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Interlocking disulfides in circular proteins: toward efficient oxidative folding of cyclotides.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi. Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för läkemedelskemi, Avdelningen för farmakognosi.
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2011 (Engelska)Ingår i: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 14, nr 1, s. 77-86Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2011. Vol. 14, nr 1, s. 77-86
Nationell ämneskategori
Farmaceutiska vetenskaper
Identifikatorer
URN: urn:nbn:se:uu:diva-139359DOI: 10.1089/ars.2010.3112ISI: 000284572100009PubMedID: 20486762OAI: oai:DiVA.org:uu-139359DiVA, id: diva2:381055
Tillgänglig från: 2010-12-23 Skapad: 2010-12-23 Senast uppdaterad: 2018-01-12Bibliografiskt granskad
Ingår i avhandling
1. Engineering of the Ultra-stable Cystine Knot Framework of Microproteins: Design, Chemical Synthesis and Structural Studies
Öppna denna publikation i ny flik eller fönster >>Engineering of the Ultra-stable Cystine Knot Framework of Microproteins: Design, Chemical Synthesis and Structural Studies
2011 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2011. s. 77
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 139
Nyckelord
cyclotides, cystine knot, protein engineering, NMR, solid phase, disulfide rich, ultra-stable, microprotein
Nationell ämneskategori
Farmaceutiska vetenskaper
Forskningsämne
Läkemedelskemi
Identifikatorer
urn:nbn:se:uu:diva-145749 (URN)978-91-554-8003-5 (ISBN)
Disputation
2011-03-25, B42, BMC, Husargatan 3, Uppsala, 13:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2011-03-04 Skapad: 2011-02-10 Senast uppdaterad: 2018-01-12

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