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Dissecting the Membrane Association Mechanism of Aerolysin Pores at Femtomolar Concentrations Using Water as a Probe
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  • Tereza Roesel
    Tereza Roesel
    Laboratory for Fundamental BioPhotonics (LBP), Institute of Bioengineering (IBI), and Institute of Materials Science (IMX), School of Engineering (STI), and Lausanne Centre for Ultrafast Science (LACUS), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
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  • Chan Cao
    Chan Cao
    Department of Inorganic and Analytical Chemistry, School of Chemistry and Biochemistry, University of Geneva, 1211 Geneva, Switzerland
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    Orcidhttps://orcid.org/0000-0003-2592-0690
  • Juan F. Bada Juarez
    Juan F. Bada Juarez
    Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
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    Orcidhttps://orcid.org/0000-0002-1337-6424
  • Matteo Dal Peraro*
    Matteo Dal Peraro
    Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
    *[email protected]
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    Orcidhttps://orcid.org/0000-0002-2973-3975
  • Sylvie Roke*
    Sylvie Roke
    Laboratory for Fundamental BioPhotonics (LBP), Institute of Bioengineering (IBI), and Institute of Materials Science (IMX), School of Engineering (STI), and Lausanne Centre for Ultrafast Science (LACUS), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
    *[email protected]
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    Orcidhttps://orcid.org/0000-0002-6062-7871
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Nano Letters

Cite this: Nano Lett. 2024, 24, 44, 13888–13894
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https://doi.org/10.1021/acs.nanolett.4c00035
Published October 29, 2024

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Abstract

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Aerolysin is a bacterial toxin that forms transmembrane pores at the host plasma membrane and has a narrow internal diameter and great stability. These assets make it a highly promising nanopore for detecting biopolymers such as nucleic acids and peptides. Although much is known about aerolysin from a microbiological and structural perspective, its membrane association and pore-formation mechanism are not yet fully understood. Here, we used angle-resolved second harmonic scattering (AR-SHS) and single-channel current measurements to investigate how wild-type (wt) aerolysin and its mutants interact with liposomes in aqueous solutions at femtomolar concentrations. Our AR-SHS experiments were sensitive enough to detect changes in the electrostatic properties of membrane-bound aerolysin, which were induced by variations in pH levels. We reported for the first time the membrane binding affinity of aerolysin at different stages of the pore formation mechanism: while wt aerolysin has a binding affinity as high as 20 fM, the quasi-pore and the prepore states show gradually decreasing membrane affinities, incomplete insertion, and a pore opening signature. Moreover, we quantitatively characterized the membrane affinity of mutants relevant for applications to nanopore sensing. Our study provides a label-free method for efficiently screening biological pores suitable for conducting molecular sensing and sequencing measurements as well as for probing pore-forming processes.

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Pore-forming proteins are a class of proteins targeting the plasma membrane of a variety of organisms and are usually involved in defense or attack mechanisms. (1) The most extensively characterized pore-forming proteins are the bacterial pore-forming toxins (PFTs), which are classified as helical (α-) or beta-sheet (β)-PFTs depending on the secondary structure of their transmembrane region. (1,2) Most commonly, pore-forming proteins are expressed as soluble proteins that subsequently oligomerize upon protease activation and/or membrane receptor binding, converting to a transmembrane pore.
Aerolysin, a β-PFT produced by Aeromonas sp., is the founding member of a large superfamily that spans all of the kingdoms of life. (1,3,4) Aerolysin is expressed as an inactive precursor, proaerolysin, which contains 4 distinct domains: domain 1 (in gray, Figure 1A) is involved in binding N-linked oligosaccharides while domain 2 (in blue) is a glycosyl phosphatidylinositol (GPI)-anchored binding region, domain 3 (in yellow, called stem loop) is responsible for the oligomerization process, and domain 4 (in green) contains a C-terminal peptide (CTP, in red) that is required for folding into the soluble monomeric form. (5) Proteolysis of the CTP allows aerolysin to oligomerize in a heptameric ring-like complex that inserts into the target membrane to form the pore after passing through several distinct intermediate structures (namely prepore, post pre-pore, and quasi-pore intermediates; Figure 1B–D). (6,7) These stages are defined by a different length and completion of the β-barrel that is formed after the stem loop undergoes a conformational change upon oligomerization. First, a soluble transient pre-pore state is formed, which was captured in vitro only by the stabilizing Y221G mutation; this state is characterized by two concentric β-barrels, held together by hydrophobic interactions (4) (Figure 1B). Next, in the series of events putatively leading to pore formation, the inner β-barrel fully extends toward the membrane passing from a quasi-pore state captured by the stabilizing mutation K246C-E258C (Figure 1C) and ending in the mature aerolysin pore (Figure 1D). The structure of each of these states is known at high resolution from a combination of molecular modeling and cryo-EM experiments, (4,8) which has led to a crude understanding of the sequential steps leading to pore formation.

Figure 1

Figure 1. Structure of aerolysin in different states and how to measure its membrane association. A. Structure of monomeric pro-aerolysin (PDB: 1PRE): Domain 1 in gray, domain 2 in blue, domain 3 in yellow, and domain 4 in green. Illustration of the aerolysin structure in prepore (B), quasi-pore (C), and pore (D) states and labels of the various mutations with localization in panel A. The images were generated in UCSF ChimeraX. (9) E. Energy-level scheme and sketch of the AR-SHS experiment. P(S) refers to the polarization state of the beam parallel (perpendicular) to the scattering plane. All measurements were recorded with all beams polarized in the horizontal plane. For the single-angle experiments, the scattering angle θ was set to 45°, corresponding to the angle with maximum scattering intensity. Adapted with permission from ref (20). Copyright [2024] [American Physical Society] F. Top: Illustration of how interfacial electric fields (E⃗) orient dipolar water (indicated by the symbol p⃗) and how this is affected by aerolysin–membrane interaction. Here, charge-water interaction is the most relevant physical mechanism for creating the SH contrast. Bottom: Example SHS pattern of LUVs composed of 99:1 mol % DOPC:DOPA membranes interacting with 5 × 10–10 M wt aerolysin in aqueous solutions having pH values of 4 (black data) and 9 (red data). One mol % DOPA was used to increase the signal-to-noise ratio for these measurements. The maximum interfacial SH intensity occurs around θ = 45°. Figure S1 shows how this graph was generated.

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Recently, aerolysin has received much attention, not only because of its biological role, but also because of its potential use in biotechnological applications. (10,11) Due to its stability, easy incorporation into lipid bilayers, and a narrow internal diameter of the pore lumen (∼1 nm), aerolysin is nowadays one of the most promising biological nanopores, permitting the detection of several biopolymers such as nucleic acids, peptides, and oligosaccharides. (12−16) We and others have shown that point mutations within the pore can be performed for specific molecular sensing, thereby achieving enhanced sensitivity and selectivity. (10,17,18) At the same time, we observed that the mutations have an impact on the pore formation efficiency. This is, in fact, crucial since without efficient incorporation of the pore into the lipid bilayer and a strong lipid–pore interaction, nanopore experiments cannot be reliably conducted. Therefore, to explore new biological or de novo designed nanopore candidates, it would be highly desirable if one could estimate lipid–protein interactions at very low concentrations and without sacrificing the sample. Such a technique would be a useful tool to screen for pore mutants that have stable and strong lipid interactions and would provide insights into their membrane association mechanism. It would be instrumental in identifying promising nanopore candidates for molecular sensing experiments.
To better understand the complex aerolysin–membrane interplay, we use a recently introduced method called high-throughput angle-resolved second harmonic scattering (AR-SHS). (19) In this method, two fs pulsed near-infrared photons with frequency ω interact with an aqueous solution containing liposomes. A nonresonant second-order polarization is created in only those regions of the sample where there is an anisotropic distribution of anisotropic molecules. This polarization is composed of displaced charge density that oscillates at twice the frequency of the incoming light (2ω), and it emits photons at this second harmonic frequency, which are detected. The AR-SHS experiment is illustrated in Figure 1E. It was recently shown that for liposome–water and other membrane systems, interfacial water molecules can be used as a contrast agent. (20−24) Thus, this technique is a label-free interface-specific method with molecular sensitivity toward the orientational distribution of interfacial water. Recent studies have shown that changes in the membrane water can help understand temperature-induced phase transitions (22) and map different protein–membrane interactions, such as the interaction of α-synuclein with the aqueous environment of liposomes. (23) It has also been shown that AR-SHS enables the retrieval of the protein–membrane binding constant, which was exemplified by the interaction of perfringolysin O (PFO) with liposomes. (25) These measurements demonstrate the ability of AR-SHS to ultrasensitively detect protein–liposome interactions in an aqueous solution in the fM–pM range, corresponding to a single transmembrane protein bound to a single liposome. This permits the use of small volumes of a protein solution.
Here, we used this approach to quantify the interaction between aerolysin pore-forming proteins and lipid membranes (Figure 1). Our results clearly show that AR-SHS can capture pH-dependent changes in the surface charge of the aerolysin cap region. Combining the ultrasensitive AR-SHS measurements with single-channel current recording experiments, we quantitatively analyzed the binding affinities of different aerolysin mutants to the lipid membrane. We estimated a binding affinity of 20 fM for wt aerolysin at pH 7.4. Furthermore, we extracted the dissociation constants of two mutants─the first one mutated in the cap region (i.e., pore entry), R220A, and the second one mutated in the stem region (i.e., pore exit), K238N. We observed binding affinities varying by 2 orders of magnitude between these two mutants, with Kd going from 10–14 M to 10–12 M for K238N and R220A, respectively. Additionally, we compared the binding behavior of mutation Y221G (Figure 1B) and of mutation K246C-E258C (Figure 1C), and no complete pore opening was observed for either of these mutants. However, the quasi-pore exhibited binding behavior similar to that of wt aerolysin, but with a binding affinity more than an order of magnitude smaller.

