Collagen-Related Peptides:  Self-Assembly of Short, Single Strands into a Functional Biomaterial of Micrometer Scale

Abstract

Collagen, the most abundant protein in mammals, has fascinated scientists because of its extraordinary structural features and biological importance. In 1961, Rich and Crick suggested that collagen possesses a triple-helical structure, 1 which was later confirmed by X-ray analysis of 30-mer collagen-related peptides (CRPs), such as (Pro-Hyp-Gly) 4 Pro-Hyp-Ala-(Pro-Hyp-Gly) 5 and (Pro-Pro-Gly) 10 . 2 The large triple-helical domains of collagen consist of three peptide strands with Gly-X-Y repeating motifs, 300 nm in length (vs ∼9 nm for CRPs), with the X and Y mainly populated by Pro and Hyp, respectively. In addition, there are short telopeptide regions at the N-and C-termini, which are important for fibril assembly. 3 The rigidity of the ropelike super-helix and the assembled fibril helps provide mechanical strength to tissues, such as skin, tendons, ligaments, and blood vessels. Following vascular injury, the exposed collagen in the vessel wall promotes tissue repair by activating platelets for aggregation and adhesion. However, excessive platelet activity, such as from rupture of an atherosclerotic plaque, can lead to pathological thrombosis with attendant arterial obstruction, as in myocardial infarction or stroke. 4 The study of the structure, stability, and function of collagen triple helices has been facilitated by the use of synthetic collagen model peptides. 5 Although oligomerized CRPs, via dendrimer assembly or covalent crosslinking, can effectively induce platelet aggregation, less organized CRPs have lacked this property. 6 We sought to identify a single-stranded CRP that could spontaneously self-assemble into a bioactive material. Recently, two research groups obtained micrometer-scale CRP-based materials from the self-assembly of covalently attached triple-stranded entities by employing a cysteine knot. 7 We report on the design, synthesis, and characterization of novel single-stranded CRP 1a (Figure 1a), which self-assembles by noncovalent interactions into a triplehelical, micrometer-scale, composite fibrillar substance with noteworthy biological activity.The requirement of the telopeptide regions of collagen, 3 and specific Tyr and Phe residues within the C-terminal telopeptide chain, 8 suggests the importance of aromatic residues in the "code" for collagen self-assembly. 8 Inspired by nature, we sought to explore short, single-stranded CRPs that possess the main repeat-unit of collagen, Gly-Pro-Hyp, and contain aromatic recognition units at the N-and C-termini. Phenyl and pentafluorophenyl end-groups were considered, given the strong noncovalent aromatic-stacking interaction between benzene and hexafluorobenzene. 9 In this regard, 32-mer peptide 1a, with pentafluorophenylalanine (F 5 -Phe) and Phe residues at the N-and C-termini, respectively, was of interest. We speculated that interstrand aromatic-stacking and ordered hydro-

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

“…Peptides 1a-d formed stable triple helices by CD with a narrow range of melting temperatures (T m ϭ 56-62°C), which signifies similar thermodynamic stability. These T m values exceed that (T m ϭ 47°C) for a collagen-mimetic with three peptide strands covalently linked by disulfide bonds (14), but are lower than that (T m ϭ 70°C) for 31-mer Ac(GPO) 10 G (3) (22). By light microscopy, there was a clear difference between 1a and 1d in that 1a formed fibrils but 1d did not.…”

“…This approach proved to be successful, as observed in our preliminary work with 32-mer peptide 1a (Fig. 1), which yielded a bioactive, collagen-like material (22). However, it is important to gain a better understanding of the scope and limitations of such CMP self-assembly, and to develop a correlation between structure and function.…”

“…In an earlier study (22), we found that 1a forms high-order aggregates of micrometer-size by using DLS, whereas 31-mer Ac(GPO) 10 G (3), which lacks special end-groups, does not. Thus, we sought to analyze 32-mers 1a-d comparatively by DLS, but attempts to differentiate the peptides were unsuccessful, possibly related to inherent variability in this type of measurement.…”

“…We speculated that ordered hydrophobic interactions ought to encourage propagation of 32-mer building blocks, by end-to-end stacking, into lengthy strands akin to the fibrils of certain native collagens (5,6). This hypothesis was first tested theoretically via molecular mechanics calculations on 29-mer homologues of 1a-c with one less GPO (n ϭ 9; 1aЈ-cЈ) as head-to-tail triple-helical homodimers with three -stacking interactions and one salt bridge (22). We reexamined the interface of the triplex homodimer of 1aЈ [(1aЈ) 3 /(1aЈ) 3 ] and concluded that three salt bridges and three -stacking interactions could be established in the context of a ''six-point model.''…”

“…Energy-minimized (1aЈ) 3 /(1aЈ) 3 retained the six key interactions and had a total binding energy (in vacuo) of Ϫ83.5 kcal/mol, which is a 50% increase in stabilization energy relative to having just one salt bridge (22). In the final model ( Fig.…”