Diversity in the disulfide folding pathways of cystine knot peptides

Letters in Peptide Science 01/2003; 10(5):523-531. DOI: 10.1007/BF02442584

ABSTRACT The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif (CCK). This unique
family was discovered only recently but contains over 50 known sequences to date. Various biological activities are associated
with these peptides including antimicrobial and insecticidal activity. The knotted topology and cyclic nature of the cyclotides
poses interesting questions about the folding mechanisms and how the knotted arrangement of disulfide bonds is formed. Some
studies have been performed on related inhibitor cystine knot (ICK) containing peptides, but little is known about the folding
mechanisms of CCK molecules. We have examined the oxidative refolding and reductive unfolding of the prototypic member of
the cyclotide family, kalata B1. Analysis of the rates of formation of the intermediates along the reductive unfolding pathway
highlights the stability conferred by the cystine knot motif. Significant differences are observed between the folding of
kalata B1 and an acyclic cystine knot protein, EETI-II, suggesting that the circular backbone has a significant influence
in directing the folding pathway.

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    ABSTRACT: Cyclotides are a recently discovered family of mini-proteins that have a head-to-tail cyclised backbone stabilized by a knotted arrangement of three disulfide bonds. They have a wide range of biological activities, including uterotonic, anti-bacterial, anti-HIV, and anti-tumour activity but their insecticidal activities suggest that their natural function is in plant defense. They are exceptionally resistant to chemical, enzymatic and thermal treatments because of their unique structural scaffold. This stability and resistance to proteolysis makes them a potentially valuable protein engineering tool at the interface of chemistry and biology: they have the structure of proteins but the stability and biophysical properties of organic molecules. In this review the role of NMR in defining the structures of cyclotides is described.
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    ABSTRACT: Cyclic cystine knot proteins are small but topologically complex molecules that occur naturally in plants and have a wide range of bioactivities that make them interesting from a pharmaceutical perspective. Their remarkable stability is dependent on the correct formation of a knotted arrangement of disulfide bonds. This review reports on studies that have deciphered the pathways to the "tying of the knot." These studies have involved a range of biophysical techniques and suggest that the major intermediate species presented on these pathways are two disulfide native species, which are not necessarily the precursors of the native protein. Structural elucidations of one analogue and one such intermediate have been reported, and they both show highly native-like conformation and native disulfide bond connectivity. Cyclic cystine knot formation has also been shown to be assisted by protein disulfide isomerase. The points summarized in this review will be important to consider in the design of novel pharmaceutically interesting biomolecules based on the cyclic cystine knot motif, which has shown potential as a molecular scaffold because of its exceptional stability.
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    ABSTRACT: The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.
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