Cis-trans isomerization of peptoid residues in the collagen triple-helix.

Cis-peptide bonds are rare in proteins, and building blocks less favorable to the trans-conformer have been considered destabilizing. Although proline tolerates the cis-conformer modestly among all amino acids, for collagen, the most prevalent proline-abundant protein, all peptide bonds must be trans to form its hallmark triple-helix structure. Here, using host-guest collagen mimetic peptides (CMPs), we discover that surprisingly, even the cis-enforcing peptoid residues (N-substituted glycines) form stable triple-helices. Our interrogations establish that these peptoid residues entropically stabilize the triple-helix by pre-organizing individual peptides into a polyproline-II helix. Moreover, noting that the cis-demanding peptoid residues drastically reduce the folding rate, we design a CMP whose triple-helix formation can be controlled by peptoid cis-trans isomerization, enabling direct targeting of fibrotic remodeling in myocardial infarction in vivo. These findings elucidate the principles of peptoid cis-trans isomerization in protein folding and showcase the exploitation of cis-amide-favoring residues in building programmable and functional peptidomimetics.

Terminal repeats impact collagen triple-helix stability through hydrogen bonding.

Nearly 30% of human proteins have tandem repeating sequences. Structural understanding of the terminal repeats is well-established for many repeat proteins with the common α-helix and ß-sheet foldings. By contrast, the sequence-structure interplay of the terminal repeats of the collagen triple-helix remains to be fully explored. As the most abundant human repeat protein and the most prevalent structural component of the extracellular matrix, collagen features a hallmark triple-helix formed by three supercoiled polypeptide chains of long repeating sequences of the Gly-X-Y triplets. Here, with CD characterization of 28 collagen-mimetic peptides (CMPs) featuring various terminal motifs, as well as DSC measurements, crystal structure analysis, and computational simulations, we show that CMPs only differing in terminal repeat may have distinct end structures and stabilities. We reveal that the cross-chain hydrogen bonding mediated by the terminal repeat is key to maintaining the triple-helix's end structure, and that disruption of it with a single amide to carboxylate substitution can lead to destabilization as drastic as 19 °C. We further demonstrate that the terminal repeat also impacts how strong the CMP strands form hybrid triple-helices with unfolded natural collagen chains in tissue. Our findings provide a spatial profile of hydrogen bonding within the CMP triple-helix, marking a critical guideline for future crystallographic or NMR studies of collagen, and algorithms for predicting triple-helix stability, as well as peptide-based collagen assemblies and materials. This study will also inspire new understanding of the sequence-structure relationship of many other complex structural proteins with repeating sequences.