NANO LETTERS
Structure of Peptides Investigated by Nanopore Analysis
2004 Vol. 4, No. 7 1273-1277
Todd C. Sutherland,†,‡ Yi-Tao Long,†,‡ Radu-Ioan Stefureac,†,‡ Irene Bediako-Amoa,† Heinz-Bernhard Kraatz,*,† and Jeremy S. Lee*,‡ Department of Chemistry, UniVersity of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada, and Department of Biochemistry, UniVersity of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada Received April 21, 2004; Revised Manuscript Received May 13, 2004
ABSTRACT Bacterial r-hemolysin forms a pore in a lipid membrane with well-behaved single-channel conductance so that the transit of single molecules can be studied. Here we demonstrate that the structure of individual peptides can be analyzed. Peptides were synthesized containing repeats of the sequence (Gly−Pro−Pro), which exist as mixtures of single, double, or collagen-like triple helices. The structure of individual peptides could be identified as they traverse the pore based on signature transit times and blockage currents.
A current goal of nanotechnology is the analysis of single molecules; not only because of increased sensitivity but also because bulk sampling leads to an averaging of the information.1 For example, by analyzing single molecules the thermodynamics and kinetics of unfolding or self-assembly can be elucidated. Single-molecule detection systems include atomic force microscopy, video fluorescence microscopy and “laser tweezers”.2-6 Nanopores or ion channels can also be used to analyze single molecules because there is a change in the current as the molecule passes through or interacts with the pore. Bacterial R-hemolysin (R-HL) a heptameric protein that spontaneously assembles in a lipid membrane has proven useful. The crystal structure of R-HL was solved in 1996 and consists of a vestibule with a diameter of about 30 Å which leads into a constricted pore with an internal diameter of 15 Å (Figure 1a), thereby allowing relatively large linear polymers to transport.7 In a planar lipid bilayer membrane, R-HL has well-behaved single-channel conductance. In the pioneering work of Deamer, Branton, and colleagues, single-stranded DNA or RNA was studied in detail and the effects of length, sequence, temperature, and applied potential on the transport kinetics have all been reported.8-13 Of particular importance was the observation that poly(A) tracts gave larger blockade currents than poly(U) tracts, demonstrating the potential of this technique for nucleic acid sequencing. There has been much less work on doublestranded DNA (ds-DNA) transportation through a nanopore14,15 and most involves either tethered systems16-18 or * Corresponding author. † Department of Chemistry. ‡ Department of Biochemistry. 10.1021/nl049413e CCC: $27.50 Published on Web 06/02/2004
© 2004 American Chemical Society
single-stranded DNA (ss-DNA) that forms hairpin loops.19,20 It was shown that the DNA hairpins had to unfold in order to transit the pore. The less stable the hairpin, the faster the rate of transit. Therefore, the nanopore technique also offers the possibility of gathering structural information.21 Here we demonstrate that peptide transport through nanopores can also be analyzed. As shown in Figure 1b, peptides were synthesized containing one, two, or three repeats of the collagen-like sequence (Gly-Pro-Pro)n (n ) 1 P1, n ) 2 P2, and n ) 3 P3) that are terminated in ferrocene (Fc). Extended repeats of this sequence form a stable collagen triple helix with a crystallographically determined22 diameter of 12 Å, which is expected to give a large blockade current. The shorter repeats also form dimers and monomers, which should exhibit smaller blockade currents and shorter transit times. Originally, the ferrocene groups were added for surface electrochemical studies, but by electrochemical oxidation, to form Fc+, the individual peptides become charged so that the resulting tertiary structures are electrostatically retarded through the nanopore under an applied positive bias. In general, the two transport parameters of blockade current (iblock) and duration (tblock), see Figure 1c, yield sequence and structural information, which would be difficult to obtain by bulk spectroscopic techniques, such as CD or NMR. R-Hemolysin (R-HL) was purchased from Sigma (St. Louis, MO) and used without purification. Diphytanoylphosphatidyl choline in CHCl3 was purchased from Avanti Polar lipids. Collagens, (Gly-Pro-Pro)n (n ) 1 P1, n ) 2 P2, and n ) 3 P3) that are terminated in ferrocene (Fc) were synthesized and the characterization of these compounds is described elsewhere.23 Unless otherwise noted, additional
Figure 1. Representation of the R-hemolysin pore, the structure of the peptides tested, and example current transients as the peptides interact with the pore. (a) A schematic of the structure of the R-HL inserted into a planar lipid bilayer with a linear collagen-like peptide traversing the 15 Å diameter pore. (b) The structure and labeling scheme of the collagen-like peptides used in this study. Typical current transients of peptides P1, P2, and P3 are shown by the open blue circles. (c) Idealized blockage behavior represented by the black line iblock is equal to the difference between the baseline open current and the average amplitude of the blockage current. Similarly, the duration of the blockage, tblock, was found from the idealized current trace.
