Structural optimization of cyclic peptides that efficiently detect denatured collagen.

To develop a facile method for detecting denatured collagen, we investigated the structure-activity relationship of cyclic collagen-mimetic peptides (cCMPs). Reported cCMP prototypes tend to self-assemble and they must be disassembled just before use. Introducing charge repulsion and a deformation in the peptide backbone structure enabled cCMPs to detect denatured collagen without a pre-treatment for disassembly. Using the optimized cCMP, types I-V collagen were detected by western blotting and denatured collagen fibrils were visualized in a cell culture system.

Cyclic Peptides for Efficient Detection of Collagen.

We report here a new class of collagen-binding peptides, cyclic collagen-mimetic peptides (cCMPs), that efficiently hybridize with the triple-helix-forming portions of collagen. cCMPs are composed of two parallel collagen-like (Xaa-Yaa-Gly)n strands with both termini tethered by covalent linkages. Enzyme-linked immunosorbent assays and western blotting analysis showed that cCMPs exhibit more potent affinity toward collagen than reported collagen-binding peptides and can specifically detect different collagen polypeptides in a mixture of proteins. Collagen secreted from cultured cells was detected by confocal microscopy with fluorescein-labeled cCMP. The cCMP is also shown to detect sensitively folding intermediates in the endoplasmic reticulum, something that was difficult to visualize with conventional collagen detectors. Molecular-dynamics simulations suggested that a cCMP forms a more stably hybridized product than its single-chain counterpart; this could explain why cCMP has higher affinity toward denatured collagen. These results indicate the usefulness of cCMPs as tools for detecting denatured collagen.