The Single Disulfide-Directed β-Hairpin Fold. Dynamics, Stability, and Engineering

Abstract

Grafting bioactive peptide sequences onto small cysteine-rich scaffolds is a promising strategy for enhancing their stability and value as novel peptide-based therapeutics. However, correctly folded disulfide-rich peptides can be challenging to produce by either recombinant or synthetic means. The single disulfide-directed β-hairpin (SDH) fold, first observed in contryphan-Vc1, provides a potential alternative to complex disulfide-rich scaffolds. We have undertaken recombinant production of full-length contryphan-Vc1 (rCon-Vc1[Z1Q]) and a truncated analogue (rCon-Vc1[Z1Q]), analyzed the backbone dynamics of rCon-Vc1[Z1Q], and probed the conformational and proteolytic stability of these peptides to evaluate the potential of contryphan-Vc1 as a molecular scaffold. Backbone N relaxation measurements for rCon-Vc1[Z1Q] indicate that the N-terminal domain of the peptide is ordered up to Thr19, whereas the remainder of the C-terminal region is highly flexible. The solution structure of truncated rCon-Vc1[Z1Q] was similar to that of the full-length peptide, indicating that the flexible C-terminus does not have any effect on the structured domain of the peptide. Contryphan-Vc1 exhibited excellent proteolytic stability against trypsin and chymotrypsin but was susceptible to pepsin digestion. We have investigated whether contryphan-Vc1 can accept a bioactive epitope while maintaining the structure of the peptide by introducing peptide sequences based on the DINNN motif of inducible nitric oxide synthase. We show that sCon-Vc1[NNN] binds to the iNOS-binding protein SPSB2 with an affinity of 1.3 μM while maintaining the SDH fold. This study serves as a starting point in utilizing the SDH fold as a peptide scaffold.

Characterisation of Elevenin-Vc1 from the Venom of Conus victoriae: A Structural Analogue of α-Conotoxins

“…Most conotoxins have multiple disulfide bonds, although there are some that lack disulfide bonds [ 2 , 43 , 44 , 45 , 46 ]. Those peptides with multiple disulfide bonds are relatively stable compared to those lacking them [ 44 ].…”

Modulation of Lymphocyte Potassium Channel KV1.3 by Membrane-Penetrating, Joint-Targeting Immunomodulatory Plant Defensin

“…Globular peptides are known to be stable to pepsin at low pH, hence contryphan-Vc1 was used as a control substrate for pepsin. 88 Reactions were conducted in triplicate (n = 3). Peptide stability was also determined in rat and human plasma as described in the Supporting Information.…”

“…Peptide digestions were quantified by LC–MS (Shimadzu, Kyoto, Japan). Globular peptides are known to be stable to pepsin at low pH, hence contryphan-Vc1 was used as a control substrate for pepsin . Reactions were conducted in triplicate ( n = 3).…”