Surface Charge Detection upon pH Changing Conditions

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Figure 2A shows the recorded change (ΔS) in the normalized SH intensity as a function of the pH. This plot was created by recording PPP-polarized SH intensities at the angle of maximum intensity (θmax = 45°, Figure 1F and Figure S1) of liposome solutions containing 0.5 nM wt aerolysin and of identical solutions that contained no liposomes. Normalized SHS intensities were then computed via eq S1 and compared to liposome solutions containing no aerolysin, resulting in ΔS values, which were measured as a function of pH. ΔS is defined as the absolute difference between the normalized SH intensity of liposomes with a given concentration of aerolysin in the solution and liposomes without added aerolysin (eq S2, Materials and Methods)

Figure 2

Figure 2. pH-dependent surface charge and membrane binding affinities. A. Second harmonic intensity difference relative to bulk water (S) at the scattering angle with maximum intensity (θmax = 45°) vs pH of the aqueous solution. The measurements were performed with DOPC liposomes and wt aerolysin. The blue and red highlighted areas indicate the positive and negative surface charges on the cap region of aerolysin. B. Electrostatic potential mapped on the cross sections of the aerolysin cap domain obtained via the APBS Web server (26) depicting the charge distribution of the pore (blue: positive charge; red: negative charge) visualized in PyMol. (27) C, D. A representative single-channel current recording traces of the aerolysin-DOPC free-standing membrane system in aqueous solution at pH 5 (C) and 7.4 (D), with the corresponding histogram current distribution, where each step indicating that another pore incorporation is counted (numbered by #). E, F. Normalized SH intensity difference (ΔS) at the angle with the maximum intensity (θmax = 45°) vs wt aerolysin concentration on the logarithmic scale at pH 5 (E) and 7.4 (F). The data are fitted using eq S2, giving a dissociation constant of Kd = (6.2 ± 0.4) × 10–14 M for pH 5 and Kd = (2.0 ± 0.2) 10–14 M for pH 7.4 (represented by a dashed line). The error bars were determined as the standard deviation from 100 measurements for all of the AR-SHS measurements. The AR-SHS measurements were performed with liposomes composed of 99:1 mol % DOPC:DOPS liposomes interacting with wt aerolysin.

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The interaction of charge with dipolar water molecules results in orientational directionality (Figure 1F top). Water molecules facing a positively charged surface will tend to orient with the O atoms toward the surface. This mechanism can be used to determine the electrostatic surface potential. (20) As such, the SH response is very sensitive to the amino acid charges of the protein. Note that the DOPC membrane is, on average, charge neutral. At pH 4, the normalized SH intensity (eq S1, Materials and Methods) is the lowest at ΔS = 0.02. At pH 4.5, the normalized SH intensity sharply increases by 0.06, after which it smoothly increases until pH 8, at which point it rapidly increases to 0.2. The electrostatic potential on the surface of the extracellular part of the wt aerolysin over pH change is computed with the help of the APBS Web server (26) (see Method F in the SI) and reported in Figure 2B. This result shows how the protonation state of the amino acids present in the cap domain significantly changes with pH, producing a mostly positively charged pore at pH 4 and a negatively charged one at pH 9. It is interesting that significant variations in the normalized intensity occur in correspondence with the pKa values of the relevant titratable residues (i.e., Asp/Glu, His and Arg/Lys) within the pH range explored (Table S1 shows the pKa values). When the pH is lower than 4, Asp and Glu residues are mostly protonated, which reduces the negative charge of the pore cap domain (Figure 2B). On increasing the pH, the net charge of the cap domain becomes more negative with significant steps at pH 6 (deprotonation of His) and pH 9 (deprotonation of Arg and Lys).
The trend observed in Figure 2A can be explained by considering the changes in the orientation of the water molecules that interact with aerolysin. It was previously shown that DOPC liposomes in an aqueous solution have water molecules weakly but preferentially oriented with their H atoms toward the membrane surface. (20) At pH 4, the inserted aerolysin is positively charged, which forces some water molecules to orient their O atoms toward the aerolysin surface, balancing out the effect of the water orientation induced by the DOPC membrane and resulting in a minimal SH intensity. Between pH 4.5 and 8, the aerolysin cap domain is mostly neutral or slightly negative. In this pH range, the orientation of water is primarily determined by water–DOPC interactions, resulting in a weak preference for H atoms to face the surface. Above pH 8, the aerolysin becomes more negatively charged, and more water molecules reorient with their H atoms toward the surface, causing an increase in the SH intensity. Therefore, the AR-SHS response is a very sensitive marker of the protonation state of the amino acids of aerolysin. We expect this to be a general trend for all transmembrane proteins possessing titratable moieties under variable pH conditions.

Membrane Binding Affinity of wt Aerolysin Pores

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Subsequently, the binding of aerolysin to the membrane was probed using AR-SHS and single-channel current measurements at pH 5.0 and 7.4. In single-channel current recording experiments (Figure 2C and D), the incorporation of an aerolysin pore into a DOPC free-standing membrane was followed by a rapid increase in current when a voltage was applied. This current is proportional to the number of pores present in the membrane. For the aerolysin–DOPC–membrane system in an aqueous solution at pH 5.0, three aerolysin pores were observed in a 20 s recording time, whereas at pH 7.4, many more pores were incorporated into the bilayer under the same conditions and equal recording time. Figure 2E and F shows the coherent SH intensity difference ΔS as a function of the wt aerolysin concentration in the solution. ΔS increased rapidly between 10 and 100 fM aerolysin, after which it stayed constant. This increase occurred at lower concentrations at pH 7.4 compared to pH 5.0. The leveling off represents the saturation of the interactions, where no additional proteins could insert into the membrane. Using eq S2, we obtained wt aerolysin dissociation constants of Kd = (6.2 ± 0.4) × 10–14 M and Kd = (2.0 ± 0.2) × 10–14 M for pH 5 and pH 7.4, respectively. The single-channel current and AR-SHS measurements together demonstrate that wt aerolysin has a slightly lower membrane affinity under acidic conditions compared to neutral pH, and this is reflected by less efficient incorporation into lipid bilayers (Table S2).

