Ireland, David C.Colgrave, Michelle L.Nguyencong, PhilipDaly, Norelle L.Craik, David J.2026-06-112026-06-110022-2836PubMed:16488428ORCID:/0009-0005-7721-4140/work/217149686https://hdl.handle.net/1885/733810527Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.D.C.I. is supported by a University of Queensland Graduate School Scholarship. This work was supported by a grant from the Australian Research Council (ARC). D.J.C. is an ARC Professorial Fellow. We thank Dr. Jason Mulvenna for assistance with primer design and Dr Josh Mylne for comments on the manuscript.14enCircular peptidesCyclic cystine knotCyclotidesViola odorataViolacin A2006-04-1410.1016/j.jmb.2006.01.05133645047649