15N Cyclotides by Whole Plant Labeling

Cyclotides are 28-37 amino acid peptides incorporating three disulfide bonds and a cyclic backbone. Their cyclic and knotted topology renders them immune to denaturation by heat or organic solvents and highly resistant to proteolysis. They have a range of interesting and potentially useful pharmaceutical properties and have been proposed as scaffolds within which peptides with drug activities can be stabilized for delivery. Some members of the family also have agricultural applications deriving from their potent insecticidal activity. Labeling peptides with the NMR-active and stable 15N isotope facilitates a range of studies by NMR, including structural and dynamics studies and their use as tracers. However, owing to their head-to-tail cyclized peptide backbone labeled cyclotides are not amenable to conventional recombinant labeling strategies. We have developed an approach to overcome this limitation by growing the cyclotide-bearing plant Oldenlandia affinis on nitrogen-free agar media supplemented with 15N salts and obtaining complete labeling at no detriment to plant biomass. We purified the insecticidal cyclotides kalata B1 and kalata B2 as examples and provide heteronuclear single quantum coherence (HSQC) NMR spectra for each. This method of labeling cyclotides involves only a fraction of the cost of uniform labeling by solid-phase peptide synthesis.