Membrane Binding Affinities of Aerolysin Mutants

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Having the ability to detect protonation changes of the amino acids of wt aerolysin, we aimed to detect the changes caused by mutating amino acids, particularly those in the cap or the stem region. We used two single point mutations defined by distinctive steric hindrance and electrostatics. (10) By replacing R220 in the cap region with alanine (R220A), it was shown that the pore diameter becomes two times wider while the insertion efficiency is lowered. (10) A mutation at the constriction point in the stem region, where K238 is replaced with an asparagine (K238N), was shown instead to prolong the dwell time for DNA sensing with respect to the wt pore. (10)
We investigated the insertion of these mutants in comparison with the wt aerolysin at pH 7.4, employing single-channel current recording and AR-SHS measurements (Figure 3). From the single-channel current recordings at pH 7.4, we observed that two R220A pores and five K238N pores are formed on the membrane (Figure 3A and B). Using the same pores, we performed AR-SHS measurements to obtain the dissociation constant and compared it to that of wt aerolysin. We observed that in the case of R220A, the increase was more gradual than for the wt, starting at less than 1 pM in the mutant concentration and increasing to 100 pM, where it leveled off (Figure 3C). In the case of K238N, the SH intensity difference increased in a similar concentration range (10 fM–1 pM), but more gradually than in the case of wt. It saturated at around 1 pM, without a further change in SH intensity (Figure 3D). Using eq S2, we obtained dissociation constants of Kd = (2.3 ± 0.3) × 10–12 M and Kd = (4.6 ± 0.6) × 10–14 M for R220A and K238N, respectively. The extracted dissociation constant of K238N at pH 7.4 is slightly higher than that of the wt aerolysin, revealing that K238N has a slightly less favorable interaction with the membrane, confirmed by less incorporation into the membrane. The R220A dissociation constant is 2 orders of magnitude larger than that of wt aerolysin, reflecting the notion that it is less effective at creating pores in the membrane, a finding that is confirmed by the current measurements (Figure 3A, Table S2). Comparing the number of aerolysin pore incorporations in the free-standing bilayer with the dissociation constants obtained from AR-SHS shows that the two techniques return a consistent and quantitative description of the membrane association process.

Figure 3

Figure 3. Aerolysin single-point mutant-lipid membrane binding in aqueous solution. A, B. A representative single-channel current recording measurement of R220A (A) and K238N (B) measured with a DOPC free-standing membrane at pH 7.4. C, D. SH intensity difference (ΔS) at the angle with maximum intensity (θmax = 45°) vs R220A (C) and K238N (D) concentration on the logarithmic scale at pH 7.4. The data are fitted using eq S2, giving dissociation constants of Kd = (2.3 ± 0.3) 10–12 M and Kd = (4.6 ± 0.6) 10–14 M for R220A and K238N, respectively (the dashed line represents the dissociation constant). The data in parts C and D were measured with DOPC doped with 1% DOPS liposomes. The error bars were determined as the standard deviation from 100 measurements for all of the AR-SHS measurements. The dotted line represents the fitted Kd value for each aerolysin we tested in A and B.

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Dissecting Membrane Association throughout the Pore-Forming Process

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To dissect more directly the mechanism of pore formation, we investigated how different pore intermediates interact with the membrane (Figure 4A and B). The Y221G mutant is known to form a prepore state in which the prestem loop is prevented from moving away from the five-stranded β-sheet in the same domain and thereby blocks the hemolytic activity of the toxin (8,28) while still being able to transiently interact with the target membrane even though it is fully hydrophilic. (28) The K246-E258C mutations block the pore formation in a later stage of the formation of full transmembrane β-barrel due to the creation of a disulfide bridge between mutated residues 246 and 258. (5)

Figure 4

Figure 4. Membrane properties of aerolysin mutants blocked at different stages of pore formation. A, B. Structural model of the aerolysin quasi-pore (A) and prepore (B). The images were generated in UCSF ChimeraX. (9) C, D. Representative single-channel current recording measurements of the quasi-pore (C) and prepore (D) at pH 7.4 on a DOPC free-standing membrane. E, F. SH intensity difference (ΔS) recorded at the scattering angle having the maximum intensity (θmax = 45°) vs K246C-E258C (E) and Y221G (F) concentration on a logarithmic scale. The data were fitted using eq S2, giving a dissociation constant of Kd = (4.1 ± 0.3) × 10–13 M for K246C-E258C (quasi-pore). These data were measured with DOPC doped with 1% DOPS liposomes at pH 7.4. The error bars were determined as a standard deviation from 100 measurements for all of the AR-SHS measurements. The dotted line represents the fitted Kd value for each aerolysin we tested in A.

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Figure 4C and D shows zero current during the entire time of the single-channel current measurement, thereby proving that there are no (or very few) pores inserted into the membrane (Table S2). The SH intensity difference (ΔS) as a function of the concentration of aerolysin mutants at pH 7.4 is depicted in Figure 4E and F. For the quasi-pore, ΔS increased from 10 fM to 10 pM, at which point it saturated. The extracted dissociation constant for the quasi-pore is Kd = (4.1 ± 0.3) × 10–13 M, thus more than 1 order of magnitude higher than for wt aerolysin (Figure 4E). The molecular interpretation of these results is that quasi-pores can bind efficiently to the membrane because they have an almost complete β-barrel that can insert into the membrane, but they cannot fully pierce the membrane and are thereby unable to form conducting pores, leading to a decreased binding affinity with respect to the wt pore. The combination of these results implies that the K246C-E258C mutant inserts into the membrane but is unable to transition into a mature pore state, a finding suggested previously. (28) In the case of Y221G, ΔS increased but did not follow the same trend (Figure 4F). The fitting procedure indeed failed for this data set, as indicated by the dashed line in Figure 4F. This could mean that the mutant is transiently interacting with the membrane but not fully anchored. The absence of detectable current in the single-channel recordings agrees with this assessment and is consistent with the fact that the Y221G prepore mutation is known to form fully soluble pores and the barrel region is only partially folded, with its hydrophobic stem region still being protected (Figure 4B).
In summary, by using single-channel current experiments on free-standing membranes and AR-SHS measurements on aerolysin–LUV interactions in aqueous solutions, we have shown that different mutants of aerolysin have distinct types of interaction with the membrane, leading to various degrees of pore formation and targeted insertion. Thanks to the high interfacial and charge sensitivity of AR-SHS, we were able to observe changes in the surface charge of the cap region of aerolysin bound to the LUV for different pH conditions. The charge of the cap region changes from positive at pH 4 to highly negative at pH 9, which is consistent with the predicted electrostatic properties of the pore. This combination of techniques provides a unique method to assess the electrostatic properties of a pore’s surface, which is of special interest for nanopore applications since the changes in the surface can induce a strong electroosmotic flow, enabling the capture of all kinds of biomolecules regardless of their charges. (29)
By combining AR-SHS and single-channel current measurements, we compared the binding affinities of wt aerolysin at different pH values. We extracted the dissociation constants Kd = (6.2 ± 0.4) × 10–14 M and Kd = (2.0 ± 0.2) × 10–14 M for pH 5.0 and 7.4, respectively. These are 2 orders of magnitude higher than for the interaction of PFO with a cholesterol-rich lipid membrane. (25) Additionally, we studied the interactions of aerolysin mutants, R220A and K238N, and observed a difference of around 2 orders of magnitude in the Kd of these two mutants, going from 10–14 to 10–12 M for K238N and R220A, respectively. Lastly, we examined the different binding behavior of aerolysin mutants at different stages of pore formation. No open pore was observed for either quasi-pore or prepore mutants in single-channel current measurements, but the binding of the quasi-pore was measured and demonstrated the extreme sensitivity of the AR-SHS technique, which could be measured at as low as 10 fM.
The combination of using low volumes (10 μL) and low protein concentrations and with the possibility of varying the temperature, pH, or membrane composition means that the AR-SHS method can distinguish which aerolysin pores are efficiently incorporated into the lipid bilayer. Thereby, it provides the opportunity to dissect the molecular features of membrane association along the pore formation pathway or use it for the screening of pore variants with enhanced membrane incorporation and sensitivity. Furthermore, as shown here, AR-SHS alone can successfully characterize (membrane) protein interactions in a label-free environment, and its combination with single-channel current recordings provides a promising method for bionanotechnology.

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.4c00035.

