Efforts to sequence single protein molecules in nanopores1,2,3,4,5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.
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Experimental raw data, all input files necessary to rerun the MD simulations, the simulation trajectories and raw SEM current data are available at Illinois Data Bank, https://doi.org/10.13012/B2IDB-4905767_V1.
Experimental data acquisition was controlled using Clampex 10.2 software (Molecular Devices). Experimental data were analyzed using Igor Pro 6.12A software (WaveMetrics) and in-house developed procedures, which are available at https://github.com/hadjerouldali/blockade-detection. All MD simulation trajectories were generated using the NAMD2 software package, the source code of which is available at http://www.ks.uiuc.edu/Research/namd. Ionic current analysis of the MD trajectories was carried out using an SEM, which is described in full detail in ref. 30. A python implementation of the SEM model is available at https://gitlab.engr.illinois.edu/tbgl/tools/sem.
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This work was supported by the Agence Nationale de la Recherche (ANR) (ANR-17-CE09-0032-01 to A.O. and F.P.; ANR-17-CE09-0044-02 to P.M., J.P. and A.O.), by the Direction Générale de l’Armement (the French Defence Procurement Agency, no. 2017 60 0042 to A.O. and H.O.) and by the Region Ile-de-France in the framework of DIM ResPore (no. 2017-05 to A.O., H.O., P.M. and J.P.). F.P. was supported by Bpifrance (i-Lab 2018 Dreampore). K.S. and A.A. were supported by the National Institutes of Health grants R01-HG007406 and P41-GM104601 and the National Science Foundation grant PHY-1430124. K.S. and A.A. gratefully acknowledge supercomputer time provided through the XSEDE Allocation Grant MCA05S028 and the Blue Waters Sustained Petascale Computer System at the University of Illinois at Urbana-Champaign. T.E. was a fellow in the International Research Training Group 1642 ‘Soft Matter Science’ of the Deutsche Forschungsgemeinschaft (DFG). We thank F. Gisou van der Goot (Ecole Polytechnique Federale de Lausanne, Switzerland) for providing the pET22b-proAL plasmid containing the pro-aerolysin sequence. We thank M. Pastoriza-Gallego for producing recombinant wild-type pro-aerolysin. We thank G. Baaken, E. Zaitseva and S. Petersen for technical advice and help.
A.O., J.P. and P.M. are co-founders of DreamPore S.A.S., and F.P. is the head of research development at DreamPore S.A.S. J.C.B. is a co-founder of Ionera Technologies GmbH, Freiburg, Germany and of Nanion Technologies GmbH, Munich, Germany. All other authors have no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
Supplementary Fig. 1 Electrical signatures of real-time reduction of CR7 peptides by dithiothreitol.
Histograms of relative residual current Ib/I0 (left) and scatter plots of the blockade duration versus Ib/I0 (right) measured for CR7 peptides in the absence of the disulfide-bond reducing agent dithiothreitol (DTT) (a, n = 3311 events), 15 (b, n = 5211 events) and 30 (c, n = 5926 events) minutes after the addition of 25 mM DTT. Colored rectangles indicate the mean (centerline) and the standard deviation (widths) of the Ib/I0 values recorded individually for CR7 dimers (data from Fig. 1f–j) and recorded for CR7 monomers after disulfide bonds reduction. In absence of DTT, the main population of blockade current has a mean Ib/I0 value of 0.084 ± 0.005, which we attribute to CR7 dimers. Fifteen minutes after the DTT addition, frequency of blockades produced by CR7 dimers is reduced as another population of blockade currents of Ib/I0 = 0.383 ± 0.005 emerges, indicating reduction of CR7 dimers to CR7 monomers by DTT. After 30 minutes of the DTT activity, the 0.084 ± 0.005 population has almost entirely disappeared. In the absence of DTT, the minority clusters of events at relative residual current of around 0.4 correspond to impurities present in our sample except for the cluster of events characterized by the relative residual current of 0.383 ± 0.005, which corresponds to events induced by a minority population of CR7 monomers. The mean value (respectively uncertainty) of relative residual current of each peptide was obtained as the mean value (respectively standard deviation) of a gaussian fit of the corresponding Ib/I0 distribution; from single independent experiments. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment.
