL pore by adding from the trans chamber, which produces a reversible partial blockage of the ion current and reduces the single-channel conductance to less than half of the value in the absence of β-CD.47 It was considered that the internal barrier of the cis entrance was hindered by the side chains of Glu-111 and Lys-147.48 Thus, β-CD had no effect on R-HL when it was applied from the cis compartment. Only when entering from the trans side can β-CD reach its binding site in the lumen of R-HL. Bayley and Gu primarily investigated the interaction of β-CD with R-HL as a function of voltage and pH.47 Next in our study, we probed the effects of β-CD on Aβ42 and its effects on self-association and translocation events. For this purpose, we directly incubated βCD with Aβ42 before adding it to the cis chamber for analysis by an R-HL pore. We carried out the measurements under the same experimental conditions as described before for Aβ42-CR. Aβ42 was incubated with β-CD at various incubation times ranging from 0.5 to 16.5 h at room temperature and then analyzed by nanopore measurements. Our results show that the events with high-amplitude current display a strong time-dependent decrease (Figure 5b). Increasing the incubating time of β-CD with Aβ42 results in a decreasing number of translocation events (Figure 6), while the proportion of partial bumping events is increased (Figure 5b). By contrast, in the absence of β-CD, the proportion of bumping events caused by the collision of Aβ42 fibrils is about 40% (Figure 3b). This result is interpreted in terms of the Aβ42 peptide aggregations promoted by β-CD due to the interaction between aromatic side chain of Phe residues and β-CD.49 A distinct change occurred at an amplitude current larger than 50 pA after adding Aβ42-CD from the cis chamber (Figure 5c). On the basis of these results, we deduce that the Aβ42 aggregated procedure is promoted by β-CD, which results in a reduction of the translocation events since the aggregated Aβ42 is too large to 1750

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Analytical Chemistry translocate through the nanopore (Figure 5a). AFM and cell viability studies confirm that β-CD promotes Aβ42 peptide aggregation.33 Previous studies50,51 showed that Aβ42 has a tendency to selfaggregate into fibrils. In order to study the self-aggregation tendency of Aβ42 in our experiments, we compared the nanopore results of Aβ42 at the first day and the 25th day as shown in Figure 3b and Figure S4 in the Supporting Information, respectively. During this period, the proportion of bumping events increases due to the self-organized Aβ42 aggregate. However, the number of bumping events for Aβ42 at the 25th day is less than the number of Aβ42-CD. Consequently, the self-aggregation is not the dominant factor when analyzing the assay of Aβ42-CD.

’ CONCLUSIONS Like most other potential brain drugs, CR suffers from low blood-brain barrier permeability and raises concern for medical applications in recent years.52,53 β-CD, on the other hand, has been used to deliver drugs and has found application in clinical treatments.54 However, considering the interactions between β-CD analogues and peptides,55,56 which may lead to structural changes, there is reason for concern. In the present study, we analyzed conformational changes caused by CR and β-CD on Aβ42 using a R-HL nanopore. While in essence our studies provide information about translocation or bumping events, these are related to conformational changes caused by the interactions of CR and β-CD with Aβ42. The frequency of translocation events is a key distinguishing factor. The use of a nanopore provides a real-time approach to monitor peptide association by intermolecular hydrogen bonding interactions. Thus, our studies contribute to the understanding of Aβ42 inhibition and their aggregation in real-time at the singlemolecule level. Furthermore, our results suggest that it should be possible to evaluate the effects of small molecules on the peptide structure and aggregation, which has significant potential for drug screening. This highlights the capacity of nanopore studies as a discovery tool to study intermolecular interactions at the single molecule level with potential applications in the design of drug screening assays. ’ ASSOCIATED CONTENT

bS

Supporting Information. Representative current recording traces, distribution histogram of duration time, distribution histogram of current showing the detection of CR with the same experimental conditions; the current and duration histogram of Aβ42 incubated with β-CD at different times; and the scatter plot of Aβ42-CD conducted on the 25th day. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (Grants 20875030 and 91027035), the Fundamental Research Funds for the Central Universities (Grant No. WK 1013002), the Ministry of Health (Grant 2009

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ZX 10004-301), and the Shuguang Project of Shanghai (Grant 07SG36). This research was partially supported by NSERC.

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