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Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
2008 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, no 1, 103-113 p.Article in journal (Refereed) Published
Abstract [en]

The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide-cycloviolacin O2-and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid-phase Fmoc peptide synthesis and cyclised by thioester-mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single-step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.

Place, publisher, year, edition, pages
2008. Vol. 9, no 1, 103-113 p.
Keyword [en]
cyclotides, native chemical ligation, peptides, protein folding, synthesis, thioesters
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-98767DOI: 10.1002/cbic.200700357ISI: 000252292200017PubMedID: 18058973OAI: oai:DiVA.org:uu-98767DiVA: diva2:201121
Available from: 2009-03-03 Created: 2009-03-03 Last updated: 2012-01-23Bibliographically approved
In thesis
1. Engineering of the Ultra-stable Cystine Knot Framework of Microproteins: Design, Chemical Synthesis and Structural Studies
Open this publication in new window or tab >>Engineering of the Ultra-stable Cystine Knot Framework of Microproteins: Design, Chemical Synthesis and Structural Studies
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ultra-stable cystine knotted microproteins, in which two disulfides and their connecting backbones form a circle that is penetrated by the third disulfide bonds, have attracted high interest due to their resistance to degradation in vitro and potential for the development of peptide drugs. This thesis gives new insights into engineering of that framework of microproteins, including approaches to their chemical synthesis, backbone engineering, structural and biological evaluations.

Synthetic and oxidative folding approaches for bracelet cyclotides, a family of cyclic cystine knotted microproteins, was developed using a model peptide, cycloviolacin O2. Following assembly of the peptide chain, protected peptide was generated by mild cleavage that was subsequently thioesterified and cyclized in solution. The cyclic peptide was oxidatively folded under optimized conditions containing co-solvent and non-ionic detergent affording native cycloviolacin O2 as a major product. To gain further insights into the heterogeneity, efficiency and kinetics of cyclotides’ oxidative folding, the intermediates that accumulate in oxidative refolding pathways of all cyclotide subfamilies: Möbius, bracelet and the hybrid cyclotides were quantitatively determined under four different folding conditions. The results were used for defining major folding pathways, which indicated that Möbius cyclotides might accumulate heterogeneous folding intermediates with one-, two- and three-disulfides, whereas bracelet tend to accumulate a homogenous intermediate with three-disulfides, depending on the buffer systems used.

Furthermore, to probe the internal factors contributing to inefficiency of oxidative folding, as well as undesired bioactivities of bracelet cyclotides (e.g., cytotoxic activity), polymer-hybridized cyclotides were designed by replacing non-conserved residues with small isosteric polymers. The designed hybrid analogs in which hybridization involved replacement of loop 3 with isosteric polymers showed improved synthetic and oxidative folding properties. The cytoxicity of a model hybrid designed with replacement of loop 3 and 5 exhibited no cytotoxic activity at concentration of 128-fold relative to that of native peptide. Furthermore, 1D and 2D 1H NMR analysis of this hybrid showed that it had well structured fold.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 77 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 139
cyclotides, cystine knot, protein engineering, NMR, solid phase, disulfide rich, ultra-stable, microprotein
National Category
Pharmaceutical Sciences
Research subject
Medicinal Chemistry
urn:nbn:se:uu:diva-145749 (URN)978-91-554-8003-5 (ISBN)
Public defence
2011-03-25, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Available from: 2011-03-04 Created: 2011-02-10 Last updated: 2011-05-04

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