Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.
Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.
Gowd
Konkallu Hanumae
KH
Department of Biology, University of Utah, Salt Lake City, Utah 84108, USA.
Yarotskyy
Viktor
V
Elmslie
Keith S
KS
Skalicky
Jack J
JJ
Olivera
Baldomero M
BM
Bulaj
Grzegorz
G
D000818:Animals; D003553:Cystine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D051379:Mice; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010455:Peptides; D017510:Protein Folding; D018038:Sodium Selenite / Q000737:chemistry*; D020866:omega-Conotoxin GVIA / Q000737:chemistry*; D020960:omega-Conotoxins / Q000737:chemistry
D004220:Disulfides; D010455:Peptides; D020960:omega-Conotoxins; D003553:Cystine; D020866:omega-Conotoxin GVIA; D018038:Sodium Selenite