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J. Biol. Chem., Vol. 281, Issue 12, 8224-8232, March 24, 2006

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Knots in Rings

THE CIRCULAR KNOTTED PROTEIN MOMORDICA COCHINCHINENSIS TRYPSIN INHIBITOR-II FOLDS VIA A STABLE TWO-DISULFIDE INTERMEDIATE^*

Maa emaar¹, Norelle L. Daly², Sara Häggblad, Kai Pong Lo, Ernie Yulyaningsih, and David J. Craik³

From the Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane 4072 QLD, Australia

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys¹-Cys¹⁸ disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.

Received for publication, December 16, 2005 , and in revised form, January 18, 2006.

^* The studies described herein have been supported by a grant from the Australian Research Council. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data.

¹ Australian Research Council postdoctoral fellow.

² A National Health and Medical Research Council industry fellow.

³ Australian Research Council professorial fellow. To whom correspondence should be addressed: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia. Tel.: 61-7-3346-2019; Fax: 61-7-3346-2029; E-mail: d.craik{at}imb.uq.edu.au.

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