Engineering a Transmembrane Nanopore Ion Channel from a

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Letter

Engineering a Transmembrane Nanopore Ion Channel from a Membrane Breaker Peptide Muralikrishna Lella, and Radhakrishnan Mahalakshmi J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b00987 • Publication Date (Web): 03 Jun 2016 Downloaded from http://pubs.acs.org on June 4, 2016

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The Journal of Physical Chemistry Letters

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Engineering a Transmembrane Nanopore Ion Channel from a

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Membrane Breaker Peptide

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Muralikrishna Lella[a] and Radhakrishnan Mahalakshmi*[a]

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[a]

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Education and Research, ITI Building, Govindpura, Bhopal – 462023. India. E-mail:

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[email protected].

Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science

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ACS Paragon Plus Environment


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ABSTRACT

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Re-engineering nature’s molecules is an ideal strategy to obtain explicit functionality such

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as synthetic molecular machines. Yet, novel strategies for producing engineered molecular channels

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are few. Here, we report a peptide engineering strategy through sequence reversal, which we

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applied on the first transmembrane peptide of the mycobacteriophage membranoporin protein holin.

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We have successfully re-designed the membrane rupture property of this peptide to form specific

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nanopore ion channels. We report the structural characterization and electrophysiology

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measurements of a library of 28-residue engineered membrane peptides, with remarkable ion

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channel behavior. We further identify that key residues at the peptide terminus, the central proline,

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charge distribution, and hydropathy index of the peptide together contribute to the channel

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properties that we measure. Our sequence reversal strategy for peptide engineering to successfully

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obtain nanopore channels can pave the way for better bio-based design of controlled nanopores,

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using only natural amino acids.

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TOC GRAPHIC:

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KEYWORDS: Peptide engineering; Ion channels; Nanopore; Membranes; Electrophysiology;

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Spectroscopy.

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Protein engineering, or protein design, is an ingenious method for generating bioactive and

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functionally efficient molecules with therapeutic applications. The production of peptide-based

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small molecular machines as functional effectors that heavily borrow from biomolecules available

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in nature, has always been of considerable interest.1-4 Such re-design to mimic natural systems

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poses challenges requiring innovative solutions;5 however, reports1,

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provides assurance that re-designed functional proteins can be realistically achieved.

6-9

of success in this field

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The current interest is in generating membrane-spanning synthetic molecular channels;10, 11 a

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key problem with the design of membrane channels is the need for complex chemistry involving

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unnatural residues or the incorporation of bulky tags for channel assembly. It was recently shown

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that naturally occurring membrane proteins can be engineered to function as nanopores.9, 12-14 These

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proteins, however, have the limitation that they are large in size. We asked whether nanopores can

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be made using small peptides derived from such membrane proteins. The mycobacteriophage D29

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transmembrane protein holin serves as a lucrative candidate for this purpose.

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D29 holin forms irreversible holes in the inner membrane of the bacterium, and causes host

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cell death.15 The first transmembrane domain (TM1) spans residues K4-A31. This is the longest

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known chameleonic sequence to show α-helix ↔ β-hairpin conformational interconversion in

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lipidic micelles.16 Recent studies have shown that TM1 is sufficient to elicit bacterial host cell death

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and disrupt lipid bilayer membranes within seconds.17 Further, TM1 is small, with a length of 28

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residues, unlike engineered membrane proteins that are considerably large in size. Hence, TM1

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serves as a good candidate for pore engineering. In this study, we report the engineering of the

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membrane-breaker properties of TM1 into transmembrane nanopore ion channels, by simply

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reversing the peptide sequence (Figure 1).

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To apply the concept of biomolecular re-design, we first considered the following features

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of the TM1 peptide. (i) The peptide shows a structural switch assisted by a centrally located proline

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residue. (ii) The peptide shows uncontrolled association in membranes. (iii) The residues towards

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the N-terminus (K4-V16) show a greater propensity for ϕ-ψ values of β-strands.16 To re-design TM1, 3



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we addressed both the structural and functional characteristics of the peptide. For this, we assumed

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that the unique features of TM1 are embedded in the primary protein sequence. Hence, we adopted

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a heuristic approach, where we followed a residue–swap strategy. We swapped residues at the

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terminal (tTM1), semi-strand (sTM1) or carried out a near-complete reversal (rTM1) of the TM1

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peptide (Figure 1A-B), and examined how these residues play a role in TM1 behavior.