reagents were purchased from Aldrich and used as received. Millipore water (18 MΩ cm) was used in all solutions. CT7n was obtained from the Alberta Peptide Research Institute. Once a stable single pore insertion was detected, 10 µL of peptide (1 mM) solution was added to the trans chamber, proximal to the aperture, and 100 mV potential was applied. The experiments were carried out at 22 ( 1 °C. Analysis of all data was performed by ClampFit 9.0 (Axon Instruments, Foster City, CA) and Origin 7.0 (OriginLab Corporation). The data set was converted to a matrix of blockage current and blockage duration to make contour plots. Unless otherwise noted, the bin increments were set at 0.5 pA and 0.025 ms for the blockage current and the duration, respectively. The blockage current population was obtained by fitting the blockage current distribution with the Gaussian function. The lifetime data was obtained by fitting the blockage duration distribution with a double exponential function. The synthesis and structural characterization of these peptides have been published elsewhere.23 Each peptide has a distinct circular dichroism (CD) spectrum with P2 and P3 having a negative band at 209 nm, which is characteristic of collagen-like helices,22,24 whereas P1 shows essentially no secondary/tertiary structure. From the magnitude of the negative band at 209 nm it was estimated that P3 is mostly in a collagen-like conformation, P2 adopts a partial collagenlike structure, and P1 is in a random conformation. Although CD can provide some structural information, the spectra are an average of all the species that are present. Therefore, detailed analysis is difficult when multiple species coexist. On the other hand, the passage of a molecule through a nanopore is a single-molecule event, which can be 1274
described in terms of the magnitude of the blockade current and its duration. An unobstructed R-HL pore has a constant current of 100 pA under the conditions used in the peptide analysis. Figure 1c shows a series of typical events for the transit of P1 to P3 through a single R-hemolysin pore. For each peptide, thousands of events were measured and analyzed as a contour map of blockade duration versus blockade current shown in Figure 2. The use of 2-dimensional (2-D) binning in the contour plots allows for analysis of coupled parameters (iblock and tblock).25 1-D binning of blockage duration or blockage current can be misleading as short duration events may only be occurring at large blockage current or vice versa. Treatment of the data in a 2-D array, represented by the contour plots of Figure 2, allows for a more precise description of the population. For P1 (Figure 2a) there are two populations; the major one, labeled 2, has an iblock equal to -52.3(0.1) (standard deviation) pA and the minor population, labeled 1, has an iblock of about -45 pA. The iblock population is a measure of the degree to which the peptide occupies the narrowest portion of the pore. A horizontal profile showing the distribution of iblock events is at the top of Figure 2a and gives a visual indication of the relative sizes of the two populations. The vertical profile of the major blockage duration is shown at the right of Figure 2a and is fit to two exponentials that result in lifetimes of τ1 of 72(10) µs and τ2 of 408(16) µs. The same analysis was carried out on P2, P3, and oxidized P3, and the curve fitting results are shown in Table 1. In general, there were too few events for the minor populations to allow detailed analysis and, therefore, only the lifetimes for the major populations are described. The shorter lifetime parameter (τ1 in Table 1) is attributed to the time necessary to organize the peptide in Nano Lett., Vol. 4, No. 7, 2004
Figure 2. Contour plots of current transients of P1 (a), P2 (b), P3 (c) and oxidized P3 (d). The horizontal and vertical red lines represent cross-sections of the contour plots. Each horizontal cross-section was fit to a Gaussian distribution, and each vertical cross-section was fit to a double exponential function. (a) P1 has a blockage current (iblock) distribution positioned at -52.3(0.1) pA with a width at halfmaximum (w1/2) of 3.1 pA, and the blockage duration (tblock) distribution is described by lifetimes of 72(10) µs and 408(16) µs. (b) P2 has a iblock distribution positioned at -57.6(0.2) pA with a w1/2 of 7.1 pA, and the tblock distribution is described by lifetimes of 39(14) µs and 547(16) µs. (c) P3 has a iblock distribution positioned at -75.4(0.2) pA with a w1/2 of 6.2 pA, and the tblock distribution is described by lifetimes of 33(5) µs and 591(17) µs. (d) P3 oxidized has an iblock distribution positioned at -69 (1) pA with a w1/2 of 7.4 pA, and the tblock distribution is described by lifetimes of 83(20) µs and 649(15) µs. Bin widths used for a-d: Current blockage 0.5 pA and blockage duration 0.025 ms. Values in parentheses are the standard deviations.
