Autonomously Assembled Synthetic Transmembrane Peptide Pore

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An Autonomously Assembled Synthetic Transmembrane Peptide Pore Smrithi Krishnan R, Remya Satheesan, Neethu Puthumadathil, K. Santhosh Kumar, Poornendhu Jayasree, and Kozhinjampara R Mahendran J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09973 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Journal of the American Chemical Society

Submitted to JACS

An Autonomously Assembled Synthetic Transmembrane Peptide Pore Smrithi Krishnan R1, Remya Satheesan1, Neethu Puthumadathil1, K Santhosh Kumar1, Poornendhu Jayasree1 and Kozhinjampara R Mahendran1*

1Membrane

Biology Laboratory, Interdisciplinary Research Program, Rajiv Gandhi Centre for

Biotechnology, Thiruvananthapuram 695014, India.

*To whom correspondence should be addressed *e-mail: [email protected]

1 ACS Paragon Plus Environment

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Submitted to JACS Abstract: The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-amino acid, synthetic peptide, pPorA corresponding to porin PorACj inserts into the lipid bilayers and form well-defined pores. By electrical recordings, we measured the single channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site-specific chemical modification in single channel electrical recordings and gel electrophoresis. Based on these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic α-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large α-helix based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.

Keywords: Pore, peptides, alpha-helical, cyclodextrins, Single channel, lipid bilayer


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Submitted to JACS Introduction: Membrane proteins are found in all major groups of organisms and are involved in a wide variety of functions1. They mainly consist of two structural classes: α-helix bundles and β-barrels that are typically hydrophobic in nature2,3. Engineering membrane proteins into membrane-based biosensors for applications in biotechnology are one of the main approaches within the protein engineering field4,5,6,7,8. Previously, β-barrel heptameric α-hemolysin (αHL) pore derived from Staphylococcus aureus has been engineered extensively for the single-molecule sensing of a wide variety of analytes including nucleic acids5,7,9,10. Remarkably, Oxford Nanopore has developed a nanopore-based device containing numerous engineered β-barrel protein pores in a chip for DNA sequencing5, 11. Recently, there is considerable interest in developing better transmembrane pores for single-molecule sensing of complex biopolymers such as nucleic acid fragments, peptides, polysaccharides etc5,6. Importantly, most of the natural ion channels consist of α-helical bundles that have great potential in engineering new pores with sophisticated architecture and specific functionality12,13,14. Engineering membrane pores based on α-helices remain relatively underexplored due to their complex non-specific associations in the lipid environment15,16,17,18. Most previous efforts in this area have focused on membrane-active amphipathic α-helical antimicrobial peptides (AMPs)19,20,21. Despite this, assembly of well-defined uniform AMP pores in the lipid membranes have not been demonstrated previously, as the amphipathic helices tend to rupture membranes nonspecifically16,22,23.

Engineered membrane pores such as 12-helix ClyA pore, 8-helix FraC, de novo designed α-helical bundles including a 4-helix divalent metal-ion transporter were developed17,24,25,26. More recently, a monodisperse transmembrane barrel comprising eight parallel helices made of 35-amino-acids



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Submitted to JACS derived from the D4 domain of the Escherichia coli Wza polysaccharide translocon was constructed27,28. However, the assembly pathway of this pore consists of intermediate conducting steps, and require cyclodextrin scaffolds to support stable pore27. Until now, the use of narrow pores of few nanometer internal diameters has limited their application in nanopore technology5,6,7,29,30,31. Accordingly, transmembrane alpha helix pores having new structural motifs with larger inner diameter would expand the scope of single-molecule sensing of a wide variety of large, diverse biomolecules.

In this work, we are fabricating larger α-helical pores based on porin PorACj of Corynebacterium jeikeium32. Previous studies report that the 40 amino acid long PorACj protein expressed in Corynebacterium and E. coli strains form an anion-selective channel of varying conductance in lipid bilayers indicating the possibility of multimeric forms. The structure of PorACj is not solved till date, but the pore is predicted to be either homo-hexameric or octameric containing heptameric repeat motifs indicating the presence of α-helical structures which allow the protein to form an amphipathic helix32 (Figure 1a and Supplementary Text). Here, we synthesized the peptide, pPorA corresponding to PorACj porins and explored the pore-forming functional properties of peptides and elucidated the structural assembly pathway of the pore. We assembled a large diameter synthetic monodisperse pore with a single oligomerization state with specificity and distinct functions. This rationally designed peptide pores with refined architecture can be exploited for several applications in bio-nanotechnology and single-molecule chemistry. Additionally, contributing to our mechanistic understanding of antimicrobial peptides and pore-forming toxins. Results


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Submitted to JACS Biophysical and electrical properties of peptide pores: The 40-residue long peptide (pPorA) derived from porin PorACj of C. jeikeium was made using solid-phase peptide synthesis and purified by reversed-phase high-performance liquid chromatography (HPLC). Their molecular mass was subsequently confirmed by mass spectrometry (Figure 1a and Supplementary Figure S1).

Figure 1: Structure of pPorA, biophysical and electrical properties of the pPorA. a) Structure of the modeled pPorA and the pPorA peptide sequence. b) CD spectra at 20°C for pPorA in PBS with 1% DDM at 25 µM (blue) and 50 µM((red) peptide concentration. c) Electrical recording of multiple insertions of pPorA peptide pores into a planar bilayer at +50 mV. Each step represents the insertion of a single pore and corresponding all-points histogram is shown in inset d) Single pPorA pore insertion at +100 mV. e) Histogram of the unitary conductance at +50 mV by fitting the distribution to a Gaussian (n = 100). f) I-V curve obtained from a single pPorA 5 ACS Paragon Plus Environment


