15N cyclotides by whole plant labeling

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Products Chemistry

Background:

• Cyclotides are cyclic peptides with unique knotted structures, conferring high stability against heat, solvents, and proteolysis.
• Their pharmaceutical and agricultural applications, including drug stabilization and insecticidal activity, are significant.
• Isotopic labeling with nitrogen-15 (15N) is essential for NMR studies (structural, dynamics, tracers) but challenging for cyclotides due to their backbone.

Purpose of the Study:

• To develop an efficient and cost-effective method for uniformly labeling cyclotides with the 15N isotope.
• To overcome limitations of conventional recombinant labeling strategies for head-to-tail cyclized peptides.
• To enable advanced NMR analyses of cyclotides for research and development.

Main Methods:

• Cultivating the cyclotide-producing plant *Oldenlandia affinis* on nitrogen-free agar media.
• Supplementing the media with 15N-labeled salts to achieve complete isotopic incorporation.
• Purifying specific insecticidal cyclotides (kalata B1, kalata B2) from the labeled plants.
• Acquiring heteronuclear single quantum coherence (HSQC) NMR spectra for characterization.

Main Results:

• Successful complete labeling of cyclotides with 15N without compromising plant biomass.
• Purification of 15N-labeled kalata B1 and kalata B2.
• Generation of HSQC NMR spectra for the purified labeled cyclotides.
• Demonstration of a significantly lower cost compared to solid-phase peptide synthesis labeling.

Conclusions:

• The developed method provides a practical and economical approach for 15N-labeling of cyclotides.
• This technique facilitates crucial NMR studies, advancing research into cyclotide structure, dynamics, and function.
• The method supports the exploration of cyclotides as scaffolds for drug delivery and in agricultural applications.