Enabling Efficient Folding and High-Resolution Crystallographic Analysis of Bracelet Cyclotides

Area of Science:

• Biochemistry
• Structural Biology
• Drug Design

Background:

• Cyclotides are cyclic peptides with a unique topology, attracting interest as drug scaffolds.
• The bracelet subfamily, the most abundant and bioactive, is underutilized due to low in vitro folding yields.
• Low folding yields hinder structural and activity characterization of bracelet cyclotides.

Purpose of the Study:

• To overcome the challenge of low in vitro folding yields in bracelet cyclotides.
• To develop a facile strategy for producing bracelet cyclotides and accessing their structures.
• To enable the biotechnological application of bracelet cyclotides.

Main Methods:

• Introducing a single point mutation (Ile-11 to Leu or Gly) to enhance folding yields.
• Synthesizing mirror-image enantiomers of bracelet cyclotides.
• Utilizing quasi-racemic crystallography for structural elucidation.

Main Results:

• Substantial increases in bracelet cyclotide folding yields were achieved via the single point mutation.
• The first crystal structures of bracelet cyclotides were elucidated using quasi-racemic crystallography.
• A facile strategy for producing and structurally characterizing bracelet cyclotides was established.

Conclusions:

• A single point mutation strategy significantly enhances bracelet cyclotide folding yields.
• This method provides facile access to atomic resolution structures of bracelet cyclotides.
• The findings lay the groundwork for broader biotechnological applications of bracelet cyclotides.