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We synthesized a peptide library with chimeric segments (Figure 1A, Table S1), and

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characterized them in lipidic micelles (Figure 2). TM1 shows a structural switch that is dependent

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on the micelle-peptide ratio and temperature.16 Structural characterization of all our engineered

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chimeric constructs revealed that the reversible α-helix ↔ β-hairpin switch is abolished (Figure 2A-

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B, Figure S2). Thermal denaturation and recovery measurements further support that the engineered

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TM1 peptides sTM1 and rTM1 retain a helical structure (Figure 2B, Figure S2). The other peptides

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form a mixed α+β structure.

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Engineered TM1 sequences with swapped strand segments also exhibited an increase in

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helicity upon prolonged incubation at 4 °C (Figure 2C, Figure S3). Here, we find that reversing the

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terminal seven residues in the peptide sTM1 is adequate to enrich structural helicity. Furthermore,

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swapping three residues at the N-terminal (K1IR of TM1 versus A1DI in tTM1) is sufficient to

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abolish the conformational switch of TM1 (Figure 2A-B). Further, truncations of tTM1 (trTM127,

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trTM126; Figure 2), and substitutions with other residues (Figure S4a) do not rescue the

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conformational switch. Hence, the presence of K1IR at the N-terminus may promote a well-

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regulated α-helix ↔ β-hairpin interconversion in TM1, whereas replacing these residues abolishes

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the interconversion.

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Proline is frequently observed in transmembrane helices, wherein it is essential for helix

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packing in the membrane.18 TM1 also bears a conserved proline (P14) in the sequence, which is

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important for its structure.16 Changing the position of proline (P14-G15 in rTM1, to G14-P15 in

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rTM1GP) allows for retention of the helical conformation (Figure S4b). However, when LPro is

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replaced by DPro (see Figure 1A for sequences), we primarily observed a β-strand rich structure 4


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(Figure S4c). Put together, our spectroscopic analysis allows us to deduce that the terminal 3-7

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residues play a major role in the structure of the engineered TM1 analogs. A helical structure is

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favorably obtained as a consequence of reversing the TM1 sequence.

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TM1 peptide can rapidly break lipid bilayers within seconds of association (Figure 1C). We

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measured this process using planar lipid bilayer systems, wherein the peptide is allowed to interact

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with a single lipid bilayer painted across a 150 μm aperture separating two buffer-filled chambers.

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The ability of TM1 to disrupt membranes independent of the full-length holin protein17 suggests

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that the function is encoded in the sequence and structure of the peptide. Since the engineered TM1

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sequences show different structural characteristics, we envisioned that they should also possess

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altered functions.

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As expected, we found that rTM1 did not disrupt membrane bilayers for several hours after

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peptide insertion (Figure 1C, Figure S5). Instead, we observed the remarkable occurrence of ion

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channel-like transitions in all the engineered peptides (Figure 3, Figure S6-S7). The initial

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association of all peptides with the membrane is similar (Figure S6); yet, the ion channel behavior is

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different. For example, the peptides rTM1 and sTM1 show prominent open state conductance, while

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some DPro-containing analogs show lowered conductance states (rTM1pG in Figure 3A, Figure

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S7a,b,d). tTM1 and trTM126 show both ion channel activity and instances where it disrupts the

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membrane (Figure S7c). Hence, these peptides retain some behavior of the native TM1 peptide. In

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several other peptides, we also observed more peptide insertion (compare conductance values in

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Figure S8), membrane destabilization and rupture at higher voltages (Figures S7c-d).