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Submitted to JACS pore. g) Reverse potential obtained from the I-V curve of a single pPorA pore. h) Electrical recording of pPorA showing gating at +100 mV. The current signals were filtered at 2 kHz and sampled at 10 kHz. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4. Notably, pPorA peptide exists as insoluble aggregates in phosphate-buffered saline (PBS) that is solubilized in the detergents. Circular dichroism (CD) spectra confirmed the secondary structures of pPorA which exist in α-helical conformation in 1% n-dodecyl β-D-maltoside (DDM) micelles (Figure 1b and Supplementary Figure S2). We explored the pore-forming electrical properties of pPorA by high-resolution single channel recording in planar lipid bilayers. The pPorA peptide rapidly inserted into DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) bilayers with multiple stepwise pore-forming insertion events at +50 mV and single stepwise insertion at +100 mV (Figure 1c, 1d). Based on the numerous stepwise pore-forming events, conductance histograms were obtained (Supplementary Figure S2). The pore had a mean unitary conductance (G) of 3.0 ± 0.2 nS at +50 mV in 1M KCl, 10 mM HEPES, pH 7.4 (n = 100) that revealed the homogeneity of the pore. (Figure 1e). The pore showed slight asymmetry in the single-channel conductance, predominantly exhibiting rectification at the positive applied voltage. However, the magnitude of conductance asymmetry is negligible to conclude on the orientation of the pore (Figure 1f and Supplementary Figure S3). Next, we performed ion selectivity measurements and calculated the permeability ratio PK+/PCl- as ~ 1:3 which revealed that the pore is anion selective. (Figure 1g and Supplementary Text). Furthermore, at higher voltages (~ ±100 mV), the pore fluctuated between several conductance states (open and closed state) and showed voltage-dependent gating (Figure 1h). The gating mechanism is observed both at positive and negative voltages, and the exact molecular mechanism is not known (Supplementary Figure S3).


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Submitted to JACS Interaction of anionic cyclodextrins with pPorA pores: We examined the interaction of cyclodextrins (CDs) with pPorA peptide pores to demonstrate the functional properties of the pore. Due to the anion selectivity of the pore, the binding of differently sized anionic CDs were studied under the electrophoretic driving force applied across the pore at voltages ˂ ± 100 mV as the pore remained open for extended periods (Figure 2) 33,34.

Figure 2: Interaction of pPorA peptide pores with anionic cyclodextrins. a) Structure of s8γCD, electrical recordings of single pPorA in the absence and presence of s8γCD (1 mM, cis) at +50 mV with corresponding all-points current amplitude histogram. b) Structure of s6αCD, electrical recordings of single pPorA in the absence and presence of s6αCD (1 mM, cis) at +50 mV with corresponding all-points current amplitude histogram. c) Structure of cm8γCD, electrical recordings of single pPorA in the absence and presence of cm8γCD (1 mM, cis) at +50 7 ACS Paragon Plus Environment


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Submitted to JACS mV with corresponding all-points current amplitude histogram. d) Structure of neutral γCD and interaction with pPorA peptide pores at +50 mV (10 mM, cis). e) Structure of cationic am8γCD and interaction with pPorA peptide pores at +50 mV (10 mM, cis). The current signals were filtered at 2 kHz and sampled at 10 kHz. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4. An anionic cyclic octasaccharide sulfate, s8γCD, blocked the ion currents through pPorA peptide pores in a voltage-dependent manner. Addition of 1mM s8γCD to the cis compartment resulted in steady ion current blockade only at positive potentials (Figure 2a). For example, at +50 mV, s8γCD blocked the pore in a single step without reverting to an open state indicating strong binding and not translocation. Increase in the voltage to +75 mV produced steady current blockade rapidly whereas the pore remains in the fully open state at +20 mV (Supplementary Figure 4). Notably, by reversing the voltage to negative potential, we observed no blockage as the pore returns to its fully open state, which indicates that the applied voltage serves as a driving force to electrophoretically pull the anionic CDs to the pore (Supplementary Figure S4). In particular, s8γCD induced steady ion current blockades through WT pPorA pores does not depend on the concentration of the CDs. The residual current due to the CD interaction with the pore is same irrespective of different CD concentrations which suggest the entry of individual CD molecules into the pore (Figure 2a, Supplementary Figure S4 and Supplementary Table S1). As expected, the addition of s8γCD to the trans side resulted in steady current blockade only at negative potentials (Supplementary Figure S4). The binding of CDs suggests the distribution of positively charged residues along the pore surface, which serves as a binding site for electrostatic interaction between the anionic CDs and positively charged residues in agreement with ion selectivity measurements. In the next set of experiments, we tested the interaction of a smaller anionic cyclic hexasaccharide sulfate, s6αCD with pPorA peptide pores to elucidate the effect of size and charge on CD binding. At positive potentials, the addition of 1 mM s6αCD to the cis 8 ACS Paragon Plus Environment

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Submitted to JACS compartment resulted in steady ion current blockade and a single step pore closure indicating strong CD binding. Moreover, s6αCD induced steady ion current blockades through WT pPorA are obtained irrespective of the concentration of the CD used similar to s8γCD (Figure 2b, Supplementary Figure S5 and Supplementary Table S1). As expected, the pore is reverted to the fully open state by reversing the polarity of the voltage (Supplementary Figure S5). A similar trend of binding was obtained for the cis side addition of anionic cyclic carboxymethyl octasaccharide cm8γCD with pPorA peptide pores at positive voltages and hardly any binding at negative voltages. (Figure 2c, Supplementary Figure S6 and Supplementary Table S1). In addition, we have characterized the interaction of neutral octasaccharide γCD, hexasaccharide αCD and cationic octasaccharide am8γCD with pPorA pores. We observed no ion current blockage events even at a high concentration of (10 mM) CDs added symmetrically and asymmetrically to both sides of the membrane (Figure 2d, Figure 2e and Supplementary Figure S5) which establishes the role of positively charged residues of the pore lumen in capturing the anionic CDs.

Electrical and functional properties of pPorA cysteine mutant pores: We synthesized a cysteine peptide mutant pPorA-K24C assisted by the modeled structure of octameric porin PorACj of C. jeikeium to explore the pore structure by site-specific chemical modification. We rationally designed the mutant that the cysteine side-chains will point into the pore lumen (Figure 3a). Circular dichroism (CD) spectra showed alpha-helical conformation of pPorA-K24C in DDM micelles similar to WT pPorA (Figure 3b and Supplementary Figure S2). In single-channel electrical recordings, pPorA-K24C rapidly inserted into DPhPC bilayers with multiple stepwise pore-forming events even at a low voltage of +5 mV (Figure 3c and Supplementary Figure 7). The single step insertion of peptide pPorA-K24C was observed at +



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Submitted to JACS 50 mV confirming a highly stable well-defined pore (Figure 3d). Based on the conductance histogram, the unitary conductance of the pore was calculated to be 4.0 ± 0.2 nS at +50 mV (n = 100) which indicated the homogeneity and stability of the pore (Figure 3e). The conductance of the pore varied linearly with the applied potential (Figure 3f and Supplementary Figure 7). Next, we performed ion selectivity measurements and calculated the permeability ratio PK+/PCl- as ~ 10:1 which revealed that the pore is cation selective (n=25) (Figure 3g and Supplementary Text). Remarkably, the conductance of pPorA-K24C is much higher than that of WT revealing the large pore size of cysteine mutant.

