| 1. |
|
|
| 2. |
- Aboye, Teshome Leta, et al.
(författare)
-
Interlocking disulfides in circular proteins : toward efficient oxidative folding of cyclotides.
- 2011
-
Ingår i: Antioxidants and Redox Signaling. - : Mary Ann Liebert Inc. - 1523-0864 .- 1557-7716. ; 14:1, s. 77-86
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
|
|
| 6. |
|
|
| 7. |
- Burman, Robert, et al.
(författare)
-
Chemistry and Biology of Cyclotides : Circular Plant Peptides Outside the Box
- 2014
-
Ingår i: Journal of natural products (Print). - : American Chemical Society (ACS). - 0163-3864 .- 1520-6025. ; 77:3, s. 724-736
-
Forskningsöversikt (refereegranskat)abstract
- Cyclotides stand out as the largest family of circular proteins of plant origin hitherto known, with more than 280 sequences isolated at peptide level and many more predicted from gene sequences. Their unusual stability resulting from the signature cyclic cystine knot (CCK) motif has triggered a broad interest in these molecules for potential therapeutic and agricultural applications. Since the time of the first cyclotide discovery, our laboratory in Uppsala has been engaged in cyclotide discovery as well as the development of protocols to isolate and characterize these seamless peptides. We have also developed methods to chemically synthesize cyclotides by Fmoc-SPPS, which are useful in protein grafting applications. In this review, experience in cyclotide research over two decades and the recent literature related to their structures, synthesis, and folding as well the recent proof-of-concept findings on their use as "epitope" stabilizing scaffolds are summarized.
|
|
| 8. |
- Burman, Robert, et al.
(författare)
-
Cyclotide-membrane interactions: defining factors of membrane binding, depletion and disruption
- 2011
-
Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1808:11, s. 2665-2673
-
Tidskriftsartikel (refereegranskat)abstract
- The cyclotide family of plant-derived peptides is defined by a cyclic backbone and three disulfide bonds locked into a cyclic cystine knot. They display a diverse range of biological activities, many of which have been linked to an ability to target biological membranes. In the current work, we show that membrane binding and disrupting properties of prototypic cyclotides are dependent on lipid composition, using neutral (zwitterionic) membranes with or without cholesterol and/or anionic lipids. Cycloviolacin O2 (cyO2) caused potent membrane disruption, and showed selectivity towards anionic membranes, whereas kalata B1 and kalata B2 cyclotides were significantly less lytic towards all tested model membranes. To investigate the role of the charged amino acids of cyO2 in the membrane selectivity, these were neutralized using chemical modifications. In contrast to previous studies on the cytotoxic and antimicrobial effects of these derivatives, the Glu6 methyl ester of cyO2 was more potent than the native peptide. However, using membranes of Escherichia coil lipids gave the opposite result: the activity of the native peptide increased 50-fold. By using a combination of ellipsometry and LC-MS, we demonstrated that this unusual membrane specificity is due to native cyO2 extracting preferentially phosphatidylethanolamine-lipids from the membrane, i.e., PE-C16:0/cyC17:0 and PE-C16:0/C18:1.
|
|
| 9. |
- Burman, Robert, et al.
(författare)
-
Cyclotide proteins and precursors from the genus Gloeospermum : filling a blank spot in the cyclotide map of Violaceae
- 2010
-
Ingår i: Phytochemistry. - : Elsevier BV. - 0031-9422 .- 1873-3700. ; 71:1, s. 13-20
-
Tidskriftsartikel (refereegranskat)abstract
- Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.
|
|
| 10. |
- Burman, Robert, 1979-, et al.
(författare)
-
Cytotoxic potency of small macrocyclic knot proteins : Structure-activity and mechanistic studies of native and chemically modified cyclotides
- 2011
-
Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 9:11, s. 4306-4314
-
Tidskriftsartikel (refereegranskat)abstract
- The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability and active in a wide range of biological activities make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (Möbius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity and derivatives in which the Trp residue was modified showed low effect, suggesting the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the Möbius cyclotides. However, despite these differences, a systematic test of mixtures of cyclotides, even from both subfamilies revealed that they act in an additive way.
|
|