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We calculated the open state dwell time (Figure 3B-C, Figure S9, Table S2), which is a

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measure of the duration a channel stays open, and reflects the stability of the peptide channel. None

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of the peptides showed any significant change in the channel dwell time, except for rTM1. rTM1

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shows ~100-fold increase in dwell time at higher voltages (Figure 3B, Figure S10). We also

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calculated the channel open probability for all peptides. Here again, the open probability only for

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rTM1 increased from ~0.4 (at low voltages) to ~1.0 at high applied voltages (> ±50 mV) (Figure 5



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S11). All other peptides including sTM1 either showed lowered channel open probability or caused

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membrane rupture (Figure 3B, Figures S7d-e). Hence, rTM1 is the only peptide to form consistently

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open channels at all applied voltages from –100 mV to +100 mV. Further characterization of rTM1

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channels showed that it is non-selective to the charged state of the ion being transported, and is pH-

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and voltage-independent (Figure S12). We conclude that when the amino acid sequence of TM1 is

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presented in the reverse order (C- to N-, in rTM1), we obtain voltage-independent nanopore

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channels that show voltage dependence only for the channel dwell time.

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It is intriguing that a membrane-breaker peptide can be engineered to form ion channels by

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reversing the sequence. How is this achieved in rTM1? We compared the unique functionalities

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with the distribution of charge and hydrophobic segments in all the structures (Figure 4A-B). There

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are five charges (3+ and 2–) in TM1 and its analogs (N-terminus, K, R, D and E). When we

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consider the net charge at both the termini, TM1 and tTM1 exhibit considerable charge asymmetry

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(Figure 4A, Figure S13). This asymmetry is abolished in the channel-forming sTM1 and rTM1. A

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difference in hydropathy is also evident only in rTM1 (Figure 4B). Hence, nanopore channel

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formation could be a likely consequence of charge and hydropathy re-distribution.

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We also find that retaining the central P14-G15 and reversing only the flanking sequence

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gives the most effective ion channels (compare rTM1 with rTM1GP in Figure 3; sequences are in

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Figure 1A). Hence, when the native TM1 sequence is faithfully reversed to rTM1GP, ion channels

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form only at lower voltages (Figure 3). Further, changing the chirality of proline gives channels

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with low dwell time values (for example, rTM1pG in Figure 3). Therefore, the position and chirality

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of proline also plays a vital role in deciding ion channel formation in the TM1 chimeras.

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We have presented a simple sequence reversal strategy by which we change charge

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asymmetry and hydrophobicity, thereby converting a membrane breaker peptide to nanopore ion

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channels. We find that different channel behavior can be achieved in the TM1 peptide analogs

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simply by changing the extent to which the sequence is re-arranged. Several factors (summarized in

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Figure 4C) can together decide whether TM1 and its analogs show membrane rupture or membrane 6


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channels. We currently do not know the mechanism by which the TM1 analogs form ion channels.

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However, we speculate that TM1 analogs may follow previous models proposed for protein channel

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assembly.19

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Comparing our results with alamethicin channels, we find that unlike the uncontrolled

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alamethicin conductance states,20,

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comparatively more controlled assemblies. Further, the extra-membrane interactions needed to

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obtain single channels of alamethicin22 are not required for our model rTM1 peptide. The greatest

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advantage our engineered peptide offers is that it is made with natural L-amino acids without the

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need for extensive chemistry.

10

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our rTM1 peptide can give defined conductance and

There has been recent interest in the design of quaternary assemblies1, 3 and stable synthetic

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nanopore channels for diverse applications.1,

9, 14, 23, 24

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excellent candidates for further programing as nanopore sensors. Our finding could also open

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lucrative avenues where naturally occurring peptides can be engineered with modular channel

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functions.

Our engineered chimeras might also form

15

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ASSOCIATED CONTENT

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Supporting Information

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Peptide synthesis and characterization are detailed in the Supporting Information. Structural studies:

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Changes with micelle concentration, time, temperature, headgroup. Functional (Electrophysiology)

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studies: channel activity (-100 to +100 mV), insertion currents, conductance at each voltage,

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G/G10, dwell time, open probability, channel activity with pH, ion gradient. The complete details

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and data are included in the Supporting Information. The Supporting Information is available free

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of charge on the ACS Publications website.

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AUTHOR INFORMATION

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Corresponding Author

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*E-mail: [email protected].

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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M.L. thanks IISER Bhopal for a research fellowship. R.M. is a Wellcome Trust/ DBT India

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Alliance Intermediate Fellow. This work is supported by intramural funds.

