Abstract
The Collagen Toolkits are libraries of 56 and 57 triple-helical synthetic peptides spanning the length of the collagen II and collagen III helices. These have been used in solid-phase binding assays to locate sites where collagen receptors and extracellular matrix components bind to collagens. Truncation and substitution allowed exact binding sites to be identified, and corresponding minimal peptides to be synthesised for use in structural and functional studies. 170 sites where over 30 proteins bind to collagen II have been mapped, providing firm conclusions about the amino acid distribution within such binding sites. Protein binding to collagen II is not random, but displays a periodicity of approximately 28 nm, with several prominent nodes where multiple proteins bind. Notably, the vicinity of the collagenase-cleavage site in Toolkit peptide II-44 is highly promiscuous, binding over 20 different proteins. This may reflect either the diverse chemistry of that locus or its diverse function, together with the interplay between regulatory binding partners. Peptides derived from Toolkit studies have been used to determine atomic level resolution of interactions between collagen and several of its binding partners and are finding practical application in tissue engineering.
Highlights
4430 words (Summary to Acknowledgments)The human collagens are a family of 28 triple-helical proteins that form, collectively, the most abundant protein group in vertebrates
Synthetic peptides derived from the Toolkits underpin structural studies and provide ligands to manipulate cell and protein function, and will in future find application in tissue engineering and regenerative medicine
We have argued previously that concealment of crucial binding sites, for VWF and for integrin a2b1 located in D1 to D3, for example, would render them unable to perform their essential roles in haemostasis[50]
Summary
The human collagens are a family of 28 triple-helical proteins that form, collectively, the most abundant protein group in vertebrates. Collagens contribute to the cellular niche, a nidus which offers the cell both anchorage and survival, and within which connective tissue cells fulfil their normal functions Such regulatory roles of collagen require engagement of diverse cell surface receptors[3], and the evolution of complex signatures within the primary Gxx’ sequence of the collagen a-chains in concert with the maintenance of triple helix stability. The resulting defined linear fragments (CB peptides) would be separated chromatographically, if sufficiently long to be thermally stable, reassembled as triple helices, and their capacity to bind target proteins established. This reductive approach allowed protein binding to be mapped, but those sites containing M (e.g. some integrin sites and the von Willebrand factor (VWF) locus – see below) were disrupted and so not identifiable. Rotary shadowing yielded relatively low-resolution data, but, unlike CB peptide mapping, could be applied to intact heterotrimeric species such as collagen I
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