Supplementary Fig. 2 Dependence of the mean relative residual current and the blockade duration on the molecular weight or the volume of amino acid X.
Mean relative residual current 〈Ib/I0〉 and its standard deviation (a,b) and mean blockade duration (c,d) for the XR7 peptide versus molecular weight (a,c) or volume (b,d) of amino acid X (n = 34025 (RR7), 1328 (WR7), 6228 (FR7), 1969 (KR7), 2319 (LR7), 3411 (YR7), 5185 (IR7), 1449 (HR7), 2317 (MR7), 3822 (QR7), 5369 (VR7), 1681 (PR7), 1655 (ER7), 3165 (NR7), 2978 (TR7), 1866 (DR7), 1717 (AR7), 3311 (CR7), 8169 (SR7), 3266 (GR7), 2626 ((NO2)-YR7), 2374 ((sulfoxide)-M R7) events). Figure 1k from the main text is reproduced here as panel b to facilitate comparison of the two dependences. The mean value (respectively uncertainty) of relative residual current of each peptide was obtained as the mean value (respectively standard deviation) of a gaussian fit of the corresponding Ib/I0 distribution; from single independent experiments. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment.
(a) Equilibration of the lipid bilayer. During this 0.5 ns simulation, all non-hydrogen atoms of the aerolysin channel were restrained to their initial coordinates while preventing water molecules from entering the lipid-protein interface. The aerolysin channel is shown as a gray cut-away molecular surface, the head groups and tails of the DPhPC lipid bilayer are shown in red and cyan; 1 M KCl solution is not shown. (b) Free equilibration of the aerolysin system. The last frame of the equilibration trajectory was used as the starting structure for subsequent electric field simulations. (c) Root-mean-square deviation (RMSD) of the protein’s alpha-carbon atoms from their initial coordinates during the equilibration simulations. Each data point was averaged over the protein’s 2976 alpha-carbon atoms. (d) The simulated current-voltage curve of the aerolysin nanopore. Each data point derives from an independent MD simulation of the aerolysin system, each lasting from 13 to 37 ns, at the specified transmembrane voltage and 1 M KCl concentration. The average current was obtained by averaging instantaneous ionic current over the course of the MD trajectory. The number of data points used for calculating the average ionic current of each system is as follows: 4521 (-1.2 V), 6193 (-0.5 V), 15084 (-0.25 V), 12733 (-0.1 V), 5914 (0.5 V), 5925 (1 V).
Histogram of relative residual current Ib/I0 (left) and scatter plot of the blockade duration versus Ib/I0 (right) obtained from nanopore experiment performed using an equimolar mixture of QR7, NR7, TR7, and SR7 peptides under a –25 mV (a), –50 mV (b), –100 mV (c), –150 mV (d) and –200 mV (e) bias applied to the trans compartment. The increase of the voltage magnitude reduces discrimination of the blockade current populations, shifts the Ib/I0 values and decreases the blockade duration. In panels a and b, solid rectangles indicate the location of distributions produced by the QR7 and SR7 peptides; the striped rectangles indicate overlapping distributions of the NR7 and TR7 peptides. In panels c-e, the dashed rectangle indicates the expected location of the QR7, NR7, TR7, and SR7 distributions. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment. For each histogram, at least 1000 events were analyzed.
(a,b) Force exerted on the peptide during the two SMD simulations versus the z coordinate of the peptide’s CoM. Hereafter Run 1 refers to the simulation reported in the main text whereas Run 2 refers to the replicate simulation. Instantaneous SMD forces are shown in gray; and their running average (5 Å window) is shown in black. (c) The z coordinate of the peptide’s CoM versus simulation time for Run 1 and Run 2; the two dependences follow closely one another. (d,e) Relative residual current versus the CoM coordinate of RR7, RR6, RR5 and RR4 peptides for Run 1 (d) and Run 2 (e). The peptides’ coordinates were derived from the conformations of RR7 sampled during the SMD simulation. The currents were computed using the steric exclusion model and averaged using a 10 Å running average. (f) Simulated versus experimental average relative currents produced by the arginine peptides within the sensing region of aerolysin. (g-i) Same as d-f but for GR7, AR7, TR7, HR7, and RR7 peptides; the experimental values are reproduced from Fig. 1. For panels f & i, the average simulated current was calculated from 130 relative residual current values for runs 1 and 2, corresponding to the presence of arginine peptides in the sensing region. For panels g & h, the currents were averaged using a 10 Å (50 values) running average.
