Trigonal tryptophane zipper as a novel building block for pH-responsive peptide nano-assemblies

Abstract

A novel C₃-symmetric peptide conjugate bearing tryptophane zipper-forming peptides showed pH-responding self-assembly into nanospheres and nanofibers in water.

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The tryptophane zipper has been attracting much attention as a secondary structure motif, which forms a stable twisted β-hairpin structure due to the interaction between tryptophane residues.¹ The main focus in the study of the tryptophane zipper has been its structural features, and there is no report that applied the tryptophane zipper as a building block for nano-assemblies. We have designed C₃-symmetric β-sheet forming peptide^2a,b and glutathione^2c,d conjugates and demonstrated that they self-assembled into nanospheres^2a,c,d and nanofibers.^2b These studies have shown that preorganizing the spatial arrangement of peptides enhanced the self-assembling ability and affords unique assemblies.²

Here, we report self-assembly of a novel C₃-symmetric peptide conjugate, Trigonal-WTW (Fig. 1), which possesses three tryptophane zipper-forming peptides. Since the present C₃-symmetric Trigonal-WTW possesses a unique arrangement of charged residues, we expect that the novel peptide conjugate will exert pH-responsive self-assembly. The distinctive twisted β-structure of the tryptophane zipper may also afford unique nano-assemblies. To date, many peptide nano-assemblies from controlled secondary structures,³ such as amphiphilic α-helixes,⁴ coiled-coils,⁵ and β-sheets,⁶ have been reported. In many cases, self-assembly of one peptide conjugate afforded only one kind of morphology of assembly (sphere or fiber). In this paper, we show Trigonal-WTW afforded two different morphologies of assembly (sphere and fiber) depending on pH.

Fig. 1 Structures of trigonal peptide conjugates at various pH.

The 8-mer peptide H-CKTWTWTE-OH was designed based on the intramolecular tryptophane zipper (β-hairpin) forming peptide reported by Cochran et al.^1a It was synthesized by a standard Fmoc-protected solid-phase method. The peptide CKTWTWTE possesses two amino groups (N-terminal and K residue) and two carboxyl groups (C-terminal and E residue), respectively. When the peptides form an intermolecular tryptophane zipper in water at neutral pH, it is expected that the antiparallel arrangement predominantly forms due to electrostatic complementarity of the peptide. Trigonal-WTW was prepared by coupling the 8-mer peptides with C₃-symmetric iodoacetoamidated core molecule 1 (Chart 1) in the presence of DIPEA, purified by reverse-phase HPLC, and confirmed by MALDI-TOF-MS (m/z = 3619 [M+H]⁺) and ¹H NMR (see Supplementary Information). As a control molecule, Trigonal-FTF (Fig. 1) in which two tryptophanes of Trigonal-WTW were replaced with phenylalanines, was also prepared by a similar method.

Chart 1 Structure of C3-symmetric iodoacetoamidated core molecule 1

It has been reported that tryptophane zipper-forming β-hairpin peptide showed an exciton-coupling type circular dichroism (CD) spectrum (positive peak at 229 nm and negative peak at 215 nm) along with small peaks at 285–295 nm based on the interaction between Trp residues.^1c In citrate buffer (pH 3), the CD spectrum of Trigonal-WTW showed a negative peak at 216 nm and a positive peak at 240 nm, which is mainly ascribed to β-sheet structure (Fig. 2, red line). The CD intensity of the positive peak at 234 nm increased in aqueous NaOH solution (pH 11, blue line), indicating an increase in the content of tryptophane zipper. In phosphate buffer (pH 7.0), the CD spectrum of Trigonal-WTW showed a positive peak at 234 nm and a negative peak at 216 nm together with weak peaks at 280–300 nm (Fig. 2, green line). These observations indicate that Trigonal-WTW adopts mixed secondary structures of tryptophane zipper and normal β-sheet structures at pH 7. In contrast, the CD spectrum of the precursor peptide CKTWTWTE revealed that the peptide adopted a random-coil structure at pH 7 (Fig. 2, black line). Apparently, the formation of secondary structures (normal β-sheet and tryptophane zipper) from the peptide CKTWTWTE is promoted by the C₃-symmetric preorganization.

Fig. 2 CD spectra of aqueous solution of Trigonal-WTW (10 μM) at 25 °C in 20 mM citrate buffer (pH 3.0, red line), 20 mM phosphate buffer (pH 7.0, green line), and aqueous NaOH solution (pH 11, blue line). Black line shows a CD spectrum of aqueous peptide CKTWTWTE (30 μM) in 20 mM phosphate buffer (pH 7.0) at 25 °C.

