title: Cyclotide isolation and characterization.
abstract: Cyclotides are disulfide-rich cyclic peptides produced by plants with the presumed natural function of defense agents against insect pests. They are present in a wide range of plant tissues, being ribosomally synthesized via precursor proteins that are posttranslationally processed to produce mature peptides with a characteristic cyclic backbone and cystine knot motif associated with their six conserved cysteine residues. Their processing is not fully understood but involves asparaginyl endoproteinase activity. In addition to interest in their defense roles and their unique topologies, cyclotides have attracted attention as potential templates in peptide-based drug design applications. This chapter provides protocols for the isolation of cyclotides from plants, their detection and sequencing by mass spectrometry, and their structural analysis by NMR, as well as describing methods for the isolation of nucleic acid sequences that encode their precursor proteins. Assays to assess their membrane-binding interactions are also described. These protocols provide a "starter kit" for researchers entering the cyclotide field.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. d.craik@imb.uq.edu.au
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.forename: Sonia Troeira
authors.initials: ST
authors.lastname: Henriques
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Mylne
authors.forename: Conan K
authors.initials: CK
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D047169:Cyclotides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010944:Plants / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053835:Unilamellar Liposomes / Q000378:metabolism
chemical_list: D047169:Cyclotides; D010940:Plant Proteins; D011498:Protein Precursors; D018749:RNA, Plant; D053835:Unilamellar Liposomes; D003545:Cysteine