Figure 3: Electrical properties and interaction of mutant pPorA pores with cyclodextrins a) Structure of the modeled pPorA-K24C showing the mutant peptide sequence. b) CD spectra at 20°C for pPorA-K24C in PBS with 1% DDM at 25 µM (blue) and 50 µM (red) peptide concentration. c) Electrical recording of multiple insertions of pPorA-K24C peptide pores into a 10 ACS Paragon Plus Environment

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Submitted to JACS planar bilayer at +5 mV d) Single pPorA-K24C pore insertion at +50 mV. e) Histogram of the unitary conductance at +50mV obtained by fitting the distribution to a Gaussian (n = 100). f) IV curve obtained from a single pPorA-K24C pore. g) Reverse potential obtained from the I-V curve of a single pPorA pore. h) Structure of th8γCD, i) the interaction of pPorA-K24C with th8γCD (2.5 µM, cis) at -25 mV, j) -50 mV and k) -75 mV. The corresponding dwell time histogram fitted with a monoexponential probability function is shown in the inset. The current signals were filtered at 2 kHz and sampled at 10 kHz. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4.

Next, we characterized the functional properties of pores by investigating the interaction of thiolated CDs with pPorA-K24C peptide pores harboring cysteine residues (Figure 3h). Addition of 2.5 μM thiolated cyclic octasaccharide, th8γCD to the cis compartment resulted in a timeresolved ion current blockage in a voltage-dependent manner (Figure 3h and Supplementary Table S1). Th8γCD blocked the pPorA-K24C pore with mean dwell times (off) of 0.6 ± 0.05 ms (n = 3) at -25 mV (Figure 3i), 0.5 ± 0.06 ms (n = 3) at -50 mV (Figure 3j) and 0.48 ± 0.5 ms (n = 3) at -75 mV (Figure 3k) indicating weak binding due to decrease in the dissociation rate constant (koff) with increasing voltages (Figure 3h and Supplementary Figure S8). Similar results were obtained for the smaller thiolated cyclic hexasaccharide, th6αCD, which also blocked the pPorAK24C pore (Supplementary Figure S9). As a control, we examined the interaction of th8γCD with WT pPorA pores that did not produce any ion current blockages even at high concentrations of CDs (Supplementary Figure S10). This data confirms that larger pore size and cysteine residues in the pPorA-K24C facilitate the binding of the thiolated CDs with the pore resulting in time-resolved ion current blockages.

Subsequently, we examined the interaction of differently sized and charged cationic CDs with cationic selective K24C peptide pore to elucidate the charge pattern and functional properties of 11 ACS Paragon Plus Environment


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Submitted to JACS the pore at the single-molecule level. Addition of 2.5 µM octasaccharide, am8γCD to the cis side resulted in time-resolved ion current blockages that increased with increase in the voltage at negative potentials (Figure 4a). The reversal of the voltage to the positive potential produced no blockage events which indicate that the applied voltage serves as a driving force to pull the charged CDs facilitating their interaction with negatively charged residues in the pore (Supplementary Figure S11).

Figure 4: Interaction of mutant pPorA peptide pores with cationic cyclodextrins. a) Structure of am8γCD, the interaction of mutant pPorA with am8γCD (1µM, cis) at -25 mV and -50 mV and plot of koff versus voltage for cis side addition of am8γCD (Mean values (±s.d.) from three independent experiments are shown). b) Structure of am7βCD, interaction of mutant pPorA with am7βCD (1 µM, cis) at -25 mV and -50 mV, and plot of koff versus voltage for cis side addition of am7βCD (Mean values (±s.d.) from three independent experiments are shown) c) Structure of 12 ACS Paragon Plus Environment

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Submitted to JACS am6αCD, interaction of pPorA with am6αCD (1 µM, cis) at -25 mV and -50 mV and plot of koff versus voltage for cis side addition of am6αCD (Mean values (±s.d.) from three independent experiments are shown) d) Structure of anionic s8γCD and interaction with pPorA peptide pores at +50 mV (10 mM, cis). e) Structure of neutral γCD and interaction with pPorA peptide pores at +50 mV (10 mM, cis). The current signals were filtered at 10 kHz and sampled at 50 kHz. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4.

The kinetics of the CD binding as a function of the applied voltage shows that the dissociation rate (koff) increased with the increase in the voltage indicating the translocation of CDs through the pore (Figure 4a). As expected, the addition of 2.5 µM am8γCD to the trans side induced ion current blockages at positive potentials and not at negative potentials confirming the electrostatic interaction of the CDs with the pore (Supplementary Figure S12). Similar ion current blockage events were obtained for the smaller heptasaccharide, am7βCD and hexasaccharide, am6αCD (Figure 4b, 4c, Supplementary Figure S12). As a control, we characterized the interaction of neutral octasaccharide γCD and anionic octasaccharide s8γCD with pPorA pores that did not produce any ion current blockage events irrespective of the side of the addition, the concentration of the CDs and the applied voltage (Figure 4d and Figure 4e). Additionally, we studied the interaction of small cationic molecule, spermine with the pPorA-K24C that produced very short ion current blockage events of ~ 100 µs closer to the resolution limit of the instrument due to its smaller size compared to CDs (Supplementary figure S13).

Site-specific chemical modification and subunit stoichiometry: We examined chemical modification of the cysteine side-chains of pPorA-K24C with thiolated polyethylene glycol, monomethoxy poly (ethylene glycol)-o-pyridyl disulfide) (MePEG-OPSS) to deduce the pore structure in single channel electrical recordings (Figure 5a)27,35. Addition of 1 13 ACS Paragon Plus Environment


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Submitted to JACS mM MePEG-OPSS-1K reagent to the cis side of the pore resulted in stepwise ion current blockages at +50 mV over 50 min (n = 15) indicating covalent modification of cysteine residues in the pore lumen with thiolated PEG reagent (Figure 5b).

Figure 5: Subunit stoichiometry of the pPorA pores a) Schematic and b) electrical recordings showing the reaction of 1 mM MePEG-OPSS-1K with the pPorA-K24C at +50 mV. c) Subsequent addition of 10 mM DTT (trans) resulted in the cleavage of PEG chains from the pore. The current signals were digitally filtered at 200 Hz using an 8-pole Bessel digital filter d) The pPorA peptides run on an SDS-PAGE. Arrow indicates the autonomously assembled pPorA-K24C oligomers. e) Electrical recording of insertion of a gel extracted single and f) double pPorA-K24C pores at +50mV and +100mV g) Histogram of the unitary conductance at +50 mV by fitting the distribution to a Gaussian (n = 50). h) I-V curve obtained from a single gel extracted pPorA-K24C pore. The current signals were filtered at 2 kHz and sampled at 10 kHz. i) Interaction of gel extracted single octameric pPorA-K24C pores with 14 ACS Paragon Plus Environment

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Submitted to JACS am8γCD (2.5 µM, cis) at -50 mV. The corresponding dwell time histogram fitted with a monoexponential probability function and ion current recording at expanded time scale is shown in the inset. The current signals were filtered at 10 kHz and sampled at 50 kHz. Electrolyte: 1 M KCl, 10 mM HEPES, pH 7.4.

