Heterogeneous‐Backbone Foldamer Mimics of a Computationally Designed, Disulfide‐Rich Miniprotein

Abstract

Disulfide-rich peptides have found widespread use in the development of bioactive agents; however, low proteolytic stability and difficulty exerting synthetic control over chain topology present barriers to applications in some systems. Here, we report a method that enables creation of artificial backbone (“foldamer”) mimics of compact, disulfide-rich tertiary folds. Systematic replacement of a subset of natural α-residues with various artificial building blocks in the context of a computationally designed prototype sequence leads to “heterogeneous-backbone” variants that undergo clean oxidative folding, adopt tertiary structures indistinguishable from the prototype, and enjoy proteolytic protection beyond that inherent to the topologically constrained scaffold. Collectively, these results demonstrate systematic backbone substitution as a viable method to engineer the properties of disulfide-rich sequences and expands the repertoire of protein mimicry by foldamers to an exciting new structural class.

Proteomimetic zinc finger domains with modified metal‐binding β‐turns

“…The Aib‐Gly motif in 2 also favored a type‐II turn, while the d ‐Pro‐Gly and BTD turn replacements in 3 and 4 adopted mirror image type‐II′ turns. The turn type preferences for the artificial turn mimetics seen in Sp1‐2 are in accord with prior observations in non‐metal‐binding proteins, 7,10,18,19,25 suggesting the sequence context is not influencing their behavior.…”

“…The result of such modification, when made judiciously, is a heterogeneous backbone in which artificial building blocks are interspersed among natural α‐peptide, yet the resulting chain is able to reproduce the sophisticated fold of a given prototype sequence. Such heterogeneous‐backbone protein mimics have been used to probe assembly behavior in amyloidogenic sequences, 5 reproduce diverse tertiary folding patterns, 6–11 and create agents capable of native‐like molecular recognition 6,8,11 . As various classes of artificial monomers have different conformational propensities, creating heterogenous‐backbone mimics of complex and diverse folds found in nature relies on the collective application of many different types of building blocks.…”

“…Backbone modifications could be introduced throughout the helix and strands in Sp1‐3 without compromising the folded structure or stability; however, supplementing these with any change in composition at the turn near the zinc binding site resulted in a variant unable to fold or bind metal 9 . This result was puzzling, as the artificial moieties attempted for modification of the turn ( d ‐Pro, aminoisobutyric acid [Aib], side‐chain linked ornithine [δ‐Orn]) were all well‐precedented as turn mimetics in short peptides 13–17 as well as larger proteins 7,10,11,18,19 …”

Proteomimetics as protein-inspired scaffolds with defined tertiary folding patterns

“…f, Crystal structure of a helix-turn-helix scaffold modified in the helices (PDB 4WPB) 72 . g, NMr structure of a de novo disulfide-rich scaffold harbouring modifications throughout the helix, loop, turn and sheet (PDB 6E5J) 74 .…”

“…De novo-designed tertiary structures have also served as inspiration for the design of proteomimetics, as shown by the backbone modification of a computationally designed disulfiderich scaffold to yield mimetics with identical folds and increased proteolytic stability (Fig. 3g) 74 .…”