We previously demonstrated aza-glycine considerably improves collagen’s thermal stability and self-assembly properties without changing collagen’s natural triple helix topology. We provided a firm structural basis for this stabilization with an atomic resolution crystal structure of collagen containing aza-glycine which revealed new cross-strand H-bonds within the triple helix interior. Here, using computational analysis, we show the enhanced properties of aza-glycine is a result of non-covalent forces (H-bonding) and backbone preorganization. The interplay of steric repulsion and hyperconjugative interactions in aza-glycine’s backbone directly preorganizes the collagen peptide main-chain (φ, ψ) dihedrals for triple helical assembly. The synergy of multiple structural and electronic changes which originate at the residue level in the aza-glycine backbone and culminate at the macromolecular level of the triple helix lead to increased stability and faster refolding of collagen peptides containing aza-glycine. Recent biophysical studies have demonstrated that natural L-amino acids can be replaced with non-natural achiral aza-amino acids in folded protein structures such as triple helical collagen. However, the structural consequences of achiral aza-amino acid incorporation has not been elucidated in the context of any relevant folded biomolecule. Here, we use X-ray crystallography to provide the first atomic resolution crystal structure of an achiral aza-amino acid residue embedded within a folded protein structure, definitively illustrating that achiral aza-proline has the capacity to effectively mimic the stereochemistry of natural amino acids within the context of triple helical collagen. We show an aza-proline residue exhibits identical thermal stability to proline when substituted in the Xaa position of the (Xaa-Yaa-Gly)n collagen repeating unit but is detrimental to stability in the Yaa position. Aza-proline showcases the preorganizing power of a neighboring hydroxyproline residue on collagen stability and self-assembly. The inclusion of multiple configurationally labile aza-proline residues adversely affects collagen self-assembly due to the increased entropy present in aza-proline compared to a fixed proline stereocenter. These studies define aza-proline as a uniquely privileged amino acid to probe biomolecular systems and demonstrate the critical importance of stereochemical preorganization in biopolymer assembly and folding processes.

Impact of aza-glycine and aza-proline residues on triple helical collagen self-assembly and stability

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.