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. Antioxid. Redox Signal. 14, 77–86.
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References
1.
BarbetaBL, MarshallAT, GillonAD, CraikDJ, AndersonMA. Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc Natl Acad Sci U S A, 105:1221–1225. 2008.
2.
BarryDG, DalyNL, BokeschHR, GustafsonKR, CraikDJ. Solution structure of the cyclotide palicourein: implications for the development of a pharmaceutical framework. Structure, 12:85–94. 2004.
3.
ChayenNE, SaridakisE. Protein crystallization: from purified protein to diffraction-quality crystal. Nat Methods, 5:147–153. 2008.
4.
ClarkRJ, DalyNL, CraikDJ. Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design. Biochem J, 394:85–93. 2006.
5.
CraikDJ, ClarkRJ, DalyNL. Potential therapeutic applications of the cyclotides and related cystine knot mini-proteins. Expert Opin Investig Drugs, 16:595–604. 2007.
6.
CraikDJ, DalyNL, BondT, WaineC. Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J Mol Biol, 294:1327–1336. 1999.
7.
CraikDJ, DalyNL, MulvennaJ, PlanMR, TrabiM. Discovery, structure and biological activities of the cyclotides. Curr Protein Pept Sci, 5:297–315. 2004.
8.
CraikDJ, SimonsenS, DalyNL. The cyclotides: novel macrocyclic peptides as scaffolds in drug design. Curr Opin Drug Discov Devel, 5:251–260. 2002.
9.
DalyN, ClarkR, GöranssonU, CraikD. Diversity in the disulfide folding pathways of cystine knot peptides. Lett Pept Sci, 10:523–531. 2003.
10.
DalyNL, ClarkRJ, CraikDJ. Disulfide folding pathways of cystine knot proteins. Tying the knot within the circular backbone of the cyclotides. J Biol Chem, 278:6314–6322. 2003.
11.
DalyNL, ClarkRJ, PlanMR, CraikDJ. Kalata B8, a novel antiviral circular protein, exhibits conformational flexibility in the cystine knot motif. Biochem J, 393:619–626. 2006.
12.
DalyNL, KoltayA, GustafsonKR, BoydMR, Casas-FinetJR, CraikDJ. Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity. J Mol Biol, 285:333–345. 1999.
13.
DalyNL, LoveS, AlewoodPF, CraikDJ. Chemical synthesis and folding pathways of large cyclic polypeptides: studies of the cystine knot polypeptide kalata B1. Biochemistry, 38:10606–10614. 1999.
14.
DawsonPE, MuirTW, Clark-LewisI, KentSB. Synthesis of proteins by native chemical ligation. Science, 266:776–779. 1994.
15.
GillonAD, SaskaI, JenningsCV, GuarinoRF, CraikDJ, AndersonMA. Biosynthesis of circular proteins in plants. Plant J, 53:505–515. 2008.
16.
GöranssonU, HerrmannA, BurmanR, Haugaard-JonssonLM, RosengrenKJ. The conserved glu in the cyclotide cycloviolacin O2 has a key structural role. Chembiochem, 10:2354–2360. 2009.
17.
GöranssonU, SjögrenM, SvangårdE, ClaesonP, BohlinL. Reversible antifouling effect of the cyclotide cycloviolacin O2 against barnacles. J Nat Prod, 67:1287–1290. 2004.
18.
GranL. Oxytocic principles of Oldenlandia affinis. Lloydia, 36:174–178. 1973.
19.
GranL, SandbergF, SlettenK. Oldenlandia affinis (R&S) DC—a plant containing uteroactive peptides used in African traditional medicine. J Ethnopharmacol, 70:197–203. 2000.
20.
GunasekeraS, DalyN, ClarkR, CraikDJ. Dissecting the oxidative folding of circular cystine knot miniproteins. Antioxid Redox Signal, 11:971–980. 2008.
21.
GunasekeraS, FoleyFM, ClarkRJ, SandoL, FabriLJ, CraikDJ, DalyNL. Engineering stabilized vascular endothelial growth factor-A antagonists: synthesis, structural characterization, and bioactivity of grafted analogues of cyclotides. J Med Chem, 51:7697–7704. 2008.
22.
GustafsonKR, SowderRCII, HendersonLE, ParsonsIC, KashmanY, CardellinaJHII, McMahonJB, BuckheitRW, PannellLK, BoydMR. Circulins A and B: novel HIV-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia. J Am Chem Soc, 116:9337–9338. 1994.
HerrmannA, BurmanR, MylneJS, KarlssonG, GullboJ, CraikDJ, ClarkRJ, GöranssonU. The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity. Phytochemistry, 69:939–952. 2008.