Specimens for scanning electron microscopy (SEM) of Trigonal-WTW were prepared by placing a drop (10 μL) of sample solutions (10 μM) on a fleshly cleaved mica substrate. At pH 3, only irregular aggregates were observed in the SEM image (Fig. 3A). In phosphate buffer (pH 7), Trigonal-WTW selectively formed spherical assemblies with the size of 20–30 nm (Fig. 3B), which is in good agreement with the average diameter (19.8 ± 1.9 nm) obtained from dynamic light scattering (Fig. 3D). At pH 11, the SEM image revealed that Trigonal-WTW formed a mixture of spherical assemblies with the size of 30–50 nm and fibrous assemblies with the width of about 5 nm (Fig. 3C, beads on string structure). These fibrous structures are typically observed for parallel β-sheet structures formed by face-to-face aggregation of trigonal molecules.^2b

Fig. 3 (A) SEM images of Trigonal-WTW (10 μM) in 20 mM citrate buffer (pH 3.0), (B) in 20 mM phosphate buffer (pH 7.0), and (C) in aqueous NaOH solution (pH 11) on mica substrate. SEM samples on mica were coated with 7–9 nm Pt. (D) Size-distribution obtained from DLS of Trigonal-WTW (10 μM) in 20 mM phosphate buffer (pH 7.0) at 25 °C.

The pH dependence of the secondary and self-assembled structure of Trigonal-WTW could be ascribed to the difference in peptide charges. By using pK_a values of each dissociation group, the isoelectric point (pI) is estimated as 7.1, thus Trigonal-WTW possesses zwitterionic structure at pH 7 (Fig. 1). It appears that Trigonal-WTW formed the nanospheres by forming intermolecular antiparallel β-sheet-like (including tryptophane zipper) structures due to the attractive ionic complementarity. It is of note that nanofibers were little observed at pH 7. It suggests that Trigonal-WTW possesses a higher tendency to form antiparallel β-sheets. Although Trigonal-WTW at pH 11 possesses 6 anionic charges near the periphery, the fibrous assemblies were formed probably because of the stacking interactions between the hydrophobic cores. It is probable that Trigonal-WTW is twisted in the fibrous assembly due to the charge repulsion. In contrast, Trigonal-WTW at pH 3 possesses 6 cationic charges close to the core, which may inhibit formation of stacked fibrous structures due to the electrostatic repulsions.

In contrast to Trigonal-WTW, CD spectra of the control molecule Trigonal-FTF revealed that the conjugate adopted random-coil structures at each pH condition (Fig. S1). In the SEM image of the aqueous solutions of Trigonal-FTF, only deformed aggregates were observed (Fig. S2). Therefore, it seems that Trigonal-WTW has higher self-assembling ability than Trigonal-FTF. The difference in self-assembling behaviour shows a good correlation with the difference in formation energy of antiparallel dimers of peptides in water at pH 7, which is estimated by molecular mechanics calculation (MacroModel) to be ΔE = −966 kJ mol⁻¹ for CKTWTWTE and ΔE = −52 kJ mol⁻¹ for CKTFTFTE (Fig. 4).

Fig. 4 Most stable conformations and total energies of peptides and their dimers with parallel and antiparallel arrangements expected by molecular mechanics calculation (MacroModel). (A) CKTWTWTE monomer, (B) CKTWTWTE antiparallel dimer, (C) CKTFTFTE monomer, and (D) CKTFTFTE antiparallel dimer.

In conclusion, we have developed a novel C₃-symmetric peptide conjugate, Trigonal-WTW, bearing three tryptophane zipper-forming peptides. The conjugate self-assembled into nanospheres via formation of tryptophane zipper at pH 7, but into a mixture of nanofibers and nanospheres at pH 11, and irregular aggregates at pH 3. Thus, we have succeeded in switching the morphology of peptide nano-assemblies by changing the pH of solution of only one peptide conjugate. This study is the first example of nano-assemblies applying a tryptophane zipper peptide as a building block. We think the unique self-assembling behavior arises from the feature of tryptophane zipper structure and C₃-symmetric conjugation. The present molecular design provides novel guidelines for the design of pH-sensitive peptide-based nano-assemblies.

The present work is supported by PRESTO, JST, by a Grant-in-Aid for Scientific Research (B) (No. 22350075) from JSPS, and by a Grant-in-Aid for the Global COE Program, “Science for Future Molecular Systems” from MEXT.

Notes and references

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Footnotes

† This article is part of the ‘Emerging Investigators’ themed issue for ChemComm.

‡ Electronic supplementary information (ESI) available: Experimental section, Scheme S1, Fig. S1 and S2. See DOI: 10.1039/c0cc01324b

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