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REFERENCES

2

1.

Zaccai, N. R.; Chi, B.; Thomson, A. R.; Boyle, A. L.; Bartlett, G. J.; Bruning, M.; Linden,

3

N.; Sessions, R. B.; Booth, P. J.; Brady, R. L., et al. A De Novo Peptide Hexamer with a

4

Mutable Channel. Nat. Chem. Biol. 2011, 7, 935-941.

5

2.

Rufo, C. M.; Moroz, Y. S.; Moroz, O. V.; Stohr, J.; Smith, T. A.; Hu, X.; DeGrado, W. F.;

6

Korendovych, I. V. Short Peptides Self-Assemble to Produce Catalytic Amyloids. Nat.

7

Chem. 2014, 6, 303-309.

8

3.

Collie, G. W.; Pulka-Ziach, K.; Lombardo, C. M.; Fremaux, J.; Rosu, F.; Decossas, M.;

9

Mauran, L.; Lambert, O.; Gabelica, V.; Mackereth, C. D., et al. Shaping Quaternary

10

Assemblies of Water-Soluble Non-Peptide Helical Foldamers by Sequence Manipulation.

11

Nat. Chem. 2015, 7, 871-878.

12

4.

13 14

Coiled-Coil Peptide Nanotubes. Angew. Chem. Int. Ed. 2016, 55, 987-991. 5.

15 16

Thomas, F.; Burgess, N. C.; Thomson, A. R.; Woolfson, D. N. Controlling the Assembly of

Cook, S. A.; Hill, E. A.; Borovik, A. S. Lessons from Nature: A Bio-Inspired Approach to Molecular Design. Biochemistry 2015, 54, 4167-4180.

6.

Reig, A. J.; Pires, M. M.; Snyder, R. A.; Wu, Y.; Jo, H.; Kulp, D. W.; Butch, S. E.; Calhoun,

17

J. R.; Szyperski, T.; Solomon, E. I., et al. Alteration of the Oxygen-Dependent Reactivity of

18

De Novo Due Ferri Proteins. Nat. Chem. 2012, 4, 900-906.

19

7.

20 21

Mahajan, M.; Bhattacharjya, S. Beta-Hairpin Peptides: Heme Binding, Catalysis, and Structure in Detergent Micelles. Angew. Chem. Int. Ed. 2013, 52, 6430-6434.

8.

Yu, F.; Cangelosi, V. M.; Zastrow, M. L.; Tegoni, M.; Plegaria, J. S.; Tebo, A. G.; Mocny,

22

C. S.; Ruckthong, L.; Qayyum, H.; Pecoraro, V. L. Protein Design: Toward Functional

23

Metalloenzymes. Chem. Rev. 2014, 114, 3495-3578.

24 25

9.

Farimani, A. B.; Heiranian, M.; Aluru, N. R. Electromechanical Signatures for DNA Sequencing through a Mechanosensitive Nanopore. J. Phys. Chem. Lett. 2015, 6, 650-657.

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

10.

Page 10 of 21

Burns, J. R.; Seifert, A.; Fertig, N.; Howorka, S. A Biomimetic DNA-Based Channel for the

2

Ligand-Controlled Transport of Charged Molecular Cargo across a Biological Membrane.

3

Nat. Nanotechnol. 2016, 11, 152-156.

4

11.

5 6

Soc. Rev. 2016. 12.

7 8

Liu, A.; Zhao, Q.; Krishantha, D. M.; Guan, X. Unzipping of Double-Stranded DNA in Engineered Alpha-Hemolysin Pores. J. Phys. Chem. Lett. 2011, 2, 1372-1376.

13.

9 10

Watson, M. A.; Cockroft, S. L. Man-Made Molecular Machines: Membrane Bound. Chem.

Zhuang, T.; Tamm, L. K. Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions. Angew. Chem. Int. Ed. 2014, 53, 5897-5902.

14.

Piguet, F.; Discala, F.; Breton, M. F.; Pelta, J.; Bacri, L.; Oukhaled, A. Electroosmosis

11

through Alpha-Hemolysin That Depends on Alkali Cation Type. J. Phys. Chem. Lett. 2014,

12

5, 4362-4367.

