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

Area of Science:

• Biochemistry
• Structural Biology
• Medicinal Chemistry

Background:

• Grafting bioactive peptides onto scaffolds enhances therapeutic stability.
• Disulfide-rich peptides are difficult to produce.
• The single disulfide-directed β-hairpin (SDH) fold is a potential alternative scaffold.

Purpose of the Study:

• Evaluate contryphan-Vc1 as a peptide scaffold.
• Analyze recombinant contryphan-Vc1 dynamics and stability.
• Assess the SDH fold's ability to incorporate bioactive epitopes.

Main Methods:

• Recombinant production of contryphan-Vc1 variants.
• Backbone dynamics analysis using 15N relaxation.
• Solution structure determination of truncated contryphan-Vc1.
• Proteolytic stability assays (trypsin, chymotrypsin, pepsin).
• Epitope grafting and binding affinity measurements (SPSB2).

Main Results:

• Recombinant contryphan-Vc1 shows an ordered N-terminal domain and flexible C-terminus.
• Truncated and full-length structures are similar.
• Contryphan-Vc1 is stable to trypsin/chymotrypsin but not pepsin.
• Grafted NNN epitope peptide binds SPSB2 with 1.3 μM affinity, retaining SDH fold.

Conclusions:

• The SDH fold of contryphan-Vc1 is a viable scaffold for therapeutic peptides.
• Bioactive epitopes can be incorporated while maintaining scaffold integrity.
• This work pioneers the use of SDH folds as versatile peptide scaffolds.