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Results and DiscussionLinear cycloviolacin O2 (30 residues) was synthesized by manual solid phase peptidesynthesis (SPPS) followed by cyclisation and oxidative folding [4]. Folding conditionswere optimized to achieve maximum yield of correctly folded product (N). Concentrationsof cosolvents, detergents, redox agents [5], salt, as well as the effects of temperature andduration of reaction, were examined. Under most conditions misfolded products, mainlynon-native 3 SS species with I-II, III-IV and V-VI disulfide bridges, were predominant.However, using 35% DMSO as cosolvent gave a yield of ~40% N, which was furtheroptimized with redox agents to ~52% N.To have deeper understanding of the influence of various folding conditions oncyclotides, selected subfamilies (Möbius, hybrid and bracelet) were reduced and subjectedto various oxidative refolding conditions. Samples were then collected at different timepoints, NEM alkylated, and relative quantities of heterogenous intermediates, i.e, 1SS, 2SSand 3SS species were determined by LC-MS. The result shows that some folding buffersfavour non-native 3SS conformations that flip to the native fold directly or throughpartially oxidatively folded species depending on cyclotide subfamilies. Folding pathwaysof Möbius cyclotides to native species mainly dominated by 1SS/2SS while that of braceletcyclotides dominated by non-native 3SS (usually without accumulating 1SS/2SS)(Figure 2).These results are a significant step to the overall goal for biomolecular engineering onthe cyclotide scaffold for utilization of their diversity and extraordinary structural stabilityand for the understanding of the major heterogeneous folding intermediates existing inoxidative folding pathways.Fig. 2. Overview of folding pathways of cyclotides. Some folding buffers seem to favournon-native 3SS conformations that directly can flip to the native fold or go back throughpartly reduced species and then to the native structure. Path A is mainly and path B ispartly favoured in the folding pathway of Möbius cyclotides, whereas path B (usuallywithout accumulating 1SS and 2SS) is the dominant folding pathway in the folding of cyO2.AcknowledgmentsWe thank Swedish International Development Cooperation Agency (SIDA), the Department forResearch Cooperation (SAREC) (T.L.A. and U.G.), The Royal Swedish Academy of Sciences and theDisciplinary Domain of Medicine and Pharmacy, Uppsala University (U.G.). Work on cyclotides at theUniversity of Queensland is supported by grants from the Australian Research Council and theNational Health and Medical Research Council.References1. Craik, DJ., Cemazar, M., Daly, N.L. Curr. Opin. Drug Discov. Devel. 9, 251-260 (2006).2. Colgrave, M.L., Craik, D.J. Biochemistry 43, 5965-5975 (2004).3. Gunasekera, S, Foley, F.M., Clark, R.J., Sando, L., Fabri, L.J, Craik, D.J., Daly, N.L. J. Med. Chem.51, 7697-704 (2008).4. Aboye, T.L., Clark, R.J., Craik, D.J., Göransson, U. ChemBioChem 9, 103-113 (2008); Aboye, T.L., Clark, R.J., Burman, R, Craik, D.J., Göransson, U. Antioxidants and redox signalling in press,doi:10.1089/ars.2010.3112.5. DeLa Cruz, R., Whitby, F.G., Buczek, O., Bulaj, G. J. Pept. Res. 61, 202-212 (2003).143