The pyridyl disulfide groups in the PEG reacts with the cysteine side chains to form disulfide bond resulting in the channel closure. The pore closure showed a distribution of 4 to 8 resolved ion current blockage steps indicating that the pore formed by pPorA comprises at least three subunits and a maximum of eight subunits (n= 15) (Supplementary Figure S14). In most cases, all pointsamplitude histogram corresponding to stepwise ion current blockages revealed the distribution of 8 steps. In addition, the histogram of the ion current blocking steps based on 15 independent experiments showed a predominant distribution at 8 (Supplementary Figure S14). The pore reopened in ~20 min at +50 mV after the addition of 10 mM DTT to the trans compartment resulting in the cleavage of disulfide bond confirming the reversible chemical modification (Figure 5c). In the next experiments, larger MePEG-OPSS-5K (1mM) was added to the cis side of the pore that resulted in a single-step closure of the pore that reverted to its open state in the presence of 10 mM dithiothreitol (DTT) confirming the covalent chemical modification (Supplementary Figure 15). We propose that pPorA-K24C can accommodate only a single large MePEG-OPSS-5K that hinders other larger PEGs to enter the pore.

Next, we performed SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to examine the subunit composition of detergent-solubilized pPorA peptides. The electrophoretic mobility of WT pPorA peptides in SDS-PAGE revealed a single ~ 4.5 kDa band corresponding to peptide monomer as expected (Figure 5d). Surprisingly, the electrophoretic mobility of pPorA-K24C peptides in SDSPAGE revealed three bands and we calculated the molecular masses based on denatured protein 15 ACS Paragon Plus Environment


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Submitted to JACS standards. As expected, lower ~ 4.5 kDa band corresponds to monomeric peptides and ~ 9 kDa band corresponds to dimeric pPorA-K24C peptides. Remarkably, we observed a higher ~ 35 kDa band which might correspond to pPorA-K24C octamers that are stable in SDS (Figure 5d). The data obtained from gel electrophoresis indicates that the pPorA-K24C autonomously assemble to form a highly stable oligomer. Finally, to confirm the oligomeric structures, we extracted the slowly migrated ~35 kDa band corresponding to high oligomer peptides and tested the poreforming properties in single channel electrical recordings. The extracted peptide spontaneously inserted into bilayers in a single step at +50 mV forming a stable pore (Figure 5e). We also observed a two-step insertion of the pore at +100 mV confirming a highly stable well-defined pore that showed voltage-dependent gating (Figure 5f and Supplementary Figure S16). The unitary conductance of the pore based on the conductance histogram was calculated to be 4.0 ± 0.2 nS at +50 mV (n = 50) resembling the pore conductance obtained by the direct addition of cysteine peptides to the bilayer (Figure 5g). This data suggests that the pPorA-K24C forms a stable uniform octameric pore in the lipid bilayers and the conductance of the pore varied linearly with the applied potential (Figure 5h). Further, am8γCD and th8γCD interacted with the gel extracted octameric pore resulting in time-resolved ion current blockages at different voltages confirming the functionality of the pore (Figure 5i and Supplementary Figure S17). Furthermore, we synthesized and characterized another cysteine peptide mutant pPorA-W36C, which inserted into planar lipid bilayers and formed a pore. (Supplementary figure S18). The pPorA-W36C fluctuated between two single-channel conductance states of ~1.8 nS and ~2.7 nS with frequent gating events and ion selectivity measurements revealed that the pore is slightly anion selective. The electrophoretic mobility of pPorA-W36C peptides in SDS-PAGE revealed bands corresponding to monomer, dimer but not higher oligomers as obtained for pPorA-K24C peptides


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Submitted to JACS (Supplementary Figure S18). This data demonstrates the sequence specificity of pPorA-K24C resulting in pre-oligomerized peptides in the SDS-PAGE and forming large conductance pores in bilayers. Additionally, we characterized the single channel electrical properties of the peptide pPorA-K24A in planar lipid bilayers. The pPorA-K24A formed a stable cation-selective pore of unitary conductance ~ 3.0 nS in 1M KCl similar to WT pPorA (Supplementary Figure S19). This establishes the specificity of cysteine mutant pPorA-K24C in forming the large pores of unitary conductance ~ 4 nS (1M KCl) in lipid bilayers. Notably, the electrophoretic mobility of pPorAK24A peptides in SDS-PAGE revealed bands corresponding to only monomers.

The molecular model for the assembly of pPorA peptide pores: Based on the experimental results obtained in single channel recordings, we propose a molecular model elucidating the assembly pathway of pPorA pores in lipid membranes. The pPorA-K24C peptides are α-helical in detergent micelles and formed a monodisperse pore in planar lipid bilayers. The pores are functional, cation-selective and are blocked by cationic cyclic oligosaccharides. We propose that pPorA-K24C most likely forms an octameric pore, which is supported by site-specific chemical modification of the pore in single channel electrical recordings to count the number of subunits (Figure 6a). For example, the interaction of pPorA-K24C with MePEG-OPSS-1K resulted in maximum eight-step closure of the pore. The 8-fold reacted pore is not completely closed which suggest that additional polymers can still enter the pore to react with the cysteines as there is no steric hindrance (Figure 5a and Supplementary Figure S14). We did not observe more than eight steps in 15 individual experiments which indicates that the pPorAK24C possibly forms an octameric pore (Figure 6a). In particular, the peptide pores bind and are blocked by the cyclic octasaccharide which has the same C8 symmetry proposed for the pore.



