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Abstract 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.
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Combining simulations and experiments for the molecular engineering of multifunctional collagen mimetic peptide-based materials
Assembling peptides allow the creation of structurally complex materials, where amino acid selection influences resulting properties. We present a synergistic approach of experiments and simulations for examining the influence of natural and non-natural amino acid substitutions via incorporation of charged residues and a reactive handle on the thermal stability and assembly of multifunctional collagen mimetic peptides (CMPs). Experimentally, we observed inclusion of charged residues significantly decreased the melting temperature of CMP triple helices with further destabilization upon inclusion of the reactive handle. Atomistic simulations of a single CMP triple helix in explicit water showed increased residue-level and helical structural fluctuations caused by the inclusion of the reactive handle; however, these atomistic simulations cannot be used to predict changes in CMP melting transition. Coarse-grained (CG) simulations of CMPs at experimentally relevant solution conditions, showed, qualitatively, the same trends as experiments in CMP melting transition temperature with CMP design. These simulations show that when charged residues are included electrostatic repulsions significantly destabilize the CMP triple helix and that an additional inclusion of a reactive handle does not significantly change the melting transition. Based on findings from both experiments and simulations, the sequence design was refined for increased CMP triple helix thermal stability, and the reactive handle was utilized for the incorporation of the assembled CMPs within covalently crosslinked hydrogels. Overall, a unique approach was established for predicting stability of CMP triple helices for various sequences prior to synthesis, providing molecular insights for sequence design towards the creation of bulk nanostructured soft biomaterials.
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- Award ID(s):
- 1703402
- PAR ID:
- 10275721
- Date Published:
- Journal Name:
- Soft Matter
- Volume:
- 17
- Issue:
- 7
- ISSN:
- 1744-683X
- Page Range / eLocation ID:
- 1985 to 1998
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Collagen, the major structural protein in connective tissue, adopts a right‐handed triple helix composed of peptide chains featuring repeating Gly‐Xaa‐Yaa tripeptide motifs. While the cyclic residues proline (Pro) and hydroxyproline (Hyp) are prevalent in the Xaa and Yaa positions due to their PPII‐favoring conformational properties, diverse acyclic peptoid (N‐alkylated Gly) residues can also stabilize the collagen fold. Here, we investigated the effects of N‐aminoglycine (aGly) derivatives—so‐called “azapeptoid” residues—on the thermal stability of collagen mimetic peptides (CMPs). Substitution of Pro at the central Xaa11 position with aGly resulted in destabilization of the triple helix, yet the introduction of select N′‐alkyl groups (isopropyl, butyl) partially restored thermal stability. Moreover, the N‐amino group of azapeptoid residues enhanced thermal CMP stability relative to an unsubstituted Gly analog. Kinetic studies revealed that the introduction of the hydrazide bonds in aGly and (iPr)aGly CMPs did not significantly impact triple helix refolding rates. Their modular late‐stage derivatization and tunable properties highlight azapeptoid residues as potentially valuable tools for engineering CMPs and probing the structural determinants of collagen folding.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0SM01562H
