title: Oxidative folding of conotoxins sharing an identical disulfide bridging framework.
abstract: Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100,000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.
authors.affiliation: Cognetix Inc, Salt Lake City, UT 84112, USA.
authors.forename: Erika
authors.initials: E
authors.lastname: Fuller
authors.forename: Brad R
authors.initials: BR
authors.lastname: Green
authors.forename: Phil
authors.initials: P
authors.lastname: Catlin
authors.forename: Olga
authors.initials: O
authors.lastname: Buczek
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Nielsen
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 / Q000235:genetics / Q000378:metabolism*; D003553:Cystine / Q000235:genetics / Q000378:metabolism*; D008974:Mollusca / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D017510:Protein Folding
chemical_list: D020916:Conotoxins; D003553:Cystine