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Submitted to JACS

Figure 6: Model for membrane insertion and pPorA pore formation. a) The pPorA-K24C exists as monomeric and pre-oligomers which associate with membrane and inserts to form high conductance cation-selective pores of octameric subunit composition. The pore is blocked by cationic am8γCD. b) The WT pPorA peptide monomers associate with the membrane and inserts to form a high conductance anion-selective pore of hexameric subunit composition. The pore is blocked by anionic s6αCD. Further, SDS PAGE revealed that pPorA-K24C peptides exist in multimeric conformations including monomers, dimers and autonomously assembled pre-oligomers (Figure 5b). The SDS stable pPorA-K24C pre-oligomers corresponding to ~35 kDa formed a stable pore of singlechannel conductance ~ 4.0 nS similar to pore formed by the direct addition of pPorA-K24C


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Submitted to JACS peptides to the bilayer supporting the octameric subunit composition. Importantly, pPorA-K24C peptide oligomers are stable in the presence of 2-Mercaptoethanol in SDS-PAGE indicating the role of strong hydrophobic interaction in the assembly of monomer peptides into higher oligomers (Supplementary Figure S18).

WT pPorA peptides are α-helical in detergent micelles and do not assemble into pre-oligomers as suggested by the CD spectra and SDS PAGE. The monomeric peptides spontaneously selfassemble in the membrane resulting in the formation of the uniform pores of a steady conductance state. The pores are functional, selective for anions and blocked by anionic cyclodextrins. We speculate that WT peptide most likely forms a hexameric pore in the membrane based on the conductance (Figure 6b). Notably, the biological porin, PorACj derived from Corynebacterium jeikeium formed channels with two different conductance states indicating the formation of two kinds of pores either hexamer or octamer32. Further, we deduced the peptide pore diameter based on the experimental single-channel conductance obtained at 1M KCl by presuming that the pPorA is an electrolyte-loaded cylinder of length 40Å (Supplementary Text). Accordingly, the relative pore diameter of pPorA-K24C was estimated to be ~ 1.4 nm and WT pPorA to be ~1.2 nm36,37. The lower unitary conductance and pore diameter of WT pPorA compared to pPorA-K24C could be due to different subunit composition. The pPorA-K24A and WT pPorA exhibited similar conductance which demonstrate that the different charge distribution at the constriction of the peptide pore did not alter conductance states of the pores. Accordingly, we propose that the substitution of lysine specifically for cysteine makes the pPorA-K24C pore bigger and most likely the oligomeric state of the pore has changed compared to WT pPorA. Importantly, our study demonstrates how synthetic peptide monomers assemble consistently to form a stable uniform pore



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Submitted to JACS which gives insights into the assembly mechanism of many pore-forming proteins that damage their targets by assembling and creating pores in the target cell membranes2,38,39. Discussion C. jeikeium, a mycolic acid-containing gram-positive bacteria produces a 40 amino acid length alpha-helical porin, PorACj which is considered as an ancient cell wall channel of Corynebacterium32. The porin is anion selective and formed channels with two different conductance states indicating variable oligomerization and assembly states32. Notably, the high proportion of hydrophobic residues makes it a potential candidate for membrane protein engineering for applications in nanobiotechnology. Here, we explored the structural and functional properties of the synthetic peptide corresponding to the PorACj porin by peptide redesign, chemical, and biophysical techniques. Peptide pore consists of forty amino acid length monomeric units, which spontaneously self-assembles to generate the transmembrane peptide pores. We showed that the pore is ion-selective, functional, as conductance was blocked via non-covalent binding of cyclodextrin blockers that defines the pathway of ion flow through pPorA pores. Importantly, we have characterized the assembly pathway and the mechanism by which pore inserts across the membrane.

Recently, it has been shown that the synthetic peptide corresponding to the C-terminal domain of the polysaccharide transporter Wza forms an octameric α-helix barrel that spans the outer membrane of Gram-negative bacteria27. Remarkably, synthetic peptides corresponding to D4 domain insert into lipid bilayers to form non-selective functional monodisperse pores containing eight parallel monomeric units. However, the pore fluctuated between several conductance states with discrete assembly intermediates during pore formation. Moreover, the diameter of the pore is


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Submitted to JACS restricted which limits its application in nanotechnology and single-molecule sensing28. Here in this work, we present synthetic pPorA peptides, which is autonomously assembled to form functional ion-selective pores with higher conductance indicating the larger diameter of the pore with a single oligomerization state. In particular, hydrophobic residues of the pPorA are scattered throughout the sequence and thus maintain a higher membrane insertion possibility.

Engineered α-helical peptide pores can find applications in medicine40,41,42. Previous reports suggest that antimicrobial peptides invariably form pores with diverse conductance states43,44. For example, tilted insertion of an antimicrobial peptide results in the formation of transient monolayer pores of ~ 2 nm size that is visualized in reconstituted lipid bilayers and live bacterial cells45. Some antimicrobial peptides forms pore which continue to grow to become large holes ranging in ~20 nm in diameter46. The large sized homogenous peptide pores demonstrated in this study can be used as a potential antimicrobial agent that can disrupt and kill the bacterial cells. The higher conductance is an added advantage for pPorA to put forward as a potential antimicrobial agent. Furthermore, our study elucidates the assembly pathway of a stable transmembrane α-helical peptide pore with a discrete structure, which may be useful for understanding the possible modes of action of antimicrobial peptides. We believe that the pPorA peptide might represent a stable version of one of many possible states, and therefore might be useful to study in the context of antimicrobial peptides. Similarly, designed peptides can be used for targeting cancer cells for apoptotic death and also can be used to carry specific active cargo to tumors47. Despite the longterm effort, synthetic protein pores with stable structures have rarely been obtained for applications in nanopore technology27,48,49,50. The key finding of our study is that pPorA peptide pore is uniform instead of being composed of multiple oligomers, which is essential to build new peptide-based



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Submitted to JACS sensors. Our studies open possibilities for using de novo designed α-helical peptides as membranespanning sensors for the applications in single molecule sensing of complex biomolecules5,51,52,53. The pores may be made by solid-phase synthesis that will enable us to incorporate unnatural amino acids for exploring sensitive chemical reactions at the single-molecule level49. Future studies will be focused on the design of synthetic peptide to control the pore geometry to build large stable monodisperse structures for applications in nanopore proteomics54. Similarly, ion selectivity, as observed in natural ion channels can be established in engineered pPorA pores to conduct selectively specific ions. Conclusion: Here, we form a transmembrane pore, pPorA from 40-amino-acid synthetic α-helical peptides derived from the porin PorACj of C. jeikeium. We investigated its structure and functional properties and elucidated the defined assembly pathway by single-channel current recording. The pore is ion-selective and is functionally blocked by the cyclic oligosaccharides. Our observation that short synthetic alpha-helical peptides can autonomously assemble to form large synthetic transmembrane uniform pores is noteworthy and unique. The knowledge acquired here is useful for understanding the actions of antimicrobial peptides and provides a novel platform for developing highly versatile synthetic nanostructures for applications in biotechnology and singlemolecule chemistry. Experimental Section: Single channel electrical recordings Planar lipid bilayer recordings were carried out by using bilayers of 1,2-diphytanoyl-sn-glycero3-phosphocholine (DPhPC, Avanti Polar Lipids) formed across an aperture (~40 µm in diameter) in a 25-µm thick polytetrafluoroethylene (Teflon) film (Goodfellow, Cambridge), which separated the apparatus into cis and trans compartments (500 µL each)55. Bilayers were formed by first pre22 ACS Paragon Plus Environment

