Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata 132, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.