NMR-Based Mapping of Disulfide Bridges in Cysteine-Rich Peptides: Application to the μ-Conotoxin SxIIIA
Aleksandra Walewska,†‡ Jack J. Skalicky,§ Darrell R. Davis,∥ Min-Min Zhang,† Estuardo Lopez-Vera,†⊥ Maren Watkins,# Tiffany S. Han,† Doju Yoshikami,† Baldomero M. Olivera,† and Grzegorz Bulaj*∥
Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, μ-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. μ-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with 15N/13C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance (1H, 13C, 15N) NMR experiments and 2D [13C, 1H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of μ-conotoxins. μ-SxIIIA was found to be a potent blocker of the sodium channel subtype NaV1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure−function studies of many bioactive peptides.