  • Additional experimental details and materials and methods for protein expression, single-channel recordings and AR-SHS experiments including Figure S1 showing the polar plots of raw data and resultant S(θ) values, Table S1 containing the number of amino acids in aerolysin, and Table S2 containing the number of experiments performed for each mutant (PDF)

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Author Information

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  • Corresponding Authors
    • Matteo Dal Peraro - Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland;  Orcidhttps://orcid.org/0000-0002-2973-3975;  Email: [email protected]
    • Sylvie Roke - Laboratory for Fundamental BioPhotonics (LBP), Institute of Bioengineering (IBI), and Institute of Materials Science (IMX), School of Engineering (STI), and Lausanne Centre for Ultrafast Science (LACUS), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland;  Orcidhttps://orcid.org/0000-0002-6062-7871;  Email: [email protected]
  • Authors
    • Tereza Roesel - Laboratory for Fundamental BioPhotonics (LBP), Institute of Bioengineering (IBI), and Institute of Materials Science (IMX), School of Engineering (STI), and Lausanne Centre for Ultrafast Science (LACUS), École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
    • Chan Cao - Department of Inorganic and Analytical Chemistry, School of Chemistry and Biochemistry, University of Geneva, 1211 Geneva, Switzerland;  Orcidhttps://orcid.org/0000-0003-2592-0690
    • Juan F. Bada Juarez - Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland;  Orcidhttps://orcid.org/0000-0002-1337-6424
  • Author Contributions

    T.R. and C.C. contributed equally

  • Funding

    S.R. acknowledges support from the Julia Jacobi Foundation and European Research Council Grant Agreement No. 951324 (no. H2020, R2-tension). M.D.P. acknowledges support from the Swiss National Science Foundation (nos. 200021L_212128 and 205321_192371). C.C. acknowledges support from the Swiss National Science Foundation (PR00P3_193090) and the Novartis Foundation for Medical-Biological Research.

  • Notes
    The authors declare no competing financial interest.