the vestibule before transit occurs. τ2, on the other hand, is the lifetime for the peptide to cross the channel and, as expected, τ2 is dependent on the structure and length of the peptide. As shown to the right of Figure 2, there are four major structures that the peptides might adopt, and these can be identified based on iblock and τ2. P1, peak 1, is identified as a single linear peptide with a small iblock of about -45 pA. The major peak with a blockage current centered at -52.3(0.1) pA and with a lifetime of 408(16) µs (population 2 of Figure 2a) can be assigned to a monomeric peptide folded onto itself into a U-shape during transit. For P2 (Figure 2b) there are minor populations with iblock values from about -45 pA to -55 pA, which are assigned to linear and folded monomeric peptides. The major peak for P2 (populaNano Lett., Vol. 4, No. 7, 2004
tion 3 of Figure 2b) exhibits a small increase in iblock compared to P1 with a peak centered at -57.6(0.2) pA, but the lifetime of 547(16) µs is increased substantially, which is consistent with a dimeric linear molecule. For P3 (Figure 2c) the major population (population 4) has a larger blockade current of -75.4(0.2) pA and a longer lifetime of 591(17) µs, consistent with a structure that is a linear collagen-like triple helix. Furthermore, many events are seen at short durations spanning the full blockage current range that can be assigned to dimeric and monomeric linear-extended conformations or even folded monomers as in the case of P1 (populations 1, 2, and 3). These populations are of low probability and overlap so that no clear peaks are discernible; but they are labeled to be consistent with the peaks of Figures 2a,b. For P3ox (Figure 2d) the shape of the contour plot is 1275
Figure 3. Contour plot of CT7n current transients. Region 2 describes the events that are short-lived and do not traverse the pore, whereas Region 1 describes the events that do transit the pore. Here, n ) 4059 and current blockage and blockage duration used bin widths of 1 pA and 0.05 ms, respectively. Table 1. Nonlinear Curve Fitting Results to Profiles Shown in Figure 3a peptide
τ1/µs
τ2/µs
P1 (n ) 4349) P2 (n ) 4487) P3 (n ) 4890) P3ox (n ) 6132)
72(20) 39(20) 33(20) 83(20)
408(16) 547(16) 591(17) 649(15)
a
iblock /pA major iblock /pA minor -52.3(0.1) -57.6(0.2) -75.4(0.2) -69.0(0.2)
-45 -40 to -55 -45, -55, -65 -40, -50, -60
Values in parentheses are the standard deviations.
very similar to that of P3 except that the blockade durations are increased: 649(15) µs for the major peak compared to 591(17) µs for unoxidized P3. Since the ferrocenium ion is positively charged, P3ox must diffuse against the applied field, which is present across the membrane, resulting in longer transit times. As well, there is a significant decrease in blockade current for P3ox compared to P3, which is presumably caused by changes in solvation. Assuming the major peptides of P2 and P3 can be approximated in shape to a rod, the blockage current can be related to the diameter of the rod, thus allowing calculation of diffusion coefficients through a pore one molecule at a time. Thus, P2 and P3 have effective diffusion coefficients of 1.2(0.2) × 10-11 cm2 s-1 and 1.7(0.2) × 10-11 cm2 s-1, respectively. The diffusion rates determined by cyclic voltammetry for these peptides are many orders of magnitude larger,23 suggesting that large frictional forces act on the peptides as they traverse the pore. The neutral peptides traverse the pore at a speed of 0.06(0.02) Å µs-1, which is an order of magnitude slower than that of a polynucleotide.11 We also investigated another simple peptide, CT7n, having the sequence Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)5Gly. The heptapeptide is found as a tandem repeat at the C-terminus of RNA polymerse II from eucaryotes and provides sites for protein-protein interactions that regulate mRNA synthesis.26 The structure of CT7n is thought to be 1276
extended, very flexible, and plastic in the sense that the conformation it adopts is determined by the protein, which binds to it.27,28 The current transients for CT7n are shown in Figure 3 and the shape of the contour plot is very different from that observed for the Pn peptides. Distinct regions are apparent from the data. Region 2 (Figure 3) are the shortduration events that vary in blockage current over the complete range of values. These events are attributed to the peptide interacting with the vestibule area and block the passage of ions but do not necessarily traverse the pore. Region 1 (Figure 3) are the events that have a longer interaction with the pore and are ascribed to peptides that traverse the pore. One striking feature is that the blockade duration is very short (
