Biomimetic organization: Octapeptide self-assembly into nanotubes of viral capsid-like dimension

Abstract

The controlled self-assembly of complex molecules into well defined hierarchical structures is a promising route for fabricating nanostructures. These nanoscale structures can be realized by naturally occurring proteins such as tobacco mosaic virus, capsid proteins, tubulin, actin, etc. Here, we report a simple alternative method based on self-assembling nanotubes formed by a synthetic therapeutic octapeptide, Lanreotide in water. We used a multidisciplinary approach involving optical and electron microscopies, vibrational spectroscopies, and small and wide angle x-ray scattering to elucidate the hierarchy of structures exhibited by this system. The results revealed the hexagonal packing of nanotubes, and high degree of monodispersity in the tube diameter (244 A) and wall thickness (approximately equal to 18 A). Moreover, the diameter is tunable by suitable modifications in the molecular structure. The self-assembly of the nanotubes occurs through the association of beta-sheets driven by amphiphilicity and a systematic aromatic/aliphatic side chain segregation. This original and simple system is a unique example for the study of complex self-assembling processes generated by de novo molecules or amyloid peptides.

Fig. 1.

Optical textures of hexagonal columnar phases of Lanreotide (a and b) and its derivative (c and d) observed between cross-polarizers (±45°) through thin preparations (magnification, ×2,500). A color plate is added in b. (c) Texture growing from isotropic liquid (magnification, ×1,250). The observed fan-shape textures are characteristic deformations of parallel planes in developable surfaces (arrows). These deformations are compatible with hexagonal columnar liquid-crystal phases (or lamellar phases).

Fig. 2.

Characterization of Lanreotide nanotubes. (a and b) Freeze-fracture electron micrographs of a 14% wt/wt Lanreotide acetate-water sample. The planes of fracture are perpendicular (a) and parallel (b) to the director of the tightly packed thin tubes. (Insets) A ×2 enlargement of the corresponding micrographs. (c) SAXS with sample-detector distance of 6 m. Superimposed lines indicate calculated values in the case of a 2D columnar hexagonal phase with a packing parameter a_hex of 365 Å, which corresponds to the distance between the centers of the nanotubes. (d) SAXS with a sample-detector distance of 1.5 m. The diffraction peaks are not resolved, but their envelope (form factor) is observed. The zeros are in agreement with a Bessel function (J₀(q·r₀)/q·r₀)² (dashed curve), which corresponds to the form factor of a monodisperse cylinder of radius r₀ = 122 Å. The wall thickness of the nanotubes, estimated from the position of the last oscillation, is ≈18 Å.

Fig. 3.

Conformation of Lanreotide in the nanotubes determined by vibrational spectroscopies. (a) Fourier transform (FT)-Raman spectroscopy. The figure shows the frequency range of disulfide bond vibrations. The 506-cm^-1 vibration (arrow) indicates a gauche-gauche-gauche disulfide bridge. The 519-cm^-1 vibration is characteristic of the Naphthalene ring of the d-naphthylalanine residue. (b) Fourier transform infrared spectrum of the amide I region (vibrations of the carbonyl groups) after subtraction of the water contribution. The percentages of backbone carbonyls involved in hydrogen bonds in different conformations have been estimated after deconvolution, i.e., 35% of antiparallel β-sheet (1,618 and 1,689 cm^-1), 15% of turn (1,663 cm^-1), and 50% of random (1,639 cm^-1).

Fig. 4.

Crystalline structure of the wall of Lanreotide nanotubes. (a) High-resolution fiber diffraction of the Lanreotide derivative at 10% wt/wt (acetate) in water. (b and c) Simulations of diffuse scattering at wide angles (WAXS) of selected zones (rectangles in a). (d) Two-dimensional Patterson function indicating the main electron density variations of the nanotube wall. (e) Zoom of the unit cell and definition of the cell vectors i and j. The black circles indicate the 2-fold symmetry axes. The β-hairpin backbone of Lanreotide is drawn on the zoom to fit the regions of high (red) and low (blue) electron densities. Note the alternation of low and high electron density areas along j and the continuity of these areas along i.

Fig. 5.

Schematic view of the different hierarchical levels in the self-assembly of Lanreotide-acetate nanotubes in water. (a)(Left) The Lanreotide molecule in the β-hairpin planar conformation, which is stabilized by the disulfide bridge, the turn, and intramolecular hydrogen bonds. (Right) Interaction between two Lanreotide molecules within the wall (bilayer) of the nanotubes. (Bottom) CPK models of a conformation in agreement with experimental data. The segregation of aromatic residues (red) from aliphatic residues (blue) and from hydrophilic region (green) is remarkable. (b) The structure of a filament with two different β-sheet fibers superimposed with their C₂ 2-fold axes (black circles) meeting together. The segregation between aliphatic/aromatic residues is conserved within the filament organization. (Inset) Packing of the aromatic residues within the β-sheet fibers. (c) Self-assembly of 26 filaments to form a nanotube. (d) Liquid crystalline hexagonal columnar phase formed by the nanotubes.