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Submitted to JACS treating the aperture with hexadecane in n-pentane (1 μL, 5 mg mL-1) on each side. Both compartments were then filled with the electrolyte solution (1 M KCl, 10 mM HEPES, pH 7.4) and DPhPC in n-pentane (2 μL, 5 mg mL-1) was added to both sides after that the solvent evaporated. A bilayer was formed when the electrolyte was raised bringing the two lipid surface monolayers together at the aperture. The pPorA pores were formed by adding a solution of the peptide (cis side) in 0.1% DDM (1 µL, 100 µg mL-1) under an applied potential of +100 mV. 1µM DTT was added to the cis side to facilitate faster insertion of cysteine pPorA peptides into the lipid bilayer even at low voltages (+5 mV). The cis compartment was connected to the grounded electrode and the trans compartment was attached to the working electrode. A potential difference was applied through a pair of Ag/AgCl electrodes, set in 3% agarose containing 3.0 M KCl. We investigated the ion selectivity of pores using KCl salt gradient applied across the bilayer chambers (1M cis /0.15M trans) (Supplementary text). The current was amplified by using an Axopatch 200B amplifier, digitized with a Digidata 1550B and recorded with the pClamp 10.6 acquisition software (Molecular Devices, CA) with a low-pass filter frequency of 2 kHz and a sampling frequency of 10 kHz. In the case of pPorA-K24C, blocking by cyclodextrins will be quantified using a statistical analysis of the pore in its unblocked and blocked states which allows estimation of the association rate (kon), the dissociation rate (koff) and the equilibrium binding constant (K). This data was recorded with a low-pass filter frequency of 10 kHz and a sampling frequency of 50 kHz to resolved blocking events up to 100 µS. The data were analyzed and prepared for presentation with pClamp (version 10.6, Molecular Devices, CA) and Origin 9.0. Circular dichroism spectroscopy Circular dichroism spectra were obtained using MOS-500 spectropolarimeters fitted with Peltier temperature controllers (Bio-Logic Science instruments). Peptide samples were prepared as 25 µM



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Submitted to JACS and 50 µM solutions in phosphate buffered saline (PBS, 8.2 mM sodium phosphate, 1.8 mM potassium phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride at pH 7.4) with 1% DDM. Spectra were collected using a 1 mm path length quartz cuvette at 20 °C. For each dataset (in deg), baselines from the same buffer and cuvette were subtracted, and then data points were normalized for amide bond concentration and path length to give mean residue ellipticity (MRE; deg cm2 dmol-1 res-1). Acknowledgments This work was supported by the ‘Innovative Young Biotechnologist Award’ of Department of Biotechnology, Government of India awarded to KRM (BT/010/IYBA/2016/06). KRM acknowledge the Ramalingaswami Re-entry fellowship of Department of Biotechnology, Government of India (BT/RLF/Re-entry/49/2014) for their support. KRM thanks Prof. M. Radhakrishna Pillai, the Director, RGCB, for lab facilities and support. KRM thanks Dr. Ramanathan Natesh, IISER Thiruvananthapuram for his assistance in CD spectroscopy experiments. KRM thanks Prof. Ulrich Kleinekathöfer, Jacobs University Bremen for providing the modeled structure of porin PorACj. Supporting Information Available This material is available. Materials, text, table and Figures S1–S19 are referred in the Supplementary Information. References: 1. von Heijne, G., Membrane-protein topology. Nat. Rev. Mol. Cell. Biol. 2006, 7 (12), 909-18. 2. Dal Peraro, M.; van der Goot, F. G., Pore-forming toxins: ancient, but never really out of fashion. Nat. Rev. Microbiol. 2016, 14 (2), 77-92. 3. Bayley, H.; Jayasinghe, L., Functional engineered channels and pores (Review). Mol. Membr. Biol. 2004, 21 (4), 209-20. 24 ACS Paragon Plus Environment

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Submitted to JACS 4. Bayley, H.; Cremer, P. S., Stochastic sensors inspired by biology. Nature 2001, 413 (6852), 226-30. 5. Ayub, M.; Bayley, H., Engineered transmembrane pores. Curr. Opin. Chem. Biol. 2016, 34, 117-126. 6. Howorka, S., Building membrane nanopores. Nat. Nanotechnol. 2017, 12 (7), 619-630. 7. Kasianowicz, J. J.; Balijepalli, A. K.; Ettedgui, J.; Forstater, J. H.; Wang, H.; Zhang, H.; Robertson, J. W., Analytical applications for pore-forming proteins. Biochim. Biophys. Acta. 2016, 1858 (3), 593-606. 8. Huang, P. S.; Feldmeier, K.; Parmeggiani, F.; Velasco, D. A. F.; Hocker, B.; Baker, D., De novo design of a four-fold symmetric TIM-barrel protein with atomic-level accuracy. Nat. Chem. Biol. 2016, 12 (1), 29-34. 9. Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W., Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. USA. 1996, 93 (24), 13770-3. 10. Bayley, H., Nanopore sequencing: from imagination to reality. Clin. Chem. 2015, 61 (1), 2531. 11. Quick, J.; Loman, N. J.; Duraffour, S.; Simpson, J. T.; Severi, E.; Cowley, L.; Bore, J. A.; Koundouno, R.; Dudas, G.; Mikhail, A.; Ouedraogo, N.; Afrough, B.; Bah, A.; Baum, J. H.; Becker-Ziaja, B.; Boettcher, J. P.; Cabeza-Cabrerizo, M.; Camino-Sanchez, A.; Carter, L. L.; Doerrbecker, J.; Enkirch, T.; Dorival, I. G. G.; Hetzelt, N.; Hinzmann, J.; Holm, T.; Kafetzopoulou, L. E.; Koropogui, M.; Kosgey, A.; Kuisma, E.; Logue, C. H.; Mazzarelli, A.; Meisel, S.; Mertens, M.; Michel, J.; Ngabo, D.; Nitzsche, K.; Pallash, E.; Patrono, L. V.; Portmann, J.; Repits, J. G.; Rickett, N. Y.; Sachse, A.; Singethan, K.; Vitoriano, I.; Yemanaberhan, R. L.; Zekeng, E. G.; Trina, R.; Bello, A.; Sall, A. A.; Faye, O.; Faye, O.; Magassouba, N.; Williams, C. V.; Amburgey, V.; Winona, L.; Davis, E.; Gerlach, J.; Washington, F.; Monteil, V.; Jourdain, M.; Bererd, M.; Camara, A.; Somlare, H.; Camara, A.; Gerard, M.; Bado, G.; Baillet, B.; Delaune, D.; Nebie, K. Y.; Diarra, A.; Savane, Y.; Pallawo, R. B.; Gutierrez, G. J.; Milhano, N.; Roger, I.; Williams, C. J.; Yattara, F.; Lewandowski, K.; Taylor, J.; Rachwal, P.; Turner, D.; Pollakis, G.; Hiscox, J. A.; Matthews, D. A.; O'Shea, M. K.; Johnston, A. M.; Wilson, D.; Hutley, E.; Smit, E.; Di Caro, A.; Woelfel, R.; Stoecker, K.; Fleischmann, E.; Gabriel, M.; Weller, S. A.; Koivogui, L.; Diallo, B.; Keita, S.; Rambaut, A.; Formenty, P.; Gunther, S.; Carroll, M. W., Real-time, portable genome sequencing for Ebola surveillance. Nature 2016, 530 (7589), 228-232. 12. Woolfson, D. N., Coiled-Coil Design: Updated and Upgraded. Subcell. Biochem. 2017, 82, 3561. 13. Tajkhorshid, E.; Nollert, P.; Jensen, M. O.; Miercke, L. J.; O'Connell, J.; Stroud, R. M.; Schulten, K., Control of the selectivity of the aquaporin water channel family by global orientational tuning. Science 2002, 296 (5567), 525-30. 25 ACS Paragon Plus Environment