References

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    Rossjohn, J.; Feil, S. C.; McKinstry, W. J.; Tsernoglou, D.; Van Der Goot, G.; Buckley, J. T.; Parker, M. W. Aerolysin─A Paradigm for Membrane Insertion of Beta-Sheet Protein Toxins?. J. Struct. Biol. 1998, 121 (2), 92– 100,  DOI: 10.1006/jsbi.1997.3947
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    Cirauqui, N.; Abriata, L. A.; van der Goot, F. G.; Dal Peraro, M. Structural, Physicochemical and Dynamic Features Conserved within the Aerolysin Pore-Forming Toxin Family. Sci. Rep. 2017, 7 (1), 13932,  DOI: 10.1038/s41598-017-13714-4
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    Iacovache, I.; Degiacomi, M. T.; Pernot, L.; Ho, S.; Schiltz, M.; Dal Peraro, M.; van der Goot, F. G. Dual Chaperone Role of the C-Terminal Propeptide in Folding and Oligomerization of the Pore-Forming Toxin Aerolysin. PLOS Pathog. 2011, 7 (7), e1002135  DOI: 10.1371/journal.ppat.1002135
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    Degiacomi, M. T.; Iacovache, I.; Pernot, L.; Chami, M.; Kudryashev, M.; Stahlberg, H.; van der Goot, F. G.; Dal Peraro, M. Molecular Assembly of the Aerolysin Pore Reveals a Swirling Membrane-Insertion Mechanism. Nat. Chem. Biol. 2013, 9 (10), 623– 629,  DOI: 10.1038/nchembio.1312
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    Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism
    Degiacomi, Matteo T.; Iacovache, Ioan; Pernot, Lucile; Chami, Mohamed; Kudryashev, Misha; Stahlberg, Henning; van der Goot, F. Gisou; Dal Peraro, Matteo
    Nature Chemical Biology (2013), 9 (10), 623-629CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Aerolysin is the founding member of a superfamily of β-pore-forming toxins whose pore structure is unknown. We have combined X-ray crystallog., cryo-EM, mol. dynamics and computational modeling to det. the structures of aerolysin mutants in their monomeric and heptameric forms, trapped at various stages of the pore formation process. A dynamic modeling approach based on swarm intelligence was applied, whereby the intrinsic flexibility of aerolysin extd. from new X-ray structures was used to fully exploit the cryo-EM spatial restraints. Using this integrated strategy, we obtained a radically new arrangement of the prepore conformation and a near-atomistic structure of the aerolysin pore, which is fully consistent with all of the biochem. data available so far. Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane β-barrel.
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  7. 7
    Iacovache, I.; De Carlo, S.; Cirauqui, N.; Dal Peraro, M.; van der Goot, F. G.; Zuber, B. Cryo-EM Structure of Aerolysin Variants Reveals a Novel Protein Fold and the Pore-Formation Process. Nat. Commun. 2016, 7, 12062  DOI: 10.1038/ncomms12062
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    7
    Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process
    Iacovache, Ioan; De Carlo, Sacha; Cirauqui, Nuria; Dal Peraro, Matteo; van der Goot, F. Gisou; Zuber, Benoit
    Nature Communications (2016), 7 (), 12062CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Owing to their pathogenical role and unique ability to exist both as sol. proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-sol. monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.
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  8. 8
    Tsitrin, Y.; Morton, C. J.; El Bez, C.; Paumard, P.; Velluz, M.-C.; Adrian, M.; Dubochet, J.; Parker, M. W.; Lanzavecchia, S.; van der Goot, F. G. Conversion of a Transmembrane to a Water-Soluble Protein Complex by a Single Point Mutation. Nat. Struct. Biol. 2002, 9 (10), 729– 733,  DOI: 10.1038/nsb839
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  9. 9
    Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Meng, E. C.; Couch, G. S.; Croll, T. I.; Morris, J. H.; Ferrin, T. E. UCSF ChimeraX: Structure Visualization for Researchers, Educators, and Developers. Protein Sci. 2021, 30 (1), 70– 82,  DOI: 10.1002/pro.3943
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    9
    UCSF ChimeraX: Structure visualization for researchers, educators, and developers
    Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.; Meng, Elaine C.; Couch, Gregory S.; Croll, Tristan I.; Morris, John H.; Ferrin, Thomas E.
    Protein Science (2021), 30 (1), 70-82CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    UCSF ChimeraX is the next-generation interactive visualization program from the Resource for Biocomputing, Visualization, and Informatics (RBVI), following UCSF Chimera. ChimeraX brings (a) significant performance and graphics enhancements; (b) new implementations of Chimera's most highly used tools, many with further improvements; (c) several entirely new anal. features; (d) support for new areas such as virtual reality, light-sheet microscopy, and medical imaging data; (e) major ease-of-use advances, including toolbars with icons to perform actions with a single click, basic "undo" capabilities, and more logical and consistent commands; and (f) an app store for researchers to contribute new tools. ChimeraX includes full user documentation and is free for noncommercial use, with downloads available for Windows, Linux, and macOS from .
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  10. 10
    Cao, C.; Cirauqui, N.; Marcaida, M. J.; Buglakova, E.; Duperrex, A.; Radenovic, A.; Dal Peraro, M. Single-Molecule Sensing of Peptides and Nucleic Acids by Engineered Aerolysin Nanopores. Nat. Commun. 2019, 10 (1), 4918,  DOI: 10.1038/s41467-019-12690-9
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    10
    Single-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores
    Cao Chan; Cirauqui Nuria; Marcaida Maria Jose; Buglakova Elena; Duperrex Alice; Dal Peraro Matteo; Cao Chan; Marcaida Maria Jose; Dal Peraro Matteo; Cirauqui Nuria; Buglakova Elena; Radenovic Aleksandra
    Nature communications (2019), 10 (1), 4918 ISSN:.
    Nanopore sensing is a powerful single-molecule approach for the detection of biomolecules. Recent studies have demonstrated that aerolysin is a promising candidate to improve the accuracy of DNA sequencing and to develop novel single-molecule proteomic strategies. However, the structure-function relationship between the aerolysin nanopore and its molecular sensing properties remains insufficiently explored. Herein, a set of mutated pores were rationally designed and evaluated in silico by molecular simulations and in vitro by single-channel recording and molecular translocation experiments to study the pore structural variation, ion selectivity, ionic conductance and capabilities for sensing several biomolecules. Our results show that the ion selectivity and sensing ability of aerolysin are mostly controlled by electrostatics and the narrow diameter of the double β-barrel cap. By engineering single-site mutants, a more accurate molecular detection of nucleic acids and peptides has been achieved. These findings open avenues for developing aerolysin nanopores into powerful sensing devices.
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  11. 11
    Cressiot, B.; Ouldali, H.; Pastoriza-Gallego, M.; Bacri, L.; Van der Goot, F. G.; Pelta, J. Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications. ACS Sens. 2019, 4 (3), 530– 548,  DOI: 10.1021/acssensors.8b01636
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    11
    Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications
    Cressiot, Benjamin; Ouldali, Hadjer; Pastoriza-Gallego, Manuela; Bacri, Laurent; Van der Goot, F. Gisou; Pelta, Juan
    ACS Sensors (2019), 4 (3), 530-548CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society)
    A review. The nanopore elec. approach is a breakthrough in single mol. level detection of particles as small as ions, and complex as biomols. This technique can be used for mol. anal. and characterization as well as for the understanding of confined medium dynamics in chem. or biol. reactions. Altogether, the information obtained from these kinds of expts. will allow us to address challenges in a variety of biol. fields. The sensing, design, and manuf. of nanopores is crucial to realize these objectives. For some time now, aerolysin, a pore forming toxin, and its mutants have shown high potential in real time anal. chem., size discrimination of neutral polymers, oligosaccharides, oligonucleotides and peptides at monomeric resoln., sequence identification, chem. modification on DNA, potential biomarkers detection, and protein folding anal. This review focuses on the results obtained with aerolysin nanopores on the fields of chem., biol., physics, and biotechnol. We discuss and compare as well the results obtained with other protein channel sensors.
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  12. 12
    Cao, C.; Li, M.-Y.; Cirauqui, N.; Wang, Y.-Q.; Dal Peraro, M.; Tian, H.; Long, Y.-T. Mapping the Sensing Spots of Aerolysin for Single Oligonucleotides Analysis. Nat. Commun. 2018, 9 (1), 2823,  DOI: 10.1038/s41467-018-05108-5
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    12
    Mapping the sensing spots of aerolysin for single oligonucleotides analysis
    Cao Chan; Li Meng-Yin; Wang Ya-Qian; Tian He; Long Yi-Tao; Cao Chan; Cirauqui Nuria; Dal Peraro Matteo; Cirauqui Nuria
    Nature communications (2018), 9 (1), 2823 ISSN:.
    Nanopore sensing is a powerful single-molecule method for DNA and protein sequencing. Recent studies have demonstrated that aerolysin exhibits a high sensitivity for single-molecule detection. However, the lack of the atomic resolution structure of aerolysin pore has hindered the understanding of its sensing capabilities. Herein, we integrate nanopore experimental results and molecular simulations based on a recent pore structural model to precisely map the sensing spots of this toxin for ssDNA translocation. Rationally probing ssDNA length and composition upon pore translocation provides new important insights for molecular determinants of the aerolysin nanopore. Computational and experimental results reveal two critical sensing spots (R220, K238) generating two constriction points along the pore lumen. Taking advantage of the sensing spots, all four nucleobases, cytosine methylation and oxidation of guanine can be clearly identified in a mixture sample. The results provide evidence for the potential of aerolysin as a nanosensor for DNA sequencing.
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  13. 13
    Fennouri, A.; Przybylski, C.; Pastoriza-Gallego, M.; Bacri, L.; Auvray, L.; Daniel, R. Single Molecule Detection of Glycosaminoglycan Hyaluronic Acid Oligosaccharides and Depolymerization Enzyme Activity Using a Protein Nanopore. ACS Nano 2012, 6 (11), 9672– 9678,  DOI: 10.1021/nn3031047
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    13
    Single Molecule Detection of Glycosaminoglycan Hyaluronic Acid Oligosaccharides and Depolymerization Enzyme Activity Using a Protein Nanopore
    Fennouri, Aziz; Przybylski, Cedric; Pastoriza-Gallego, Manuela; Bacri, Laurent; Auvray, Loic; Daniel, Regis
