Disulfide mapping of the cyclotide kalata B1 - Chemical proof of the cyclic cystine knot motif

Goransson, Ulf and Craik, David J. (2003) Disulfide mapping of the cyclotide kalata B1 - Chemical proof of the cyclic cystine knot motif. Journal of Biological Chemistry, 278 48: 48188-48196. doi:10.1074/jbc.M308771200

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Author Goransson, Ulf
Craik, David J.
Title Disulfide mapping of the cyclotide kalata B1 - Chemical proof of the cyclic cystine knot motif
Journal name Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
Publication date 2003
Sub-type Article (original research)
DOI 10.1074/jbc.M308771200
Open Access Status File (Publisher version)
Volume 278
Issue 48
Start page 48188
End page 48196
Total pages 9
Editor Herbet Tabor
Place of publication Bethesda
Publisher The American Society for Biochemistry and Molecular Biology, Inc.
Language eng
Subject C1
250302 Biological and Medical Chemistry
780105 Biological sciences
Abstract 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.
Keyword Biochemistry & Molecular Biology
Inhibitory Macrocyclic Peptides
Plant Cyclotides
Structural Motif
3-dimensional Structure
Selective Reduction
Drug Design
Q-Index Code C1

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Created: Wed, 19 Sep 2007, 18:28:45 EST