Histograms of Ib/I0 (left axis) and scatter plots of blockade duration versus Ib/I0 (right axis) obtained from nanopore experiments performed using an equimolar mixture (1 M) of VKR7, VHR7, VER7, VDR7, and VRR7 peptides (a, n = 18066 events) or an equimolar mixture (1 M) of KR7, HR7, ER7, DR7, or RR7 peptide species (b, n = 19641 events). In both cases, the addition of the mixture produces five distinct peaks matching the location of the peaks observed in the individual peptide species experiments. The addition of a V amino acid to each XR7 peptide leads to a shift of the population to lower Ib/I0 values. The mean Ib/I0 values are: 0.353 ± 0.003 (KR7) versus 0.314 ± 0.002 (VKR7); 0.362 ± 0.003 (HR7) versus 0.320 ± 0.002 (VHR7); 0.371 ± 0.004 (DR7) versus 0.336 ± 0.004 (VDR7); 0.332 ± 0.002 (RR7) versus 0.293 ± 0.001 (VRR7). The mean value (respectively uncertainty) of relative residual current of each peptide was obtained as the mean value (respectively standard deviation) of a gaussian fit of the corresponding Ib/I0 distribution; from single independent experiments. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment.
(a) Histograms of relative residual current Ib/I0 (left) and scatter plots of blockade duration versus of Ib/I0 (right) obtained from nanopore experiment using an equimolar mixture of KR7, HR7, ER7, DR7, and RR7 peptides (top row) or individual KR7, HR7, ER7, DR7, or RR7 peptide species (rows 2-6). The location of each peak observed in the mixture experiment matches the location of the peaks observed in the individual peptide species experiments, allowing us to identify populations present in the mixture. (b) Successive addition experiments. Histograms of relative residual current Ib/I0 (left) and scatter plots of blockade duration versus Ib/I0 obtained from nanopore experiment during successive addition of KR7, HR7, DR7, ER7, and RR7 peptides. Each successive addition produces a distinct peak in the Ib/I0 histogram, allowing us to associate individual peaks with the peptide species. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment. For each histogram, at least 1000 events were analyzed.
Supplementary Fig. 8 Identification of peptide species different by a single hydroxylation or a structural isoform.
Histogram of Ib/I0 (top plot of each panel) and scatter plot of blockade duration versus Ib/I0 (bottom plot of each panel) obtained from nanopore experiments during successive addition of YR7 and FR7 peptides (a,b), where Y and F differ by an hydroxyl group or LR7 and IR7 peptides (c,d), where L and I are structural isomers. In both cases, well-separated distributions can be associated to the presence of individual peptide species. For each histogram, at least 1000 events were analyzed. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment.
Supplementary Fig. 9 Theoretical assessment of peptide distinguishability from a single nanopore passage.
(a) Number of events per bin per second for LR7 and IR7 (open blue circles) derived from our experimental measurements (Fig. 3d). Gaussian fits, PL(Ib/I0) and PI(Ib/I0), to the experimental data for LR7 and IR7 are shown in black and red, respectively. The shaded area under the PL curve indicates the region of single passage distinguishability of LR7 peptides (see Supplementary Note 1). (b) PL(Ib/I0) and PI(Ib/I0) distributions when the peptide residence time is 200 ms. The shaded area under the PL curve indicates the region of single passage distinguishability. (c) Probability of distinguishing tyrosine (Y) and phenylalanine (F) from a single nanopore passage as a function of the passage duration. (d) Probability of distinguishing leucine (L) from isoleucine (I) when ion counting error is the only noise source.