    ACS Nano (2012), 6 (11), 9672-9678CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Glycosaminoglycans are biol. active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural anal. and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein anal. to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosaminoglycan and its depolymn.-derived oligosaccharides. The translocation of a glycosaminoglycan is reported using aerolysin protein nanopore. Nanopore translocation of hyaluronic acid oligosaccharides was evidenced by the direct detection of translocated mols. accumulated into the arrival compartment using high-resoln. mass spectrometry. Anionic oligosaccharides of various polymn. degrees were discriminated through measurement of the dwelling time and translocation frequency. This mol. sizing capability of the protein nanopore device allowed the real-time recording of the enzymic cleavage of hyaluronic acid polysaccharide. The time-resolved detection of enzymically produced oligosaccharides was carried out to monitor the depolymn. enzyme reaction at the single-mol. level.
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  14. 14
    Piguet, F.; Ouldali, H.; Pastoriza-Gallego, M.; Manivet, P.; Pelta, J.; Oukhaled, A. Identification of Single Amino Acid Differences in Uniformly Charged Homopolymeric Peptides with Aerolysin Nanopore. Nat. Commun. 2018, 9 (1), 966,  DOI: 10.1038/s41467-018-03418-2
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    14
    Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore
    Piguet Fabien; Ouldali Hadjer; Pastoriza-Gallego Manuela; Oukhaled Abdelghani; Manivet Philippe; Manivet Philippe; Pelta Juan
    Nature communications (2018), 9 (1), 966 ISSN:.
    There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues. Here, we demonstrate that the wild-type aerolysin nanopore enables the size-discrimination of several short uniformly charged homopeptides, mixed in solution, with a single amino acid resolution. Our system is very sensitive, allowing detecting and characterizing a few dozens of peptide impurities in a high purity commercial peptide sample, while conventional analysis techniques fail to do so.
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  15. 15
    Ouldali, H.; Sarthak, K.; Ensslen, T.; Piguet, F.; Manivet, P.; Pelta, J.; Behrends, J. C.; Aksimentiev, A.; Oukhaled, A. Electrical Recognition of the Twenty Proteinogenic Amino Acids Using an Aerolysin Nanopore. Nat. Biotechnol. 2020, 38 (2), 176– 181,  DOI: 10.1038/s41587-019-0345-2
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    15
    Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore
    Ouldali, Hadjer; Sarthak, Kumar; Ensslen, Tobias; Piguet, Fabien; Manivet, Philippe; Pelta, Juan; Behrends, Jan C.; Aksimentiev, Aleksei; Oukhaled, Abdelghani
    Nature Biotechnology (2020), 38 (2), 176-181CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
    Efforts to sequence single protein mols. in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle mol. 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 mol. dynamics simulations revealed that the aerolysin nanopore has a built-in single-mol. 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 vol. 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 chem. 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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  16. 16
    Afshar Bakshloo, M.; Kasianowicz, J. J.; Pastoriza-Gallego, M.; Mathé, J.; Daniel, R.; Piguet, F.; Oukhaled, A. Nanopore-Based Protein Identification. J. Am. Chem. Soc. 2022, 144 (6), 2716– 2725,  DOI: 10.1021/jacs.1c11758
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    16
    Nanopore-Based Protein Identification
    Afshar Bakshloo, Mazdak; Kasianowicz, John J.; Pastoriza-Gallego, Manuela; Mathe, Jerome; Daniel, Regis; Piguet, Fabien; Oukhaled, Abdelghani
    Journal of the American Chemical Society (2022), 144 (6), 2716-2725CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biol. research and clin. medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-mol. limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-sol. proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.
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  17. 17
    Bhatti, H.; Jawed, R.; Ali, I.; Iqbal, K.; Han, Y.; Lu, Z.; Liu, Q. Recent Advances in Biological Nanopores for Nanopore Sequencing, Sensing and Comparison of Functional Variations in MspA Mutants. RSC Adv. 2021, 11 (46), 28996– 29014,  DOI: 10.1039/D1RA02364K
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    Lu, S.-M.; Wu, X.-Y.; Li, M.-Y.; Ying, Y.-L.; Long, Y.-T. Diversified Exploitation of Aerolysin Nanopore in Single-Molecule Sensing and Protein Sequencing. VIEW 2020, 1 (4), 20200006  DOI: 10.1002/VIW.20200006
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    Gomopoulos, N.; Lütgebaucks, C.; Sun, Q.; Macias-Romero, C.; Roke, S. Label-Free Second Harmonic and Hyper Rayleigh Scattering with High Efficiency. Opt. Express 2013, 21 (1), 815,  DOI: 10.1364/OE.21.000815
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    19
    Label-free second harmonic and hyper Rayleigh scattering with high efficiency
    Gomopoulos, Nikolaos; Lutgebaucks, Cornelis; Sun, Qinchao; Macias-Romero, Carlos; Roke, Sylvie
    Optics Express (2013), 21 (1), 815-821CODEN: OPEXFF; ISSN:1094-4087. (Optical Society of America)
    We present a method to perform hyper Rayleigh scattering from aq. solns. and second harmonic scattering measurements from unlabeled interfaces of liposomes and nanoparticles in dil. solns. The water and interfacial response can be measured on a millisecond timescale, thus opening up the possibility to measure label-free time dependent transport processes in biol. (membrane) systems.
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  20. 20
    Lütgebaucks, C.; Gonella, G.; Roke, S. Optical Label-Free and Model-Free Probe of the Surface Potential of Nanoscale and Microscopic Objects in Aqueous Solution. Phys. Rev. B 2016, 94 (19), 195410,  DOI: 10.1103/PhysRevB.94.195410
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    20
    Optical label-free and model-free probe of the surface potential of nanoscale and microscopic objects in aqueous solution
    Luetgebaucks, Cornelis; Gonella, Grazia; Roke, Sylvie
    Physical Review B (2016), 94 (19), 195410/1-195410/6CODEN: PRBHB7; ISSN:2469-9950. (American Physical Society)
    The electrostatic environment of aq. systems is an essential ingredient for the function of any living system. To understand the electrostatic properties and their mol. foundation in soft, living, and three-dimensional systems, we developed a table-top model-free method to det. the surface potential of nano- and microscopic objects in aq. solns. Angle-resolved nonresonant second harmonic (SH) scattering measurements contain enough information to det. the surface potential unambiguously, without making assumptions on the structure of the interfacial region. The scattered SH light that is emitted from both the particle interface and the diffuse double layer can be detected in two different polarization states that have independent scattering patterns. The angular shape and intensity are detd. by the surface potential and the second-order surface susceptibility. Calibrating the response with the SH intensity of bulk water, a single. unique surface potential value can be extd. We demonstrate the method with 80 nm bare oil droplets in water and ∼50 nm dioleoylphos- phatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) liposomes at various ionic strengths.
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  21. 21
    Gonella, G.; Lütgebaucks, C.; de Beer, A. G. F.; Roke, S. Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference. J. Phys. Chem. C 2016, 120 (17), 9165– 9173,  DOI: 10.1021/acs.jpcc.5b12453
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    21
    Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference
    Gonella, Grazia; Luetgebaucks, Cornelis; de Beer, Alex G. F.; Roke, Sylvie
    Journal of Physical Chemistry C (2016), 120 (17), 9165-9173CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
    The interfacial region of aq. systems also known as the elec. double layer can be characterized on the mol. level with 2nd harmonic and sum-frequency generation (SHG/SFG). SHG and SFG are surface specific methods for isotropic liqs. Here, the authors model the SHG/SFG intensity in reflection, transmission, and scattering geometry taking into account the spatial variation of all fields. In the presence of a surface electrostatic field, interference effects, which originate from oriented H2O mols. on a length scale over which the potential decays, can strongly modify the probing depth as well as the expected intensity at ionic strengths <10-3 M. For reflection expts. this interference phenomenon leads to a significant redn. of the SHG/SFG intensity. Transmission mode expts. from aq. interfaces are hardly influenced. For SHG/SFG scattering expts. the same interference increases intensity and to modified scattering patterns. The predicted scattering patterns are verified exptl.
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  22. 22
    Schönfeldová, T.; Piller, P.; Kovacik, F.; Pabst, G.; Okur, H. I.; Roke, S. Lipid Melting Transitions Involve Structural Redistribution of Interfacial Water. J. Phys. Chem. B 2021, 125 (45), 12457– 12465,  DOI: 10.1021/acs.jpcb.1c06868
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    22
    Lipid Melting Transitions Involve Structural Redistribution of Interfacial Water
    Schonfeldova, Tereza; Piller, Paulina; Kovacik, Filip; Pabst, Georg; Okur, Halil I.; Roke, Sylvie
    Journal of Physical Chemistry B (2021), 125 (45), 12457-12465CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
    Morphol. and gel-to-liq. phase transitions of lipid membranes are generally considered to primarily depend on the structural motifs in the hydrophobic core of the bilayer. Structural changes in the aq. headgroup phase are typically not considered, primarily because they are difficult to quantify. Here, the authors study structural changes of the hydration shells around large unilamellar vesicles (LUVs) in aq. soln., using DSC, and temp.-dependent ζ-potential and high-throughput angle-resolved second harmonic scattering measurements (AR-SHS). Varying the lipid compn. from 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) to 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), to 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), surprisingly distinct behavior for the different systems that depend on the chem. compn. of the hydrated headgroups. were obsd. These differences involve changes in hydration following temp.-induced counterion redistribution, or changes in hydration following headgroup reorientation and Stern layer compression.
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  23. 23
    Dedic, J.; Rocha, S.; Okur, H. I.; Wittung-Stafshede, P.; Roke, S. Membrane–Protein–Hydration Interaction of α-Synuclein with Anionic Vesicles Probed via Angle-Resolved Second-Harmonic Scattering. J. Phys. Chem. B 2019, 123 (5), 1044– 1049,  DOI: 10.1021/acs.jpcb.8b11096
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    Membrane-protein-hydration interaction of α-synuclein with anionic vesicles probed via angle-resolved second-harmonic scattering
    Dedic, Jan; Rocha, Sandra; Okur, Halil I.; Wittung-Stafshede, Pernilla; Roke, Sylvie
    Journal of Physical Chemistry B (2019), 123 (5), 1044-1049CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
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  • Abstract

