Thrombogenic collagen-mimetic peptides: Self-assembly of triple helix-based fibrils driven by hydrophobic interactions

Abstract

Collagens are integral structural proteins in animal tissues and play key functional roles in cellular modulation. We sought to discover collagen model peptides (CMPs) that would form triple helices and self-assemble into supramolecular fibrils exhibiting collagen-like biological activity without preorganizing the peptide chains by covalent linkages. This challenging objective was accomplished by placing aromatic groups on the ends of a representative 30-mer CMP, (GPO)(10), as with l-phenylalanine and l-pentafluorophenylalanine in 32-mer 1a. Computational studies on homologous 29-mers 1a'-d' (one less GPO), as pairs of triple helices interacting head-to-tail, yielded stabilization energies in the order 1a' > 1b' > 1c' > 1d', supporting the hypothesis that hydrophobic aromatic groups can drive CMP self-assembly. Peptides 1a-d were studied comparatively relative to structural properties and ability to stimulate human platelets. Although each 32-mer formed stable triple helices (CD) spectroscopy, only 1a and 1b self-assembled into micrometer-scale fibrils. Light microscopy images for 1a depicted long collagen-like fibrils, whereas images for 1d did not. Atomic force microscopy topographical images indicated that 1a and 1b self-organize into microfibrillar species, whereas 1c and 1d do not. Peptides 1a and 1b induced the aggregation of human blood platelets with a potency similar to type I collagen, whereas 1c was much less effective, and 1d was inactive (EC(50) potency: 1a/1b >> 1c > 1d). Thus, 1a and 1b spontaneously self-assemble into thrombogenic collagen-mimetic materials because of hydrophobic aromatic interactions provided by the special end-groups. These findings have important implications for the design of biofunctional CMPs.

Fig. 1.

Structures of 1a–d and 2.

Fig. 2.

Interface from energy-minimized structure of triple-helical, head-to-tail homodimer (1a′)₃/(1a′)₃ (one blue, one yellow; standard atom-coloring scheme for N, O, F, and H) showing three aromatic stacking interactions (black double-headed arrows; A–C) and three salt bridges with hydrogen bonds (magenta dotted lines; 1–3), each involving an ammonium group (NH₃⁺) and a carboxylate group (CO₂⁻). The H-bond between an ammonium and a backbone carbonyl is also shown (magenta dotted line; 4).

Fig. 3.

CD spectral data. (A) CD curves for 1a (red), 1b (blue), 1c (green), 1d (orange), and 2 (pink). (B) CD melting curves for 1a (red), 1b (blue), 1c (green), and 1d (orange).

Fig. 4.

AC-AFM topography images for 1a (A), 1b (B), 1c (C), and 1d (D) from incubated aqueous solutions (0.1 mg/ml) deposited onto freshly cleaved mica. (Scale bars, 1 μm.)

Fig. 5.

Light microscopy image of a particle from self-assembly of 1a. (Scale bar, 1 mm.)

Fig. 6.

Platelet aggregation experiments with peptides, under different conditions, and collagen. (A) 1a, green; 1b, yellow; 1c, blue; 1d, black; 2, violet; 4, light blue (2 mg/ml in PBS, incubated at 4°C for 7 days); and collagen, red. (B) 1a, dark green; 1b, yellow-brown; and 4, black (7 mg/ml in PBS, incubated at 37°C for 80 min); 1a, green; 1b, yellow, diamond; 2, violet; and 4, blue (2 mg/ml in PBS, incubated at 4°C for 7 days); collagen (red, inverted triangle). EC₅₀ values are given in Tables S2 and S3.