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Submitted to JACS

14. Lu, P.; Min, D.; DiMaio, F.; Wei, K. Y.; Vahey, M. D.; Boyken, S. E.; Chen, Z.; Fallas, J. A.; Ueda, G.; Sheffler, W.; Mulligan, V. K.; Xu, W.; Bowie, J. U.; Baker, D., Accurate computational design of multipass transmembrane proteins. Science 2018, 359 (6379), 1042-1046. 15. Woolfson, D. N., The design of coiled-coil structures and assemblies. Adv. Protein. Chem. 2005, 70, 79-112. 16. Woolfson, D. N.; Bartlett, G. J.; Burton, A. J.; Heal, J. W.; Niitsu, A.; Thomson, A. R.; Wood, C. W., De novo protein design: how do we expand into the universe of possible protein structures? Curr. Opin. Struct. Biol. 2015, 33, 16-26. 17. Joh, N. H.; Wang, T.; Bhate, M. P.; Acharya, R.; Wu, Y.; Grabe, M.; Hong, M.; Grigoryan, G.; DeGrado, W. F., De novo design of a transmembrane Zn2+-transporting four-helix bundle. Science 2014, 346 (6216), 1520-4. 18. Lear, J. D.; Wasserman, Z. R.; DeGrado, W. F., Synthetic amphiphilic peptide models for protein ion channels. Science 1988, 240 (4856), 1177-81. 19. Brogden, K. A., Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 2005, 3 (3), 238-50. 20. Jenssen, H.; Hamill, P.; Hancock, R. E., Peptide antimicrobial agents. Clin. Microbiol. Rev. 2006, 19 (3), 491-511. 21. Boman, H. G., Antibacterial peptides: basic facts and emerging concepts. J. Intern. Med. 2003, 254 (3), 197-215. 22. Song, C.; Weichbrodt, C.; Salnikov, E. S.; Dynowski, M.; Forsberg, B. O.; Bechinger, B.; Steinem, C.; de Groot, B. L.; Zachariae, U.; Zeth, K., Crystal structure and functional mechanism of a human antimicrobial membrane channel. Proc. Natl. Acad. Sci. USA. 2013, 110 (12), 458691. 23. Rakowska, P. D.; Jiang, H.; Ray, S.; Pyne, A.; Lamarre, B.; Carr, M.; Judge, P. J.; Ravi, J.; Gerling, U. I.; Koksch, B.; Martyna, G. J.; Hoogenboom, B. W.; Watts, A.; Crain, J.; Grovenor, C. R.; Ryadnov, M. G., Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers. Proc. Natl. Acad. Sci. USA. 2013, 110 (22), 8918-23. 24. Franceschini, L.; Soskine, M.; Biesemans, A.; Maglia, G., A nanopore machine promotes the vectorial transport of DNA across membranes. Nat. Commun. 2013, 4, 2415. 25. Tanaka, K.; Caaveiro, J. M.; Morante, K.; Gonzalez-Manas, J. M.; Tsumoto, K., Structural basis for self-assembly of a cytolytic pore lined by protein and lipid. Nat. Commun. 2015, 6, 6337.


