The Single Disulfide-Directed β-Hairpin Fold: Role of Disulfide Bond in Folding and Effect of an Additional Disulfide Bond on Stability

Balasubramanyam Chittoor; Bankala Krishnarjuna; Rodrigo A. V. Morales; Raymond S. Norton

doi:10.1071/CH19386

RESEARCH ARTICLE

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The Single Disulfide-Directed β-Hairpin Fold: Role of Disulfide Bond in Folding and Effect of an Additional Disulfide Bond on Stability

Balasubramanyam Chittoor ^A , Bankala Krishnarjuna ^A ^B , Rodrigo A. V. Morales ^A ^C and Raymond S. Norton ^A ^D ^E

+ Author Affiliations

- Author Affiliations

^A Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic. 3052, Australia.

^B Current address: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.

^C Current address: CSL Limited (Bio21), 30 Flemington Road, Parkville, Vic. 3010, Australia.

^D ARC Centre for Fragment-Based Design, Monash University, Parkville, Vic. 3052, Australia.

^E Corresponding author. Email: ray.norton@monash.edu

Australian Journal of Chemistry 73(4) 312-320 https://doi.org/10.1071/CH19386
Submitted: 8 August 2019 Accepted: 27 September 2019 Published: 15 November 2019

Abstract

Disulfide bonds play a key role in the oxidative folding, conformational stability, and functional activity of many peptides. A few disulfide-rich peptides with privileged architecture such as the inhibitor cystine knot motif have garnered attention as templates in drug design. The single disulfide-directed β-hairpin (SDH), a novel fold identified more recently in contryphan-Vc1, has been shown to possess remarkable thermal, conformational, and chemical stability and can accept a short bioactive epitope without compromising the core structure of the peptide. In this study, we demonstrated that the single disulfide bond is critical in maintaining the native fold by replacing both cysteine residues with serine. We also designed an analogue with an additional, non-native disulfide bridge by replacing Gln1 and Tyr9 with Cys. Contryphan-Vc1_1–22[Q1C, Y9C] was synthesised utilising orthogonal cysteine protection and its solution structure determined using solution NMR spectroscopy. This analogue maintained the overall fold of native contryphan-Vc1. Previous studies had shown that the β-hairpin core of contryphan-Vc1 was resistant to proteolysis by trypsin and α-chymotrypsin but susceptible to cleavage by pepsin. Contryphan-Vc1_1–22[Q1C, Y9C] proved to be completely resistant to pepsin, thus confirming our design strategy. These results highlight the role of the disulfide bond in maintaining the SDH fold and provide a basis for the design of more stable analogues for peptide epitope grafting.

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The Single Disulfide-Directed β-Hairpin Fold: Role of Disulfide Bond in Folding and Effect of an Additional Disulfide Bond on Stability

Abstract

References

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