JenningsC, WestJ, WaineC, CraikDJ, AndersonM. Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis. Proc Natl Acad Sci U S A, 98:10614–10619. 2001.
27.
JenningsCV, RosengrenKJ, DalyNL, PlanM, StevensJ, ScanlonMJ, WaineC, NormanDG, AndersonMA, CraikDJ. Isolation, solution structure, and insecticidal activity of kalata B2, a circular protein with a twist: do Mobius strips exist in nature?Biochemistry, 44:851–860. 2005.
28.
KamimoriH, HallK, CraikD, AguilarM. Studies on the membrane interactions of the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon resonance. Anal Biochem, 337:149–153. 2005.
29.
Leta AboyeT, ClarkRJ, CraikDJ, GöranssonU. Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2. Chembiochem, 9:103–113. 2008.
30.
LindholmP, GöranssonU, JohanssonS, ClaesonP, GullboJ, LarssonR, BohlinL, BacklundA. Cyclotides: a novel type of cytotoxic agents. Mol Cancer Ther, 1:365–369. 2002.
31.
MulvennaJP, SandoL, CraikDJ. Processing of a 22 kDa precursor protein to produce the circular protein tricyclon A. Structure, 13:691–701. 2005.
32.
PlanMR, GöranssonU, ClarkRJ, DalyNL, ColgraveML, CraikDJ. The cyclotide fingerprint in Oldenlandia affinis: elucidation of chemically modified, linear and novel macrocyclic peptides. Chembiochem, 8:1001–1011. 2007.
33.
PlanMR, SaskaI, CagauanAG, CraikDJ. Backbone cyclised peptides from plants show molluscicidal activity against the rice pest Pomacea canaliculata (golden apple snail)J Agric Food Chem, 56:5237–5241. 2008.
34.
RosengrenKJ, DalyNL, PlanMR, WaineC, CraikDJ. Twists, knots, and rings in proteins. Structural definition of the cyclotide framework. J Biol Chem, 278:8606–8616. 2003.
35.
SaskaI, GillonAD, HatsugaiN, DietzgenRG, Hara-NishimuraI, AndersonMA, CraikDJ. An asparaginyl endopeptidase mediates in vivo protein backbone cyclization. J Biol Chem, 282:29721–29728. 2007.
36.
SchöpkeT, Hasan AghaMI, KraftR, OttoA, HillerK. Hämolytisch aktive komponenten aus Viola tricolor L. und Viola arvensis Murray. Sci Pharm, 61:145–153. 1993.
37.
ShenkarevZO, NadezhdinKD, SobolVA, SobolAG, SkjeldalL, ArsenievAS. Conformation and mode of membrane interaction in cyclotides. Spatial structure of kalata B1 bound to a dodecylphosphocholine micelle. FEBS J, 273:2658–2672. 2006.
38.
SimonsenSM, DalyNL, CraikDJ. Capped acyclic permutants of the circular protein kalata B1. FEBS Lett, 577:399–402. 2004.
TamJ, WuC, LiuW, ZhangJ. Disulfide bond formation in peptides by dimethyl-sulfoxide—scope and applications. J Am Chem Soc, 113:6657–6662. 1991.
42.
TamJP, LuYA, YangJL, ChiuKW. An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc Natl Acad Sci U S A, 96:8913–8918. 1999.
43.
ThongyooP, BonomelliC, LeatherbarrowRJ, TateEW. Potent inhibitors of beta-tryptase and human leukocyte elastase based on the MCoTI-II scaffold. J Med Chem, 52:6197–6200. 2009.
44.
ThongyooP, TateEW, LeatherbarrowRJ. Total synthesis of the macrocyclic cysteine knot microprotein MCoTI-II. Chem Commun (Camb), 2848–2850. 2006.
45.
WangCK, ColgraveML, GustafsonKR, IrelandDC, GöranssonU, CraikDJ. Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis. J Nat Prod, 71:47–52. 2008.
46.
WangCK, ColgraveML, IrelandDC, KaasQ, CraikDJ. Despite a conserved cystine knot motif, different cyclotides have different membrane binding modes. Biophys J, 97:1471–1481. 2009.
47.
WangCK, HuSH, MartinJL, SjögrenT, HajduJ, BohlinL, ClaesonP, GöranssonU, RosengrenKJ, TangJ, TanNH, CraikDJ. Combined X-ray and NMR analysis of the stability of the cyclotide cystine knot fold that underpins its insecticidal activity and potential use as a drug scaffold. J Biol Chem, 284:10672–10683. 2009.
48.
WitherupKM, BoguskyMJ, AndersonPS, RamjitH, RansomRW, WoodT, SardanaM. Cyclopsychotride A, a biologically active, 31-residue cyclic peptide isolated from Psychotria longipes. J Nat Prod, 57:1619–1625. 1994.
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