FORUM ORIGINAL RESEARCH COMMUNICATION Stabilization of a-Conotoxin AuIB: Influences of Disulfide Connectivity and Backbone Cyclization

a-Conotoxins are peptides isolated from the venom ducts of cone snails that target nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential applications for treating a range of conditions in humans, including pain. However, like all peptides, conotoxins are susceptible to degradation, and to enhance their therapeutic potential it is important to elucidate the factors contributing to instability and to develop approaches for improving stability. AuIB is a unique member of thea-conotoxin family because the nonnative ‘‘ribbon’’ disulfide isomer exhibits enhanced activity at the nAChR in rat parasympathetic neurons compared with the native ‘‘globular’’ isomer. Here we show that the ribbon isomer of AuIB is also more resistant to disulfide scrambling, despite having a nonnative connectivity and flexible structure. This resistance to disulfide scrambling does not correlate with overall stability in serum because the ribbon isomer is degraded in human serum more rapidly than the globular isomer. Cyclization via the joining of the Nand C-termini with peptide linkers of four to seven amino acids prevented degradation of the ribbon isomer in serum and stabilized the globular isomers to disulfide scrambling. The linker length used for cyclization strongly affected the relative proportions of the disulfide isomers produced by oxidative folding. Overall, the results of this study provide important insights into factors influencing the stability and oxidative folding of a-conotoxin AuIB and might be valuable in the design of more stable antagonists of nAChRs. Antioxid. Redox Signal. 14, 87–95.