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

• Published in: Biochemistry (Easton) 2017-05, Vol.56 (19), p.2455-2466
• Main Authors: Chittoor, Balasubramanyam, Krishnarjuna, Bankala, Morales, Rodrigo A. V, MacRaild, Christopher A, Sadek, Maiada, Leung, Eleanor W. W, Robinson, Samuel D, Pennington, Michael W, Norton, Raymond S
• Format: Article
• Language: English
• Subjects: Conotoxins - chemistry; Conotoxins - genetics; Conotoxins - metabolism; Cysteine - chemistry; Cystine - chemistry; Epitopes; Humans; Kinetics; Nitrogen Isotopes; Oxidation-Reduction; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Peptides, Cyclic - chemistry; Peptides, Cyclic - genetics; Peptides, Cyclic - metabolism; Protein Conformation, beta-Strand; Protein Engineering; Protein Folding; Protein Stability; Proteolysis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Solubility; Suppressor of Cytokine Signaling Proteins - chemistry; Suppressor of Cytokine Signaling Proteins - genetics; Suppressor of Cytokine Signaling Proteins - metabolism; Surface Plasmon Resonance
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.7b00120

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.