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Disulfide mapping of the cyclotide kalata B1: Chemical proof of the cystic cystine knot motif
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 48, 48188-48196 p.Article in journal (Refereed) Published
Abstract [en]

The cyclotides are a recently discovered family of plant proteins that have the fascinating structural feature of a continuous cyclic backbone and, putatively, a knotted arrangement of their three conserved disulfide bonds. We here show definite chemical proof of the I-IV, II-V, III-VI knotted disulfide connectivity of the prototypic cyclotide kalata B1. This has been achieved by a new approach for disulfide analysis, involving partial reduction and stepwise alkylation including introduction of charges and enzymatic cleavage sites by aminoethylation of cysteines. The approach overcomes the intrinsic difficulties for disulfide mapping of cyclotides, i.e. the cyclic amide backbone, lack of cleavage sites between cysteines, and a low or clustered content of basic amino acids, and allowed a direct determination of the disulfide bonds in kalata B1 using analysis by mass spectrometry. The established disulfide connectivity is unequivocally shown to be cystine knotted by a topological analysis. This is the first direct chemical determination of disulfides in native cyclotides and unambiguously confirms the unique cyclic cystine knot motif.

Place, publisher, year, edition, pages
2003. Vol. 278, no 48, 48188-48196 p.
Keyword [en]
Amino Acid Motifs, Amino Acid Sequence, Chromatography; High Pressure Liquid, Conserved Sequence, Cysteine/chemistry, Disulfides, Ethylmaleimide/chemistry, Hydrogen-Ion Concentration, Ions, Molecular Sequence Data, Oldenlandia/metabolism, Peptides; Cyclic/*chemistry, Protein Folding, Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization, Spectrum Analysis; Mass, Support; Non-U.S. Gov't, Trypsin/pharmacology
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-66816DOI: 10.1074/jbc.M308771200PubMedID: 12960160OAI: oai:DiVA.org:uu-66816DiVA: diva2:94727
Available from: 2004-09-15 Created: 2004-09-15 Last updated: 2012-01-24Bibliographically approved

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