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 (EC50 potency: 1a/1b Ͼ Ͼ 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.biomaterial ͉ platelets ͉ structure-function ͉ supramolecular triplex T he self-association of peptides and proteins into well ordered supramolecular structures is of pivotal importance in normal physiology and pathophysiology, such as in the assembly of collagen fibrils (1), actin filaments (2), and amyloid fibrils (3, 4). Collagens, which constitute a ubiquitous protein family in animals, contribute an essential matrix component to soft tissues and bones (5, 6). A structural hallmark of many collagens is a rope-like triple helix, the architecture of which derives from the interplay of three proline-rich polypeptide strands (e.g., two ␣1 and one ␣2 for type I collagen) (6-8). In the core domain of the triple helix, the amino acid sequence G-X-Y is repeated multiple times, and each glycine amide NH forms a hydrogen bond with the X-position amide carbonyl on an adjacent strand. The X-and Y-positions are often populated by L-proline and 4(R)-hydroxy-L-proline (O; Hyp), respectively, with the latter stabilizing the triple helix by stereoelectronic effects (9) and water-bridged hydrogen bonds (10).To investigate collagen's structure and function, researchers have resorted to using synthetic collagen model peptides (CMPs)

Methacrylation Induces Rapid, Temperature-Dependent, Reversible Self-Assembly of Type-I Collagen

“…Such perturbations can be caused by aromatic amino acids, which have been shown to be involved in direct contacts between triple-helical units in the higher-order assembly. 29−31 This novel observation will be studied in the future to understand the molecular determinants of the negative MRE value at 222 nm.…”

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

“…The aggregation of peptides with interruptions contrasts with the selfassociation of peptides containing perfect (Gly-XaaYaa) n sequences, which requires high concentrations and higher temperatures and occurs over a period of minutes to hours. 24,40 Although the aggregates formed by peptides with uninterrupted (Gly-XaaYaa) n sequences are likely to be composed of overlapping triple-helical molecules, 24,40,41 the nature of the fibrillar material formed from peptides containing both (Gly-Pro-Hyp) n sequences and b-aggregationprone interruption sequences is not known as the high content of imino acids and Gly residue is expected disfavor b-sheet formation. The data reported here suggest that some natural interruptions may increase the propensity of triple-helices to self-associate and thus could play a previously unsuspected role in collagen molecular self-association.…”

Artificial collagen gels via self‐assembly of de novo designed peptides

“…18 Aromatic stacking and hydrophobic interactions between the side chains of the N-and C-terminal amino acid residues of synthetic triple-helical peptides was further utilized for the formation of a higher-order fibrous structure. 19,20 This strategy allowed to create hydrated particles, which could be observed by light microscope. Recently, Raines's group and ourselves independently developed a peptide system for creating elongating triple-helical supramolecules via spontaneous intermolecular folding between trimeric collagen-like peptides with self-complementary shapes.…”