13

15.

14 15

Kamilla, S.; Jain, V. Mycobacteriophage D29 Holin C-Terminal Region Functionally Assists in Holin Aggregation and Bacterial Cell Death. FEBS J. 2016, 283, 173-190.

16.

Lella,

M.;

Mahalakshmi,

R.

Pro-Gly

Mediated

Conformational

Switch

of

16

Mycobacteriophage D29 Holin Transmembrane Domain I Is Lipid Concentration Driven.

17

Chem. Commun. 2013, 49, 9594-9596.

18

17.

Lella, M.; Kamilla, S.; Jain, V.; Mahalakshmi, R. Molecular Mechanism of Holin

19

Transmembrane Domain I in Pore Formation and Bacterial Cell Death. ACS Chem. Biol.

20

2016, 11, 910-920.

21

18.

22 23

Helices. J. Mol. Biol. 2002, 323, 951-960. 19.

24 25 26

Cordes, F. S.; Bright, J. N.; Sansom, M. S. Proline-Induced Distortions of Transmembrane

Cosentino, K.; Ros, U.; Garcia-Saez, A. J. Assembling the Puzzle: Oligomerization of Alpha-Pore Forming Proteins in Membranes. Biochim. Biophys. Acta 2016, 1858, 457-466.

20.

Sansom, M. S. Alamethicin and Related Peptaibols--Model Ion Channels. Eur. Biophys. J. 1993, 22, 105-124. 10


Page 11 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

21.

2 3


Woolley, G. A.; Wallace, B. A. Model Ion Channels: Gramicidin and Alamethicin. J. Membr. Biol. 1992, 129, 109-136.

22.

Futaki, S.; Noshiro, D.; Kiwada, T.; Asami, K. Extramembrane Control of Ion Channel

4

Peptide Assemblies, Using Alamethicin as an Example. Acc. Chem. Res. 2013, 46, 2924-

5

2933.

6

23.

7

Hou, X.; Guo, W.; Jiang, L. Biomimetic Smart Nanopores and Nanochannels. Chem. Soc. Rev. 2011, 40, 2385-2401.

8

24.

Hamley, I. W. Peptide Nanotubes. Angew. Chem. Int. Ed. 2014, 53, 6866-6881.

9

25.

Hessa, T.; Kim, H.; Bihlmaier, K.; Lundin, C.; Boekel, J.; Andersson, H.; Nilsson, I.; White,

10

S. H.; von Heijne, G. Recognition of Transmembrane Helices by the Endoplasmic

11

Reticulum Translocon. Nature 2005, 433, 377-381.

12

26.

Moon, C. P.; Fleming, K. G. Side-Chain Hydrophobicity Scale Derived from

13

Transmembrane Protein Folding into Lipid Bilayers. Proc. Natl. Acad. Sci. U. S. A. 2011,

14

108, 10174-10177.

15

27.

16 17

Kyte, J.; Doolittle, R. F. A Simple Method for Displaying the Hydropathic Character of a Protein. J. Mol. Biol. 1982, 157, 105-132.

28.

Wimley, W. C.; Creamer, T. P.; White, S. H. Solvation Energies of Amino Acid Side

18

Chains and Backbone in a Family of Host-Guest Pentapeptides. Biochemistry 1996, 35,

19

5109-5124.

20 21

11



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FIGURES AND FIGURE LEGENDS

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Figure 1. Peptide engineering and design. (A) The sequence of all the peptides is listed along with the peptide code. Data from the mass spectra are summarized in Table S1. (B) Schematic illustrating how the sequence was reversed progressively, using a heuristic approach by residue– swap and PG permutation strategy. We swapped residues at the terminal (tTM1), semi-strand (sTM1) or carried out the complete reversal (rTM1) of the TM1 domain. To assess if the terminal residues were involved in helix nucleation, we also truncated the terminal residues to generate a 26residue peptide (trTM126). While these formed our key peptides for characterization, we also generated other peptide permutants, such as those involving the central Pro-Gly segment (PG; dotted line). The N-terminal segment of TM1 is colored in blue and the C-terminal segment is in orange. This color scheme is retained in the chimera library sequence shown in (A). (C) Electrophysiology measurement of the TM1 and rTM1 peptides recorded in a planar DiPhPC phosphocholine bilayer at an applied holding voltage of +10 mV. 0 pA indicates a stable membrane and 1000 pA indicates that the membrane is disrupted. Progressive insertion of TM1 (left) causes membrane disruption within a few seconds. On the other hand, when rTM1 (right) associates with the membrane, we see ion channel-like behavior (enlarged graph in the right panel). The membrane remains stable for several hours.