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    Figure 1

    Figure 1. Structure of aerolysin in different states and how to measure its membrane association. A. Structure of monomeric pro-aerolysin (PDB: 1PRE): Domain 1 in gray, domain 2 in blue, domain 3 in yellow, and domain 4 in green. Illustration of the aerolysin structure in prepore (B), quasi-pore (C), and pore (D) states and labels of the various mutations with localization in panel A. The images were generated in UCSF ChimeraX. (9) E. Energy-level scheme and sketch of the AR-SHS experiment. P(S) refers to the polarization state of the beam parallel (perpendicular) to the scattering plane. All measurements were recorded with all beams polarized in the horizontal plane. For the single-angle experiments, the scattering angle θ was set to 45°, corresponding to the angle with maximum scattering intensity. Adapted with permission from ref (20). Copyright [2024] [American Physical Society] F. Top: Illustration of how interfacial electric fields (E⃗) orient dipolar water (indicated by the symbol p⃗) and how this is affected by aerolysin–membrane interaction. Here, charge-water interaction is the most relevant physical mechanism for creating the SH contrast. Bottom: Example SHS pattern of LUVs composed of 99:1 mol % DOPC:DOPA membranes interacting with 5 × 10–10 M wt aerolysin in aqueous solutions having pH values of 4 (black data) and 9 (red data). One mol % DOPA was used to increase the signal-to-noise ratio for these measurements. The maximum interfacial SH intensity occurs around θ = 45°. Figure S1 shows how this graph was generated.

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    Figure 2

    Figure 2. pH-dependent surface charge and membrane binding affinities. A. Second harmonic intensity difference relative to bulk water (S) at the scattering angle with maximum intensity (θmax = 45°) vs pH of the aqueous solution. The measurements were performed with DOPC liposomes and wt aerolysin. The blue and red highlighted areas indicate the positive and negative surface charges on the cap region of aerolysin. B. Electrostatic potential mapped on the cross sections of the aerolysin cap domain obtained via the APBS Web server (26) depicting the charge distribution of the pore (blue: positive charge; red: negative charge) visualized in PyMol. (27) C, D. A representative single-channel current recording traces of the aerolysin-DOPC free-standing membrane system in aqueous solution at pH 5 (C) and 7.4 (D), with the corresponding histogram current distribution, where each step indicating that another pore incorporation is counted (numbered by #). E, F. Normalized SH intensity difference (ΔS) at the angle with the maximum intensity (θmax = 45°) vs wt aerolysin concentration on the logarithmic scale at pH 5 (E) and 7.4 (F). The data are fitted using eq S2, giving a dissociation constant of Kd = (6.2 ± 0.4) × 10–14 M for pH 5 and Kd = (2.0 ± 0.2) 10–14 M for pH 7.4 (represented by a dashed line). The error bars were determined as the standard deviation from 100 measurements for all of the AR-SHS measurements. The AR-SHS measurements were performed with liposomes composed of 99:1 mol % DOPC:DOPS liposomes interacting with wt aerolysin.

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    Figure 3

    Figure 3. Aerolysin single-point mutant-lipid membrane binding in aqueous solution. A, B. A representative single-channel current recording measurement of R220A (A) and K238N (B) measured with a DOPC free-standing membrane at pH 7.4. C, D. SH intensity difference (ΔS) at the angle with maximum intensity (θmax = 45°) vs R220A (C) and K238N (D) concentration on the logarithmic scale at pH 7.4. The data are fitted using eq S2, giving dissociation constants of Kd = (2.3 ± 0.3) 10–12 M and Kd = (4.6 ± 0.6) 10–14 M for R220A and K238N, respectively (the dashed line represents the dissociation constant). The data in parts C and D were measured with DOPC doped with 1% DOPS liposomes. The error bars were determined as the standard deviation from 100 measurements for all of the AR-SHS measurements. The dotted line represents the fitted Kd value for each aerolysin we tested in A and B.

