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Engineering of the Ultra-stable Cystine Knot Framework of Microproteins: Design, Chemical Synthesis and Structural Studies
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 139
Keyword [en]
cyclotides, cystine knot, protein engineering, NMR, solid phase, disulfide rich, ultra-stable, microprotein
National Category
Pharmaceutical Sciences
Research subject
Medicinal Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-145749ISBN: 978-91-554-8003-5OAI: oai:DiVA.org:uu-145749DiVA: diva2:396748
Public defence
2011-03-25, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-03-04 Created: 2011-02-10 Last updated: 2011-05-04
List of papers
1. Discovery, synthesis, and structural determination of a toxine-like disulfide-rich peptide from the cactus Trichoserus pachanoi
Open this publication in new window or tab >>Discovery, synthesis, and structural determination of a toxine-like disulfide-rich peptide from the cactus Trichoserus pachanoi
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:uu:diva-145716 (URN)
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2011-05-04
2. Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2
Open this publication in new window or tab >>Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2
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.

Keyword
cyclotides, native chemical ligation, peptides, protein folding, synthesis, thioesters
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-98767 (URN)10.1002/cbic.200700357 (DOI)000252292200017 ()18058973 (PubMedID)
Available from: 2009-03-03 Created: 2009-03-03 Last updated: 2012-01-23Bibliographically approved
3. An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS
Open this publication in new window or tab >>An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS
2010 (English)In: International Journal of Peptide Research and Therapeutics, ISSN 1573-3149, Vol. 16, no 3, 167-176 p.Article in journal (Refereed) Published
Abstract [en]

Cyclotides are mini-proteins of approximately 30 amino acid residues that have a unique structure consisting of a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. This unique cyclotide structure provides exceptional stability to chemical, enzymatic and thermal treatments and has been implicated as an ideal drug scaffold for the development into agricultural and biotechnological agents. In the current work, we present the first method for microwave assisted Fmoc-SPPS of cyclotides. This protocol adopts a strategy that combines optimized microwave assisted chemical reactions for Fmoc-SPPS of the peptide backbone, the cleavage of the protected peptide and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. To exemplify the utility of this protocol in the synthesis of a wide array of different cyclotide sequences we synthesized representative members from the three cyclotide subfamilies-the Mobius kalata B1, the bracelet cycloviolacin O2 and the trypsin inhibitory MCoTI-II. In addition, a "one pot" reaction promoting both cyclization and oxidative folding of crude peptide thioester was adapted for kalata B1 and MCoTI-II.

Keyword
Cyclotides, Microwave chemistry, Fmoc-SPPS, Circular proteins, Cystine knot, Native chemical ligation
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-134899 (URN)10.1007/s10989-010-9221-0 (DOI)000281682600007 ()
Available from: 2010-12-02 Created: 2010-12-02 Last updated: 2016-06-15Bibliographically approved
4. Interlocking disulfides in circular proteins: toward efficient oxidative folding of cyclotides.
Open this publication in new window or tab >>Interlocking disulfides in circular proteins: toward efficient oxidative folding of cyclotides.
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2011 (English)In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 14, no 1, 77-86 p.Article in journal (Refereed) Published
Abstract [en]

Cyclotides are ultrastable plant proteins characterized by the presence of a cyclic amide backbone and three disulfide bonds that form a cystine knot. Because of their extreme stability, there has been significant interest in developing these molecules as a drug design scaffold. For this potential to be realized, efficient methods for the synthesis and oxidative folding of cyclotides need to be developed, yet we currently have only a basic understanding of the folding mechanism and the factors influencing this process. In this study, we determine the major factors influencing oxidative folding of the different subfamilies of cyclotides. The folding of all the cyclotides examined was heavily influenced by the concentration of redox reagents, with the folding rate and final yield of the native isomer greatly enhanced by high concentrations of oxidized glutathione. Addition of hydrophobic solvents to the buffer also enhanced the folding rates and appeared to alter the folding pathway. Significant deamidation and isoaspartate formation were seen when oxidation conditions were conducive to slow folding. The identification of factors that influence the folding and degradation pathways of cyclotides will facilitate the development of folding screens and optimized conditions for producing cyclotides and grafted analogs as stable peptide-based therapeutics.

National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-139359 (URN)10.1089/ars.2010.3112 (DOI)000284572100009 ()20486762 (PubMedID)
Available from: 2010-12-23 Created: 2010-12-23 Last updated: 2011-05-04Bibliographically approved
5. Design, synthesis, structural and biological evaluation of backbone-engineered cyclotides
Open this publication in new window or tab >>Design, synthesis, structural and biological evaluation of backbone-engineered cyclotides
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:uu:diva-145719 (URN)
Available from: 2011-02-10 Created: 2011-02-10 Last updated: 2011-05-04

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