title: Dissecting the oxidative folding of circular cystine knot miniproteins.
abstract: Cyclotides are plant proteins with exceptional stability owing to the presence of a cyclic backbone and three disulfide bonds arranged in a cystine knot motif. Accordingly, they have been proposed as templates to stabilize bioactive epitopes in drug-design applications. The two main subfamilies, referred to as the Möbius and bracelet cyclotides, require dramatically different in vitro folding conditions to achieve the native fold. To determine the underlying elements that influence cyclotide folding, we examined the in vitro folding of a suite of hybrid cyclotides based on combination of the Möbius cyclotide kalata B1 and the bracelet cyclotide cycloviolacin O1. The folding pathways of the two cyclotide subfamilies were found to be different and influenced by specific residues within intercysteine loops 2 and 6. Two changes in these loops, a substitution in loop 2 and an addition in loop 6, enabled the folding of a cycloviolacin O1 analogue under conditions in which folding does not occur in vitro for the native peptide. A key intermediate contains a native-like hairpin structure that appears to be a nucleation locus early in the folding process. Overall, these mechanistic findings on the folding of cyclotides are potentially valuable for the design of new drug leads.
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, Brisbane, Australia.
authors.forename: Sunithi
authors.initials: S
authors.lastname: Gunasekera
authors.forename: Norelle L
authors.initials: NL
authors.lastname: Daly
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Clark
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
chemical_list: D003545:Cysteine