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    Figure 4

    Figure 4. Membrane properties of aerolysin mutants blocked at different stages of pore formation. A, B. Structural model of the aerolysin quasi-pore (A) and prepore (B). The images were generated in UCSF ChimeraX. (9) C, D. Representative single-channel current recording measurements of the quasi-pore (C) and prepore (D) at pH 7.4 on a DOPC free-standing membrane. E, F. SH intensity difference (ΔS) recorded at the scattering angle having the maximum intensity (θmax = 45°) vs K246C-E258C (E) and Y221G (F) concentration on a logarithmic scale. The data were fitted using eq S2, giving a dissociation constant of Kd = (4.1 ± 0.3) × 10–13 M for K246C-E258C (quasi-pore). These data were measured with DOPC doped with 1% DOPS liposomes at pH 7.4. The error bars were determined as a standard deviation from 100 measurements for all of the AR-SHS measurements. The dotted line represents the fitted Kd value for each aerolysin we tested in A.

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  • References

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      Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore
      Piguet Fabien; Ouldali Hadjer; Pastoriza-Gallego Manuela; Oukhaled Abdelghani; Manivet Philippe; Manivet Philippe; Pelta Juan
      Nature communications (2018), 9 (1), 966 ISSN:.
      There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues. Here, we demonstrate that the wild-type aerolysin nanopore enables the size-discrimination of several short uniformly charged homopeptides, mixed in solution, with a single amino acid resolution. Our system is very sensitive, allowing detecting and characterizing a few dozens of peptide impurities in a high purity commercial peptide sample, while conventional analysis techniques fail to do so.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1MrotlersQ%253D%253D&md5=4cbb5ce0d16d6cf5c29c56219ff49926
    15. 15
      Ouldali, H.; Sarthak, K.; Ensslen, T.; Piguet, F.; Manivet, P.; Pelta, J.; Behrends, J. C.; Aksimentiev, A.; Oukhaled, A. Electrical Recognition of the Twenty Proteinogenic Amino Acids Using an Aerolysin Nanopore. Nat. Biotechnol. 2020, 38 (2), 176– 181,  DOI: 10.1038/s41587-019-0345-2
      15
      Electrical recognition of the twenty proteinogenic amino acids using an aerolysin nanopore
      Ouldali, Hadjer; Sarthak, Kumar; Ensslen, Tobias; Piguet, Fabien; Manivet, Philippe; Pelta, Juan; Behrends, Jan C.; Aksimentiev, Aleksei; Oukhaled, Abdelghani
      Nature Biotechnology (2020), 38 (2), 176-181CODEN: NABIF9; ISSN:1087-0156. (Nature Research)
      Efforts to sequence single protein mols. in nanopores1-5 have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle mol. 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 mol. dynamics simulations revealed that the aerolysin nanopore has a built-in single-mol. 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 vol. 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 chem. 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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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXisVSktb3J&md5=20f02aa0531855896b80e6ba5800deaa
    16. 16
      Afshar Bakshloo, M.; Kasianowicz, J. J.; Pastoriza-Gallego, M.; Mathé, J.; Daniel, R.; Piguet, F.; Oukhaled, A. Nanopore-Based Protein Identification. J. Am. Chem. Soc. 2022, 144 (6), 2716– 2725,  DOI: 10.1021/jacs.1c11758
      16
      Nanopore-Based Protein Identification
      Afshar Bakshloo, Mazdak; Kasianowicz, John J.; Pastoriza-Gallego, Manuela; Mathe, Jerome; Daniel, Regis; Piguet, Fabien; Oukhaled, Abdelghani
      Journal of the American Chemical Society (2022), 144 (6), 2716-2725CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biol. research and clin. medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-mol. limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-sol. proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XislOhs70%253D&md5=875c7ef22d4a2bdb19f4fc997df27c86
    17. 17
      Bhatti, H.; Jawed, R.; Ali, I.; Iqbal, K.; Han, Y.; Lu, Z.; Liu, Q. Recent Advances in Biological Nanopores for Nanopore Sequencing, Sensing and Comparison of Functional Variations in MspA Mutants. RSC Adv. 2021, 11 (46), 28996– 29014,  DOI: 10.1039/D1RA02364K
      There is no corresponding record for this reference.
    18. 18
      Lu, S.-M.; Wu, X.-Y.; Li, M.-Y.; Ying, Y.-L.; Long, Y.-T. Diversified Exploitation of Aerolysin Nanopore in Single-Molecule Sensing and Protein Sequencing. VIEW 2020, 1 (4), 20200006  DOI: 10.1002/VIW.20200006
      There is no corresponding record for this reference.
    19. 19
      Gomopoulos, N.; Lütgebaucks, C.; Sun, Q.; Macias-Romero, C.; Roke, S. Label-Free Second Harmonic and Hyper Rayleigh Scattering with High Efficiency. Opt. Express 2013, 21 (1), 815,  DOI: 10.1364/OE.21.000815
      19
      Label-free second harmonic and hyper Rayleigh scattering with high efficiency
      Gomopoulos, Nikolaos; Lutgebaucks, Cornelis; Sun, Qinchao; Macias-Romero, Carlos; Roke, Sylvie
      Optics Express (2013), 21 (1), 815-821CODEN: OPEXFF; ISSN:1094-4087. (Optical Society of America)
      We present a method to perform hyper Rayleigh scattering from aq. solns. and second harmonic scattering measurements from unlabeled interfaces of liposomes and nanoparticles in dil. solns. The water and interfacial response can be measured on a millisecond timescale, thus opening up the possibility to measure label-free time dependent transport processes in biol. (membrane) systems.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXivVOls7w%253D&md5=3a06ed6ae1c8ed47c8dd6ccb6ac871af
    20. 20
      Lütgebaucks, C.; Gonella, G.; Roke, S. Optical Label-Free and Model-Free Probe of the Surface Potential of Nanoscale and Microscopic Objects in Aqueous Solution. Phys. Rev. B 2016, 94 (19), 195410,  DOI: 10.1103/PhysRevB.94.195410
      20
      Optical label-free and model-free probe of the surface potential of nanoscale and microscopic objects in aqueous solution
      Luetgebaucks, Cornelis; Gonella, Grazia; Roke, Sylvie
      Physical Review B (2016), 94 (19), 195410/1-195410/6CODEN: PRBHB7; ISSN:2469-9950. (American Physical Society)
      The electrostatic environment of aq. systems is an essential ingredient for the function of any living system. To understand the electrostatic properties and their mol. foundation in soft, living, and three-dimensional systems, we developed a table-top model-free method to det. the surface potential of nano- and microscopic objects in aq. solns. Angle-resolved nonresonant second harmonic (SH) scattering measurements contain enough information to det. the surface potential unambiguously, without making assumptions on the structure of the interfacial region. The scattered SH light that is emitted from both the particle interface and the diffuse double layer can be detected in two different polarization states that have independent scattering patterns. The angular shape and intensity are detd. by the surface potential and the second-order surface susceptibility. Calibrating the response with the SH intensity of bulk water, a single. unique surface potential value can be extd. We demonstrate the method with 80 nm bare oil droplets in water and ∼50 nm dioleoylphos- phatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) liposomes at various ionic strengths.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXovVynsrk%253D&md5=614897848a7f61c36ffebc0d992b32ad
    21. 21
      Gonella, G.; Lütgebaucks, C.; de Beer, A. G. F.; Roke, S. Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference. J. Phys. Chem. C 2016, 120 (17), 9165– 9173,  DOI: 10.1021/acs.jpcc.5b12453
      21
      Second Harmonic and Sum-Frequency Generation from Aqueous Interfaces Is Modulated by Interference
      Gonella, Grazia; Luetgebaucks, Cornelis; de Beer, Alex G. F.; Roke, Sylvie
      Journal of Physical Chemistry C (2016), 120 (17), 9165-9173CODEN: JPCCCK; ISSN:1932-7447. (American Chemical Society)
      The interfacial region of aq. systems also known as the elec. double layer can be characterized on the mol. level with 2nd harmonic and sum-frequency generation (SHG/SFG). SHG and SFG are surface specific methods for isotropic liqs. Here, the authors model the SHG/SFG intensity in reflection, transmission, and scattering geometry taking into account the spatial variation of all fields. In the presence of a surface electrostatic field, interference effects, which originate from oriented H2O mols. on a length scale over which the potential decays, can strongly modify the probing depth as well as the expected intensity at ionic strengths <10-3 M. For reflection expts. this interference phenomenon leads to a significant redn. of the SHG/SFG intensity. Transmission mode expts. from aq. interfaces are hardly influenced. For SHG/SFG scattering expts. the same interference increases intensity and to modified scattering patterns. The predicted scattering patterns are verified exptl.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XlsFKqsLc%253D&md5=c7d8981e11dc280fe0ff4fe9420efbb7
    22. 22
      Schönfeldová, T.; Piller, P.; Kovacik, F.; Pabst, G.; Okur, H. I.; Roke, S. Lipid Melting Transitions Involve Structural Redistribution of Interfacial Water. J. Phys. Chem. B 2021, 125 (45), 12457– 12465,  DOI: 10.1021/acs.jpcb.1c06868
      22
      Lipid Melting Transitions Involve Structural Redistribution of Interfacial Water
      Schonfeldova, Tereza; Piller, Paulina; Kovacik, Filip; Pabst, Georg; Okur, Halil I.; Roke, Sylvie
      Journal of Physical Chemistry B (2021), 125 (45), 12457-12465CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Morphol. and gel-to-liq. phase transitions of lipid membranes are generally considered to primarily depend on the structural motifs in the hydrophobic core of the bilayer. Structural changes in the aq. headgroup phase are typically not considered, primarily because they are difficult to quantify. Here, the authors study structural changes of the hydration shells around large unilamellar vesicles (LUVs) in aq. soln., using DSC, and temp.-dependent ζ-potential and high-throughput angle-resolved second harmonic scattering measurements (AR-SHS). Varying the lipid compn. from 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) to 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), to 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS), surprisingly distinct behavior for the different systems that depend on the chem. compn. of the hydrated headgroups. were obsd. These differences involve changes in hydration following temp.-induced counterion redistribution, or changes in hydration following headgroup reorientation and Stern layer compression.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXitlygs73J&md5=75edd5a8de24fc29b4a4e1b18b025753
    23. 23
      Dedic, J.; Rocha, S.; Okur, H. I.; Wittung-Stafshede, P.; Roke, S. Membrane–Protein–Hydration Interaction of α-Synuclein with Anionic Vesicles Probed via Angle-Resolved Second-Harmonic Scattering. J. Phys. Chem. B 2019, 123 (5), 1044– 1049,  DOI: 10.1021/acs.jpcb.8b11096
      23
      Membrane-protein-hydration interaction of α-synuclein with anionic vesicles probed via angle-resolved second-harmonic scattering
      Dedic, Jan; Rocha, Sandra; Okur, Halil I.; Wittung-Stafshede, Pernilla; Roke, Sylvie
      Journal of Physical Chemistry B (2019), 123 (5), 1044-1049CODEN: JPCBFK; ISSN:1520-5207. (American Chemical Society)
      Amyloid formation of the protein, α-synuclein (αS), promotes neurodegeneration in Parkinson's disease. The normal function of αS includes synaptic vesicle transport and fusion, and the protein binds strongly to neg. charged vesicles in vitro. Here, we demonstrate that nonresonant angle-resolved 2nd-harmonic scattering detects αS binding to liposomes through changes in water orientational correlations and can thus be used as a high-accuracy and high-throughput label-free probe of protein-liposome interactions. The obtained results support a binding model in which the N-terminus of αS adopts an α-helical conformation that lies flat on the vesicle surface while the neg. charged C-terminus remains in soln.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXms1eksQ%253D%253D&md5=58523f8e865e9552b2d0669508a0fc84
    24. 24
      Roesel, D.; Eremchev, M.; Schönfeldová, T.; Lee, S.; Roke, S. Water as a Contrast Agent to Quantify Surface Chemistry and Physics Using Second Harmonic Scattering and Imaging: A Perspective. Appl. Phys. Lett. 2022, 120 (16), 160501,  DOI: 10.1063/5.0085807
      There is no corresponding record for this reference.
    25. 25
      Schönfeldová, T.; Okur, H. I.; Vezočnik, V.; Iacovache, I.; Cao, C.; Dal Peraro, M.; Maček, P.; Zuber, B.; Roke, S. Ultrasensitive Label-Free Detection of Protein–Membrane Interaction Exemplified by Toxin-Liposome Insertion. J. Phys. Chem. Lett. 2022, 13 (14), 3197– 3201,  DOI: 10.1021/acs.jpclett.1c04011
      There is no corresponding record for this reference.
    26. 26
      Jurrus, E.; Engel, D.; Star, K.; Monson, K.; Brandi, J.; Felberg, L. E.; Brookes, D. H.; Wilson, L.; Chen, J.; Liles, K.; Chun, M.; Li, P.; Gohara, D. W.; Dolinsky, T.; Konecny, R.; Koes, D. R.; Nielsen, J. E.; Head-Gordon, T.; Geng, W.; Krasny, R.; Wei, G.-W.; Holst, M. J.; McCammon, J. A.; Baker, N. A. Improvements to the APBS Biomolecular Solvation Software Suite. Protein Sci. 2018, 27 (1), 112– 128,  DOI: 10.1002/pro.3280
      26
      Improvements to the APBS biomolecular solvation software suite
      Jurrus, Elizabeth; Engel, Dave; Star, Keith; Monson, Kyle; Brandi, Juan; Felberg, Lisa E.; Brookes, David H.; Wilson, Leighton; Chen, Jiahui; Liles, Karina; Chun, Minju; Li, Peter; Gohara, David W.; Dolinsky, Todd; Konecny, Robert; Koes, David R.; Nielsen, Jens Erik; Head-Gordon, Teresa; Geng, Weihua; Krasny, Robert; Wei, Guo-Wei; Holst, Michael J.; McCammon, J. Andrew; Baker, Nathan A.
      Protein Science (2018), 27 (1), 112-128CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      The Adaptive Poisson-Boltzmann Solver (APBS) software was developed to solve the equations of continuum electrostatics for large biomol. assemblages that have provided impact in the study of a broad range of chem., biol., and biomedical applications. APBS addresses the three key technol. challenges for understanding solvation and electrostatics in biomedical applications: accurate and efficient models for biomol. solvation and electrostatics, robust and scalable software for applying those theories to biomol. systems, and mechanisms for sharing and analyzing biomol. electrostatics data in the scientific community. To address new research applications and advancing computational capabilities, we have continually updated APBS and its suite of accompanying software since its release in 2001. In this article, we discuss the models and capabilities that have recently been implemented within the APBS software package including a Poisson-Boltzmann anal. and a semi-anal. solver, an optimized boundary element solver, a geometry-based geometric flow solvation model, a graph theory-based algorithm for detg. pKa values, and an improved web-based visualization tool for viewing electrostatics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslSkt7vI&md5=99651d125e38f4a85d453fecf0f71652
    27. 27
      The PyMOL Molecular Graphics System, Version 2.9; Schrödinger, LLC.
      There is no corresponding record for this reference.
    28. 28
      Iacovache, I.; Paumard, P.; Scheib, H.; Lesieur, C.; Sakai, N.; Matile, S.; Parker, M. W.; van der Goot, F. G. A Rivet Model for Channel Formation by Aerolysin-like Pore-Forming Toxins. EMBO J. 2006, 25 (3), 457– 466,  DOI: 10.1038/sj.emboj.7600959
      There is no corresponding record for this reference.
    29. 29
      Sauciuc, A.; Morozzo della Rocca, B.; Tadema, M. J.; Chinappi, M.; Maglia, G. Translocation of Linearized Full-Length Proteins through an Engineered Nanopore under Opposing Electrophoretic Force. Nat. Biotechnol. 2024, 42 (8), 1275– 1281,  DOI: 10.1038/s41587-023-01954-x
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.4c00035.

    • Additional experimental details and materials and methods for protein expression, single-channel recordings and AR-SHS experiments including Figure S1 showing the polar plots of raw data and resultant S(θ) values, Table S1 containing the number of amino acids in aerolysin, and Table S2 containing the number of experiments performed for each mutant (PDF)


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