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

Balasubramanyam Chittoor, Bankala Krishnarjuna, Rodrigo A.V. Morales, Christopher A. Macraild, Maiada M. Sadek, Eleanor W.W. Leung, Samuel D. Robinson, Michael William Pennington, Raymond S. Norton

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2 Citations (Scopus)

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-Vc11-22[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 15N 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-Vc11-22[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-Vc11-22[NNN12-14] 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.

Original languageEnglish
Pages (from-to)2455-2466
Number of pages12
JournalBiochemistry
Volume56
Issue number19
DOIs
Publication statusPublished - 16 May 2017

Cite this

Chittoor, B., Krishnarjuna, B., Morales, R. A. V., Macraild, C. A., Sadek, M. M., Leung, E. W. W., Robinson, S. D., Pennington, M. W., & Norton, R. S. (2017). The Single Disulfide-Directed β-Hairpin Fold. Dynamics, Stability, and Engineering. Biochemistry, 56(19), 2455-2466. https://doi.org/10.1021/acs.biochem.7b00120