Self-assembly of cyclic peptide monolayers by hydrophobic supramolecular hinges

Abstract

Supramolecular polymerisation of two-dimensional (2D) materials requires monomers with non-covalent binding motifs that can control the directionality of both dimensions of growth. A tug of war between these propagation forces can bias polymerisation in either direction, ultimately determining the structure and properties of the final 2D ensemble. Deconvolution of the assembly dynamics of 2D supramolecular systems has been widely overlooked, making monomer design largely empirical. It is thus key to define new design principles for suitable monomers that allow the control of the direction and the dynamics of two-dimensional self-assembled architectures. Here, we investigate the sequential assembly mechanism of new monolayer architectures of cyclic peptide nanotubes by computational simulations and synthesised peptide sequences with selected mutations. Rationally designed cyclic peptide scaffolds are shown to undergo hierarchical self-assembly and afford monolayers of supramolecular nanotubes. The particular geometry, the rigidity and the planar conformation of cyclic peptides of alternating chirality allow the orthogonal orientation of hydrophobic domains that define lateral supramolecular contacts, and ultimately direct the propagation of the monolayers of peptide nanotubes. A flexible 'tryptophan hinge' at the hydrophobic interface was found to allow lateral dynamic interactions between cyclic peptides and thus maintain the stability of the tubular monolayer structure. These results unfold the potential of cyclic peptide scaffolds for the rational design of supramolecular polymerisation processes and hierarchical self-assembly across the different dimensions of space.

Fig. 1. Rational design and structural scheme of self-assembled 2D nanotubular monolayers of cyclic peptide monomers. (A) A cyclic octapeptide (left) with a single hydrophobic domain matches the structural requirements for a staggered bilayer assembly; increasing molecular diameter to a cyclic decapeptide (right) allows the incorporation of two hydrophobic motifs at 180° in a C₂ symmetric scaffold for the lateral assembly of nanotubular monolayers. (B) Longitudinal antiparallel β-sheet hydrogen bonded network of the stacked cyclic peptide monomers. (C) Supramolecular structure of a two-dimensional monolayer assembled from nanotubes through lateral hydrophobic contacts between aligned Trp (orange) and Leu (green) residues of CP10. (D) Lateral contact model: ‘Trp hinge’ with the aromatic residue pivoting between the two possible Leu zippers.

Fig. 2. (A) Left: atomistic representation of CP10 in parallel (top) and antiparallel (bottom) configurations. Right: MD simulations of one single nanotube made of eight CP10 units stacked in either parallel (blue) or antiparallel (yellow) configuration. The data show the total interaction energy, including both Lennard-Jones and Coulomb potentials, between the top two CP10 units of the nanotubes (in darker colour) simulated for 100 ns in aqueous solution. (B) Potential of Mean Force (PMF) profile of two 3CP10-NTs in axial assembly and antiparallel configuration. ‘d’ indicates the distance between the centre of mass of the nanotubes. (C) PMF profile of two 8CP10-NTs in lateral assembly and antiparallel configuration. (D) Global free energy (ΔG) minima, i.e. dimerisation free energy, for CP10 nanotubes in axial (red) and radial (green) association (see (B) and (C)) as a function of their oligomerisation degree in antiparallel configuration. Error bars were obtained from the PMF calculation. Because of their low probability to laterally assemble, 1CP10-NT, 2CP10-NT and 3CP10-NT have a negligible lateral (green) dimerisation ΔG. Note: 1D nanotubes are denoted as ‘XCP10-NT’, where ‘X’ indicates the oligomerisation degree [i.e. number of CP10 units constituting the nanotube (NT)].

Fig. 3. Dry-state microscopic characterisation of 2D nanosheets composed of CP10 nanotube monolayers: (A) ThT-stained epifluorescence; (B) STEM; (C) electron diffraction pattern by high-resolution TEM and its Fourier transform (inset), revealing two predominant distances: d₁ (axial CP spacing) and d₂ (lateral 2D spacing); (D) AFM with height profile.

Fig. 4. (A) Free energy surface (FES) metadynamics simulation of the spatial evolution of two 8CP10-NTs shown as free energy surface mapping as a function of Trp–Trp contacts and sum of d₁ + d₂ distance (see ESI†). (B) Energy profile between (i) and (ii) minima from (A). (C) Structural snapshots of interconverting minima (i) and (ii). Trp and Leu residues are coloured in orange and green, respectively. (D) Structures of CP10 and their analogues, 3L and LW, with highlighted Trp and Leu residues. Epifluorescence micrographs of ThT-stained samples of 3L (left) and LW (right) showing no evidence of 2D nanosheets. Arrows point at bundled 1D structures. Scale bars = 50 μm.

Fig. 5. CG-MD simulations of ten 4CP10-NTs in aqueous solution with variations in the hydrophobic strength of Trp residues (Δ_hpho; see Methods). (A) Snapshots of the native 4CP10-NT model (Δ_hpho = 0) and a customised analogue with Trp beads displaying a 50% reduction of their original hydrophobic strength (Δ_hpho = −50%). Orange beads indicate tryptophan residues. (B) Time evolution of the average cos(θ) between the longitudinal axes of all possible NT couples. (C) Time evolution of the number of 4CP10-NT clusters versus Δ_hpho. (D) Self-assembly pathway as function of Δ_hpho comparing solvophobic 4CP10-NT clustering versus relative orientation based on Trp's hydrophobic character.