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Figure 2. Complete spectroscopic analysis of the conformational behavior of reverse-engineered TM1 peptides. (A) Far-UV CD spectra of peptide structure across select TM1 chimeras obtained after thermal denaturation measurements. Data are shown in increasing concentrations of LDAO (lauryldimethylamine oxide) and DPC (n-dodecyl phosphocholine, inset). TM1pG (far left) forms a peptide β-hairpin that is independent of the detergent-to-peptide ratio (DPR), with antiparallel sheets connected by the DPro-Gly central turn segment. (LDAO concentrations as high as 200 mM do not alter the peptide CD spectrum.) We obtain an increase in the helical content as we move from TM1pG to rTM1; the latter adopts a helical structure in all DPRs of LDAO and DPC (except 4 mM LDAO). At the highest micelle concentration of 100 mM LDAO (or 50 mM DPC, inset), the increase in helicity in the peptides follows the order TM1pG < trTM126 < tTM1 < sTM1 < rTM1 (left-to-right). Note that although a similar trend is maintained in DPC, the ability of these peptides to adopt helical structures is promoted even at lower DPRs of DPC, than the corresponding DPRs of LDAO. A rainbow color scheme is maintained in each spectrum, with red and violet corresponding to the lowest and highest DPRs. Note that all the data are presented as molar ellipticity (ME) values with units of deg cm2 dmol-1, and can be converted to per-residue ellipticity (MRE with units of deg cm2 dmol-1 residue-1) by dividing the ME by the total number of residues in each peptide. (B) The change in secondary structure content upon thermal denaturation (black) and recovery from melting (red) monitored for the various peptides at every 5 °C, are plotted here at 208 nm (ME208; deg cm2 dmol-1). Shown here is the data for 30 mM LDAO, and the complete data is presented in Figure S2. TM1pG (extreme left) adopts a β-hairpin structure, while TM1UG (extreme right), with a central AibGly segment, forms an example of an ideal helical structure. The behavior of all other peptide chimeras can be compiled between these two extreme structure scaffolds. trTM1 and tTM1 show low secondary structure content at 4 °C. These peptides exhibit a gain in ME208 upon heating (note 13



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that higher negative values of ME208 corresponds to greater CD signal), but revert to α+β structures upon cooling. The process exhibits hysteresis, as the unfolding and refolding curves do not overlap. The native TM1 peptide shows an increase in helicity upon heating, and re-gains structure on cooling in a completely reversible manner, without being affected by a hysteresis loop. The higher order reverse chimeras, sTM1 and rTM1 peptides, show a considerable and sigmoidal gain in helicity upon heating, with a mid-point temperature of ~50 °C. These peptides retain their helical structure even after the sample is restored to 4 °C. (C) Time-dependent gain in peptide helicity shown as an increase in the ME222 value, is seen in the case of sTM1 and rTM1. Shown here is data for 30 mM LDAO. The process is three times slower in sTM1 than rTM1. Other peptides retain their secondary structure upon prolonged incubation, and show negligible increase in their helical content. As in the case of (B), TM1pG and TM1UG serve as control peptides for comparison. Fits (solid lines) highlight the trend in the datasets. The corresponding data in 100 mM LDAO is provided in Figure S3. TM1pG data has been reproduced from 16 with permission from The Royal Society of Chemistry.

