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J. Biol. Chem., Vol. 278, Issue 48, 48188-48196, November 28, 2003

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Disulfide Mapping of the Cyclotide Kalata B1

CHEMICAL PROOF OF THE CYCLIC CYSTINE KNOT MOTIF^*

Ulf Göransson¶ and David J. Craik||

From the Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia and Department of Medicinal Chemistry, Division of Pharmacognosy, Biomedical Centre, Uppsala University, Box 574, Uppsala S-751 23, Sweden

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.

Received for publication, August 8, 2003 , and in revised form, September 5, 2003.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a fellowship from the IF Foundation, Swedish Academy of Pharmaceutical Sciences.

^|| Australian Research Council Senior Fellow. To whom correspondence should be addressed: Inst. for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia. Tel.: 61-7-3346-2019; Fax: 61-7-3346-2029; E-mail: d.craik{at}imb.uq.edu.au.

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