title: Solving the alpha-conotoxin folding problem: efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antagonists.
abstract: Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.
authors.affiliation: Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Markus
authors.initials: M
authors.lastname: Muttenthaler
authors.forename: Simon T
authors.initials: ST
authors.lastname: Nevin
authors.forename: Anton A
authors.initials: AA
authors.lastname: Grishin
authors.forename: Shyuan T
authors.initials: ST
authors.lastname: Ngo
authors.forename: Peng T
authors.initials: PT
authors.lastname: Choy
authors.forename: Norelle L
authors.initials: NL
authors.lastname: Daly
authors.forename: Shu-Hong
authors.initials: SH
authors.lastname: Hu
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Armishaw
authors.forename: Ching-I A
authors.initials: CI
authors.lastname: Wang
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Lewis
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Martin
authors.forename: Peter G
authors.initials: PG
authors.lastname: Noakes
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
authors.forename: Paul F
authors.initials: PF
authors.lastname: Alewood
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020916:Conotoxins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003964:Diaphragm / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D018733:Nicotinic Antagonists / Q000737:chemistry* / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects; D017510:Protein Folding; D055550:Protein Stability; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D012117:Resins, Synthetic / Q000737:chemistry; D017279:Selenocysteine / Q000737:chemistry; D013329:Structure-Activity Relationship; D014981:Xenopus
chemical_list: D020916:Conotoxins; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D012117:Resins, Synthetic; C089038:alpha-conotoxin PnIA; D017279:Selenocysteine