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Figure 3. Concerted ion channel measurements of all engineered chimeras in planar lipid bilayers. (A) Representative ion channel recordings for the various peptide chimeras. Data were acquired on the planar bilayer set-up, from -100 mV to +100 mV at increments of 10 mV, for at least a 2 min window. Shown here is a 10 s representative data obtained at +20 mV. The complete data is presented in Figure S7 and Figure S10. rTM1 and sTM1 exhibit transitions across states with multiple conductance. We describe these transitions as occurring from two open states (O1 and O2; green lines) and a single low conductance state (C; red line). The low conductance state is assigned as the baseline conductance measured after peptide insertion in the membrane. The other reverse chimeras only exhibit transitions from a single open state (O) to the low conductance state. The event amplitude histograms for the open and low conductance states are plotted against the corresponding conductance (G) in nS, and are presented to the right of each recording (also see Figure S8). (B) Open state dwell times for each reverse chimera peptide were calculated for the channels at different voltages (-100 mV to +100 mV, at 10 mV increments). rTM1 is the only peptide that displays a voltage-dependent change in the channel dwell time. The dwell time increases ~100-fold between 50 mV and 100 mV at both the positive and negative voltages. All other peptide dwell times are largely insensitive to the change in voltage. The trend in each dataset is shown using the solid red line. The complete dwell time values for all peptides are presented in Table S2. (C) Representative event amplitude histograms for select peptide chimeras at ±10 mV (grey), ±30 mV (blue) and ±100 mV (red). Graphs on the left are for the positive holding voltages, and those on the right are data for the negative holding voltages. The solid lines are fits of the data to a variable metric method in exponential log probability function, with the sum of squared errors as a minimization method. The fits yielded the dwell times, shown in (B). Note how the histograms shift considerably to the right with increasing absolute voltage only for the rTM1 peptide. One complete representative dataset each for rTM1 sTM1, tTM1 and trTM126 is shown in Figure S9.

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Figure 4. Role of charge asymmetry and hydropathy on membrane-breaker versus ion channel behavior. (A) Schematic representation of how charged residues can influence peptide association in the membrane, and give rise to ion channel formation or membrane disruption. The membrane breaker peptide TM1 and to some extent tTM1 (which shows significant instances of membrane disruption in lipid bilayer measurements), show an asymmetric enrichment of net charges (+2 and 1) towards the termini. sTM1 and rTM1, which form ion channels in the membrane, have an overall similar charge distribution with one net positive charge only at the C-terminal end. It is likely that this positive charge is also offset by the net dipole moment of the helix, further lowering the charge content and generating a ‘neutral’ helical structure. We speculate that this net lowering of charge distribution could be one of the factors that play a role in ion channel formation (also see Figure S13). (B) Normalized hydropathy (least hydrophobic = 0; most hydrophobic = 1) calculated using a three amino acid window, at each position along the 28-residue peptide sequences. Four independent scales (Hessa,25 Moon-Fleming,26 Kyte-Doolittle,27 and Wimley-White 28) were chosen for the analysis. The average data across all scales is shown here. Note how the hydropathy of rTM1 is distinct from the other three peptides. (Left) Comparison of average per-residue hydropathy of rTM1 (green) with TM1 (black), highlighting the differences between both peptides. (Right) TM1, tTM1 (red), and sTM1 (blue) show a similar hydropathy index. When the rTM1 sequence is presented in reverse, a change in the local vicinity of the amino acids results in an overall change in the hydropathy index. (C) Schematic representation of an ion channel formed by the assembly of the engineered TM1 peptide (blue cylinder; a hypothetical heptamer is shown for illustration) in the membrane, indicating the factors that may be responsible for the formation of a stable ion channel.

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Figure 1. Peptide engineering and design. 88x43mm (300 x 300 DPI)



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Figure 2. Complete spectroscopic analysis of the conformational behavior of reverse-engineered TM1 peptides. 124x88mm (300 x 300 DPI)


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Figure 3. Concerted ion channel measurements of all engineered chimeras in planar lipid bilayers. 101x58mm (300 x 300 DPI)



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Figure 4. Role of charge asymmetry and hydropathy on membrane-breaker versus ion channel behavior. 81x36mm (600 x 600 DPI)


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TOC Graphic 37x28mm (600 x 600 DPI)


Engineering a Transmembrane Nanopore Ion Channel from a

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