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Submitted to JACS 26. Zaccai, N. R.; Chi, B.; Thomson, A. R.; Boyle, A. L.; Bartlett, G. J.; Bruning, M.; Linden, N.; Sessions, R. B.; Booth, P. J.; Brady, R. L.; Woolfson, D. N., A de novo peptide hexamer with a mutable channel. Nat. Chem. Biol. 2011, 7 (12), 935-41. 27. Mahendran, K. R.; Niitsu, A.; Kong, L.; Thomson, A. R.; Sessions, R. B.; Woolfson, D. N.; Bayley, H., A monodisperse transmembrane alpha-helical peptide barrel. Nat. Chem. 2017, 9 (5), 411-419. 28. Spruijt, E.; Tusk, S. E.; Bayley, H., DNA scaffolds support stable and uniform peptide nanopores. Nat. Nanotechnol. 2018. 13(8):739-745 29. Majd, S.; Yusko, E. C.; Billeh, Y. N.; Macrae, M. X.; Yang, J.; Mayer, M., Applications of biological pores in nanomedicine, sensing, and nanoelectronics. Curr. Opin. Biotechnol. 2010, 21 (4), 439-76. 30. Cao, C.; Ying, Y. L.; Hu, Z. L.; Liao, D. F.; Tian, H.; Long, Y. T., Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore. Nat. Nanotechnol. 2016, 11 (8), 713-8. 31. Danelon, C.; Nestorovich, E. M.; Winterhalter, M.; Ceccarelli, M.; Bezrukov, S. M., Interaction of zwitterionic penicillins with the OmpF channel facilitates their translocation. Biophys. J. 2006, 90 (5), 1617-27. 32. Abdali, N.; Barth, E.; Norouzy, A.; Schulz, R.; Nau, W. M.; Kleinekathofer, U.; Tauch, A.; Benz, R., Corynebacterium jeikeium jk0268 constitutes for the 40 amino acid long PorACj, which forms a homooligomeric and anion-selective cell wall channel. PloS one 2013, 8 (10), e75651. 33. Gu, L. Q.; Bayley, H., Interaction of the noncovalent molecular adapter, beta-cyclodextrin, with the staphylococcal alpha-hemolysin pore. Biophys. J. 2000, 79 (4), 1967-75. 34. van den Berg, B.; Prathyusha Bhamidimarri, S.; Dahyabhai Prajapati, J.; Kleinekathofer, U.; Winterhalter, M., Outer-membrane translocation of bulky small molecules by passive diffusion. Proc. Natl. Acad. Sci. USA. 2015, 112 (23), E2991-9. 35. Miles, G.; Movileanu, L.; Bayley, H., Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore. Prot. Sci. 2002, 11 (4), 894902. 36. Benz, R.; Schmid, A.; Hancock, R. E., Ion selectivity of gram-negative bacterial porins. J. Bacteriol. 1985, 162 (2), 722-7. 37. Smart, O. S.; Breed, J.; Smith, G. R.; Sansom, M. S., A novel method for structure-based prediction of ion channel conductance properties. Biophys. J. 1997, 72 (3), 1109-26. 38. Leung, C.; Hodel, A. W.; Brennan, A. J.; Lukoyanova, N.; Tran, S.; House, C. M.; Kondos, S. C.; Whisstock, J. C.; Dunstone, M. A.; Trapani, J. A.; Voskoboinik, I.; Saibil, H. R.; Hoogenboom, 27 ACS Paragon Plus Environment


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Submitted to JACS B. W., Real-time visualization of perforin nanopore assembly. Nat. Nanotechnol. 2017, 12 (5), 467-473. 39. Das, D.; Krantz, B. A., Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes. Proc. Natl. Acad. Sci. USA. 2016, 113 (34), 9611-6. 40. Wang, G., Human antimicrobial peptides and proteins. Pharmaceuticals 2014, 7 (5), 545-94. 41. Fjell, C. D.; Hiss, J. A.; Hancock, R. E.; Schneider, G., Designing antimicrobial peptides: form follows function. Nat. Rev. Drug. Discov. 2011, 11 (1), 37-51. 42. Nguyen, L. T.; Haney, E. F.; Vogel, H. J., The expanding scope of antimicrobial peptide structures and their modes of action. Trends. Biotechnol. 2011, 29 (9), 464-72. 43. Zasloff, M., Antimicrobial peptides of multicellular organisms. Nature 2002, 415 (6870), 38995. 44. Burian, M.; Schittek, B., The secrets of dermcidin action. Int. J. Med. Microbiol. 2015, 305 (2), 283-6. 45. Pyne, A.; Pfeil, M. P.; Bennett, I.; Ravi, J.; Iavicoli, P.; Lamarre, B.; Roethke, A.; Ray, S.; Jiang, H.; Bella, A.; Reisinger, B.; Yin, D.; Little, B.; Munoz-Garcia, J. C.; Cerasoli, E.; Judge, P. J.; Faruqui, N.; Calzolai, L.; Henrion, A.; Martyna, G. J.; Grovenor, C. R. M.; Crain, J.; Hoogenboom, B. W.; Watts, A.; Ryadnov, M. G., Engineering monolayer poration for rapid exfoliation of microbial membranes. Chem. Sci. 2017, 8 (2), 1105-1115. 46. Mularski, A.; Wilksch, J. J.; Hanssen, E.; Strugnell, R. A.; Separovic, F., Atomic force microscopy of bacteria reveals the mechanobiology of pore forming peptide action. Biochim. Biophys. Acta. 2016, 1858 (6), 1091-8. 47. Gaspar, D.; Veiga, A. S.; Castanho, M. A., From antimicrobial to anticancer peptides. A review. Front. Microbiol. 2013, 4, 294. 48. Kowalczyk, S. W.; Blosser, T. R.; Dekker, C., Biomimetic nanopores: learning from and about nature. Trends. Biotechnol. 2011, 29 (12), 607-14. 49. Lee, J.; Boersma, A. J.; Boudreau, M. A.; Cheley, S.; Daltrop, O.; Li, J.; Tamagaki, H.; Bayley, H., Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry. ACS nano 2016, 10 (9), 8843-50. 50. Lalgee, L. J.; Grierson, L.; Fairman, R. A.; Jaggernauth, G. E.; Schulte, A.; Benz, R.; Winterhalter, M., Synthetic ion transporters: pore formation in bilayers via coupled activity of nonspanning cobalt-cage amphiphiles. Biochim. Biophys. Acta. 2014, 1838 (5), 1247-54. 51. Yusko, E. C.; Bruhn, B. R.; Eggenberger, O. M.; Houghtaling, J.; Rollings, R. C.; Walsh, N. C.; Nandivada, S.; Pindrus, M.; Hall, A. R.; Sept, D.; Li, J.; Kalonia, D. S.; Mayer, M., Real-time 28 ACS Paragon Plus Environment

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Submitted to JACS shape approximation and fingerprinting of single proteins using a nanopore. Nat. Nanotechnol. 2017, 12 (4), 360-367. 52. Derrington, I. M.; Craig, J. M.; Stava, E.; Laszlo, A. H.; Ross, B. C.; Brinkerhoff, H.; Nova, I. C.; Doering, K.; Tickman, B. I.; Ronaghi, M.; Mandell, J. G.; Gunderson, K. L.; Gundlach, J. H., Subangstrom single-molecule measurements of motor proteins using a nanopore. Nat. Biotechnol. 2015, 33 (10), 1073-5. 53. Bell, N. A.; Keyser, U. F., Digitally encoded DNA nanostructures for multiplexed, singlemolecule protein sensing with nanopores. Nat. Nanotechnol. 2016, 11 (7), 645-51. 54. Rosen, C. B.; Rodriguez-Larrea, D.; Bayley, H., Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat. Biotechnol. 2014, 32 (2), 179-81. 55. Gutsmann, T.; Heimburg, T.; Keyser, U.; Mahendran, K. R.; Winterhalter, M., Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization. Nat. Protoc. 2015, 10 (1), 188-98.



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Autonomously Assembled Synthetic Transmembrane Peptide Pore

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