title: NMR-based mapping of disulfide bridges in cysteine-rich peptides: application to the mu-conotoxin SxIIIA.
abstract: 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, mu-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. Mu-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with (15)N/(13)C, 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 ((1)H, (13)C, (15)N) NMR experiments and 2D [(13)C, (1)H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of mu-conotoxins. Mu-SxIIIA was found to be a potent blocker of the sodium channel subtype Na(V)1.4 (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.
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Walewska
authors.forename: Jack J
authors.initials: JJ
authors.lastname: Skalicky
authors.forename: Darrell R
authors.initials: DR
authors.lastname: Davis
authors.forename: Min-Min
authors.initials: MM
authors.lastname: Zhang
authors.forename: Estuardo
authors.initials: E
authors.lastname: Lopez-Vera
authors.forename: Maren
authors.initials: M
authors.lastname: Watkins
authors.forename: Tiffany S
authors.initials: TS
authors.lastname: Han
authors.forename: Doju
authors.initials: D
authors.lastname: Yoshikami
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Bulaj
mesh_terms: D000818:Animals; D020916:Conotoxins / Q000737:chemistry*; D052078:Conus Snail; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D010084:Oxidation-Reduction; D010456:Peptides, Cyclic / Q000737:chemistry*; D017510:Protein Folding; D012015:Reference Standards; D013997:Time Factors
chemical_list: D020916:Conotoxins; D004220:Disulfides; D010456:Peptides, Cyclic; D003545:Cysteine