Stabilization of a miniprotein fold by an unpuckered proline surrogate

Journal article

The unique role of proline in modulating protein folding and recognition makes it an attractive target for substitution to generate new proteomimetics. The design, synthesis, and conformational analysis of non-canonical surrogates can also aid in parsing the role of prolyl stereoelectronic effects on structure. We recently described the synthesis and conformational analysis of dehydro-δ-azaproline (ΔaPro), a novel unsaturated analogue of proline featuring a planar dehydropyrazine ring. When incorporated into host sequences, this backbone N-aminated proline surrogate forms an acylhydrazone bond with an unusually high trans rotamer bias and low isomerization barrier. Here, we used CD, NMR spectroscopy, and MD simulations to evaluate the impact of ΔaPro substitution within the polyproline II (PPII) and loop regions of the avian pancreatic polypeptide (aPP). The ΔaPro residue strongly favors PPII conformation and stabilizes the aPP tertiary fold when incorporated at select positions within the miniprotein. A variant featuring three ΔaPro substitutions was found to significantly enhance the thermal stability of wild-type aPP despite compromising protein dimerization. Our results suggest that the stability of proline-rich folds relies more on backbone torsional preferences than ring puckering and informs strategies for the incorporation of ΔaPro into thermally stable and functional proteomimetics.

Authors: Wright, MM; Rajewski, BH; Gerrein, TA; Xu, Z; Smith, LJ

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Springer Nature
• Journal: Communications Chemistry
• Volume: 8
• Issue: 1
• Article number: 76
• Publication date: 2025-03-12
• Acceptance date: 2025-02-27
• DOI: 10.1038/s42004-025-01474-6
• EISSN: 2399-3669
• Pmid: 40075167
• Language: English