Heterochiral Knottin Protein: Folding and Solution Structure

Abstract

Homochirality is a general feature of biological macromolecules, and Nature includes few examples of heterochiral proteins. Herein, we report on the design, chemical synthesis, and structural characterization of heterochiral proteins possessing loops of amino acids of chirality opposite to that of the rest of a protein scaffold. Using the protein Ecballium elaterium trypsin inhibitor II, we discover that selective β-alanine substitution favors the efficient folding of our heterochiral constructs. Solution nuclear magnetic resonance spectroscopy of one such heterochiral protein reveals a homogeneous global fold. Additionally, steered molecular dynamics simulation indicate β-alanine reduces the free energy required to fold the protein. We also find these heterochiral proteins to be more resistant to proteolysis than homochiral l-proteins. This work informs the design of heterochiral protein architectures containing stretches of both d- and l-amino acids.

Xenoprotein engineering via synthetic libraries

“…In all cases (20 shown), a single major product containing three disulfide bonds was formed. These data suggest that in a combinatorial library of EETI-II variants, many will be present as folded cystine knots, which likely retain the disulfide connectivity and general tertiary structure of the native EETI-II molecule (37,42).…”

“…amenable to chemical synthesis (34), is known to oxidatively fold spontaneously despite sequence variation of the trypsin-binding loop (23,(35)(36)(37)(38), and has been successfully engineered to bind α v β 3 and α v β 5 integrins based on the Arg-Gly-Asp motif using yeast-surface display (39,40). The broad tolerance of the EETI-II molecule to loop expansion and sequence variation (38) is typical of cystine knot proteins generally (41), and we anticipated that this feature could be leveraged to prepare synthetic libraries of folded variants for the selection of novel binding proteins.…”

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

“…[18] More recently, Pentelute reported heterochiral variants of Ecballium elaterium trypsin inhibitor II, a disulfide-rich miniprotein that has been retargeted toward cell-surface receptors for tumor imaging applications. [19] Replacement of a large exposed loop in the prototype with a D-α-peptide segment led to mimics of the natural protein with similar folds but improved stability to proteolytic degradation.…”

“…Bulaj showed that the incorporation of ethylene glycol and methylene spacers in place of a Gly‐Gly motif in a loop of the μ‐conotoxin SIIIA led to hybrids exhibiting bioactivities comparable to that of the native toxin . More recently, Pentelute reported heterochiral variants of Ecballium elaterium trypsin inhibitor II, a disulfide‐rich miniprotein that has been retargeted toward cell‐surface receptors for application in tumor imaging . Replacement of a large exposed loop in the prototype with a d ‐α‐peptide segment led to mimics of the natural protein with similar folds but improved stability to proteolytic degradation.…”

Proteomimetics as protein-inspired scaffolds with defined tertiary folding patterns

“…Protein domains in which the functionally relevant segment is a surface-exposed loop can also be subject to prosthetic modification, as illustrated by the introduction of a flexible spacer in an ion-channelinhibitory conotoxin 66 and the grafting of an engineered receptorbinding peptide loop onto a disulfide-rich d-α-residue (17)-based knottin scaffold (Fig. 3d) 67 . In both cases, the modification improved stability to degradation by proteolytic enzymes.…”