Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides

Daly, N. L., Clark, R. J. and Craik, D. J. (2003) Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides. The Journal of Biological Chemistry, 278 8: 6314-6322. doi:10.1074/jbc.M210492200

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Author Daly, N. L.
Clark, R. J.
Craik, D. J.
Title Disulfide folding pathways of cystine knot proteins: Tying the knot within the circular backbone of the cyclotides
Journal name The Journal of Biological Chemistry   Check publisher's open access policy
ISSN 0021-9258
Publication date 2003-02-21
Sub-type Article (original research)
DOI 10.1074/jbc.M210492200
Open Access Status File (Publisher version)
Volume 278
Issue 8
Start page 6314
End page 6322
Total pages 9
Place of publication Bethesda
Publisher The American Society for Biochemistry and Molecular Biology
Collection year 2003
Language eng
Subject C1
250302 Biological and Medical Chemistry
780105 Biological sciences
Abstract The plant cyclotides are a fascinating family of circular proteins that contain a cyclic cystine knot motif. The knotted topology and cyclic nature of the cyclotides pose interesting questions about folding mechanisms and how the knotted arrangement of disulfide bonds is formed. In the current study we have examined the oxidative refolding and reductive unfolding of the prototypic cyclotide, kalata B1. A stable two-disulfide intermediate accumulated during oxidative refolding but not in reductive unfolding. Mass spectrometry and NMR spectroscopy were used to show that the intermediate contained a native-like structure with two native disulfide bonds topologically similar to the intermediate isolated for the related cystine knot protein EETI-II (LeNguyen, D., Heitz, A., Chiche, L., El Hajji, M., and Castro B. (1993) Protein Sci. 2, 165-174). However, the folding intermediate observed for kalata B1 is not the immediate precursor of the three-disulfide native peptide and does not accumulate in the reductive unfolding process, in contrast to the intermediate observed for EETI-II. These alternative pathways of linear and cyclic cystine knot proteins appear to be related to the constraints imposed by the cyclic backbone of kalata B1 and the different ring size of the cystine knot. The three-dimensional structure of a synthetic version of the two-disulfide intermediate of kalata B1 in which Ala residues replace the reduced Cys residues provides a structural insight into why the two-disulfide intermediate is a kinetic trap on the folding pathway.
Keyword Nmr Structure Calculation
Polypeptide Kalata B1
Inhibitory Macrocyclic Peptides
Spin Coupling-constants
Torsion Angle Dynamics
Trypsin-inhibitor
Plant Cyclotides
Momordica-cochinchinensis
Beta-sheet
Spectroscopy
Q-Index Code C1

 
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Created: Wed, 15 Aug 2007, 12:43:27 EST