Self-assembly of synthetic collagen triple helices

Abstract

Collagen is the most abundant protein in animals and the major component of connective tissues. Although collagen isolated from natural sources has long served as the basis for some biomaterials, natural collagen is difficult to modify and can engender pathogenic and immunological side effects. Collagen comprises a helix of three strands. Triple helices derived from synthetic peptides are much shorter (<10 nm) than natural collagen (Ϸ300 nm), limiting their utility. Here, we describe the synthesis of short collagen fragments in which the three strands are held in a staggered array by disulfide bonds. Data from CD spectroscopy, dynamic light scattering, analytical ultracentrifugation, atomic force microscopy, and transmission electron microscopy indicate that these ''sticky-ended'' fragments self-assemble via intermolecular triple-helix formation. The resulting fibrils resemble natural collagen, and some are longer (>400 nm) than any known collagen. We anticipate that our self-assembly strategy can provide synthetic collagen-mimetic materials for a variety of applications.biomaterial ͉ coiled-coil ͉ nanotechnology ͉ cystine knot ͉ peptide C ollagen constitutes one-third of the human proteome, including three-quarters of the dry weight of human skin. Its high natural abundance and intrinsic plasticity have spurred the development of collagen as a biomaterial (1, 2). The most common source of clinical collagen is now Bos taurus, the domestic cow. Unfortunately, bovine collagen can illicit deleterious pathological and immunological effects when transplanted into humans (3-5). Moreover, the preparation of enriched solutions of natural collagen is problematic (6), and its sitespecific covalent modification is not feasible. We suspected that synthetic chemistry could offer a solution to these problems.The quaternary structure of collagen comprises three strands that wrap around one another to form a triple helix (7). Each strand has the repeating sequence XaaYaaGly, with the most abundant triplet being ProHypGly [Hyp ϭ (2 S,4R)-4-hydroxyproline] (8). The folding of (XaaYaaGly) nՅ10 peptides into blunt-ended triple helices has been investigated thoroughly (7). Although such triple helices are biomaterial candidates (9-12), they are limited to the length of a synthetic peptide (Ͻ10 nm), which is much shorter than natural collagen (Ϸ300 nm). The polymerization of (XaaYaaGly) 10 peptides has afforded long strands that adopt triple-helical structure but have high polydispersity (13).Molecular self-assembly underlies the ''bottom-up'' approach to macromolecular design, wherein a desirable structure forms spontaneously through noncovalent interactions (14, 15). Selfassembling peptides and proteins have been designed to serve as materials for biological and nanotechnological applications (16,17). Using the self-assembly approach, Woolfson and coworkers (18,19) have produced fibers with a design based on a dimeric coiled-coil structure. Likewise, fibrous peptides based on the natural protein elastin (20) and de novo building b...

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Phosphorylation as Conformational Switch from the Native to Amyloid State: Trp-Cage as a Protein Aggregation Model

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Preparation and Characterization Urea-Solubilized Sol-Gel Type I Collagen and Its Possible Use in Applications

“…Although these model peptides yield much information about the dynamic process of the assembly and triple helix building, the information is just part of that necessary for the understanding of real collagen. Studies have shown that polymerization of these model peptides yield the triple helix form but with a high polydispersity [Kotch & Raines, 2006;Paramonov, et al, 2005]. Furthermore, it must be taken into account, the α 1 and α 2 chains, containing about 1000 amino acids each, show only 43 and 28 [Gly-Pro-Hyp] (rat Type I collagen, SP:02454 and 02466) repeats, respectively.…”

“…There are two major methods to study collagen: using isolated natural collagen [Kadler, et al, 1988] and synthetic model peptide/protein [Kotch & Raines, 2006;Malone, et al, 2005]. Due to its high complexity and high molecular weight, most structural studies have relied on model peptides.…”