INVESTIGATION OF HETEROTRIMER-FORMING COLLAGEN-MIMETIC PEPTIDES THAT INCORPORATE AZA-GLYCINE RESIDUES
Collagen plays a critical role in both the structure and the function of the extracellular matrix, and its structure and expression patterns are dysregulated in a multitude of diseases. The canonical structural motif of collagen is a triple helix that is comprised either of three identical peptide sequences (homotrimeric collagen) or non-identical sequences (A2B and ABC heterotrimeric collagen, respectively). These peptide strands, in turn, are composed of a repetitive tripeptide unit with the sequence Gly-Xaa-Yaa, most commonly glycine-proline-hydroxyproline (Gly-Pro-Hyp). The challenges associated with the study of naturally-derived collagen proteins have prompted the study of collagen-mimetic peptides (CMPs), short peptides used to simulate the structure and biology of collagen. The synthesis of heterotrimeric CMPs remains a significant challenge in the field, because a high level of control over trimer assembly is required to promote the formation of the desired heterotrimer species. One major barrier to the formation of heterotrimeric collagen stems from the fact that most CMPs do not have a strong driving force to selectively heterotrimerize, leading to the formation of high amounts of homotrimer in addition to the target heterotrimer. This work explores a sterics-based approach to the formation of heterotrimeric CMPs. We designed a series of peptide strands that incorporate two substitutions: i) a bulky imino acid at the N- or C-terminal ‘Gly’ position of the Gly-Pro-Hyp repeat, a highly destabilizing replacement, and ii) internal glycine-to-aza-glycine substitutions, a highly stabilizing replacement. We use circular dichroism, ESI-MS and SEC-MALS studies to develop CMP strands that possess a markedly reduced homotrimer stability, and to gain insight into the molecular species formed in these mixtures. We also present the application of fluorophore-tagged CMPs incorporating the aforementioned structural modifications as fluorescent probes for collagen-rich tissue samples, and measured these probes’ abilies to label collagen in tissue-binding experiments using confocal microscopy. Taken together, this work presents the ‘Gly-block’ as a novel, viable approach to the selective formation of heterotrimeric CMPs, and lays the foundation for the implementation of these sterically-encumbered CMPs as probes of naturally-occurring collagen structures.
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