Histogram of Ib/I0 (top plot of each panel) and scatter plot of blockade duration versus Ib/I0 (bottom plot of each panel) obtained from nanopore experiments during analysis of: (NO2)-YR7 peptide, where the Y amino acid is modified by an additional -(NO2) group (a, n = 5201 events); an equimolar mixture of YR7 and (NO2)-YR7 peptides (b, n = 10615), (SO3H2)-YR7 peptide, where the Y amino acid is modified by an additional -(SO3H2) group (c, n = 3183 events); an equimolar mixture of YR7 and (SO3H2)-YR7 peptides (d, n = 4227 events); (P)-YR7 peptide, where the Y amino acid is modified by an additional -(P) group (e, n = 2626); and a mixture of YR7 (1 M final concentration) and (P)-YR7 (10 M final concentration) peptides (f, n = 10281 events). The chemical modification of the peptide leads to a shift of the mean Ib/I0 value from 0.360 ± 0.002 (YR7) to 0.343 ± 0.002 ((NO2)-YR7), 0.354 ± 0.002 ((SO3H2)-YR7) and 0.355 ± 0.002 ((P)-YR7). The mean value (respectively uncertainty) of relative residual current of each peptide was obtained as the mean value (respectively standard deviation) of a gaussian fit of the corresponding Ib/I0 distribution; from single independent experiments. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment.
Histogram of Ib/I0 (top plot of each panel) and scatter plot of blockade duration versus Ib/I0 (bottom plot of each panel) obtained from nanopore experiments during analysis of: (sulfoxide)-MR7 peptide (a, n = 2374 events), where the M amino acid is modified by an additional -(sulfoxide) group; and MR7 peptide (b, n = 2317 events). The chemical modification of the peptide leads to the emergence of two main peaks of mean Ib/I0 values of 0.355 ± 0.002 and 0.369 ± 0.003, different from the mean Ib/I0 value of 0.363 ± 0.003 corresponding to MR7 peptide. The mean value (respectively uncertainty) of relative residual current of each peptide was obtained as the mean value (respectively standard deviation) of a gaussian fit of the corresponding Ib/I0 distribution; from single independent experiments. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and 20.0 ± 0.5oC, and under a –50 mV bias applied to the trans compartment.
Histograms of relative residual current Ib/I0 measured using a low-noise setup (see Supplementary Note 3). The nanopore experiments were performed using an equimolar mixture of LR7 and IR7 peptides and under the following experimental conditions: –50 mV in 4M KCl (a, n = 3474 events); –40 mV in 4M KCl (b, n = 2373 events); –40 mV in 2M KCl/2M KNO3 mixture (c, n = 3437 events). In 4M KCl, the diminution of the voltage magnitude from –50 mV to –40 mV leads to a reduction of the standard deviations of the two main populations, and a shift of the Ib/I0 values from 0.380 ± 0.0018 to 0.378 ± 0.0013 (LR7) and from 0.384 ± 0.0016 to 0.382 ± 0.0012 (IR7). At –40 mV, the substitution of 50% of the Cl– ions by 50% of NO3– ions further reduces the standard deviations of the two main populations now located at Ib/I0 values of 0.382 ± 0.0010 (LR7) and 0.386 ± 0.0010 (IR7). Continuous lines show the underlying Gaussian distributions. The mean value (respectively uncertainty) of relative residual current of each peptide was obtained as the mean value (respectively standard deviation) of a gaussian fit of the corresponding Ib/I0 distribution; from single independent experiments. The data were acquired at room temperature and under a voltage bias applied to the trans compartment.
All measurements were conducted using an equimolar mixture of LR7 and IR7 peptides under the following three conditions: 4 M KCl, –50 mV (top row); 4 M KCl, –40 mV (middle row); 2 M KCl/2 M KNO3, –40 mV (bottom row). (a) Blockade duration histograms for individual levels (grey) and resistive pulses (black) with monoexponential fits (red lines). (b) Scatterplots of current variance vs Ib/I0 for individual levels. Excess variance is determined as the difference between the center of mass of the point clouds for the blocked states and the open state (Ib/I0 ≈ 1). (c) Histograms values in terms of Ib/I0 for individual levels (grey cityscape) and resistive pulses (black cityscape). Continuous black and blue lines are the sum of two Gaussians and the underlying Gaussian distributions, respectively. For each histogram, at least 1000 events were analyzed.
(a) Representative fragment of ionic current recording showing an example of a transition from an open pore current state to a blockade current state. (b) Close-up of both levels from panel a to illustrate the increased noise of the blocked current state in comparison to the open pore current state. (c) Scatterplot (red dots) of the variance of detected current levels versus I/I0. Note the approximately threefold increase in variance in the blocked state (I/I0 ≈ 0.385) with respect to the open state (I/I0 = 1). (d) Histogram of the relative residual current I/I0 (black trace), scatterplot of blockade duration versus Ib/I0 (red dots) and a Gaussian fit of the histogram (red trace). Note that the estimated mean values get progressively closer to the population mean with longer dwell times, indicating that current noise and not inter-event-variance is responsible for the width of the Gaussian peak. For panels c & d, histograms and scatter plots are based on the analysis of 7309 events. (e) Power spectral density plots for four blocked levels and four preceding baseline levels (as in panels a and b). (f) Difference of the averages of the four blocked state and four open state spectra, respectively, from panel e. Note the flat curve between 100 Hz and 10 kHz (the cut-off frequency of the 4-pole Bessel filter used). The mean value (respectively uncertainty) of relative residual current of each peptide was obtained as the mean value (respectively standard deviation) of a gaussian fit of the corresponding Ib/I0 distribution; from single independent experiments. The data were acquired in 4 M KCl, 25 mM HEPES buffer, at 7.5 pH and at room temperature, and under a –50 mV bias applied to the trans compartment, and with a 10 kHz filter and a 1 MHz sampling rate.
Supplementary Fig. 15 Analysis of individual resistive pulses from low-noise ionic current measurements and the effect of solvent.
(a) Typical resistive pulse measured using a low-noise setup and an equimolar mixture of LR7 and IR7 peptides at –40 mV in 4 M KCl. This resistive pulse consists of the following five levels: two visits to the long-lasting level and three short visits to a deeper blocked level. The dashed red line corresponds to the current level at the midpoint between the two maxima for LR7 and IR7: the red continuous lines show the extremes of the range of mean current levels that are included in determining the resistive pulse mean 〈Ib/I0〉 value. Blue markers show start and end of resistive pulse (vertical arm) and mean current value (horizontal arm). Green and red vertical lines show onset and end of transitions as detected by the DetectiVent algorithm. Vertical (b) and horizontal (c) expansions of (a) to show details. (d) Power spectral density of three blocked (red) and unblocked (black) current levels in the presence of 2 M KCl/2 M KNO3. (e) Comparison of the difference of averages in 4 M KCl (blue, same as in Supplementary Fig. 14f) and 2 M KCl/2 M KNO3 (black).
Supplementary Figs. 1–15, Supplementary Table 1 and Supplementary Notes 1—3
Equilibration of aerolysin nanopore in DPhPC membrane. The video illustrates a 10 ns MD trajectory of the aerolysin system consisting of the aerolysin nanopore (grey, cut-away molecular surface) embedded in a DPhPC lipid bilayer (red for head groups and cyan for tails) and 1 M KCl solution (not shown for clarity).
MD simulation of open pore ionic current. The video illustrates a 2 ns fragment of an MD trajectory of the aerolysin system under a transmembrane bias of −100 mV. The aerolysin nanopore is shown using a grey, cut-away molecular surface; the head groups and tails of the DPhPC lipid bilayer are shown in red and cyan, respectively; the volume occupied by 1 M KCl electrolyte is represented by a blue translucent surface; potassium and chloride ions are shown as violet and green spheres, respectively.
SMD simulation of RR7 peptide translocation through aerolysin. The video illustrates a 100 ns MD trajectory where an RR7 peptide was driven through the aerolysin nanopore using the SMD protocol. The aerolysin nanopore is shown as a grey, cutaway molecular surface; the DPhPC lipid bilayer is shown in red (head groups) and cyan (lipid tails); the backbone of the RR7 peptide is shown in grey while the side chains of all arginine residues are shown in blue except for the last one, which is shown in red.
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Ouldali, H., Sarthak, K., Ensslen, T. et al. Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore. Nat Biotechnol 38, 176–181 (2020). https://doi.org/10.1038/s41587-019-0345-2
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